Biology, pathophysiology, and epidemiology of pancreatic cancer

CHAPTER 1

Biology, pathophysiology, and epidemiology of pancreatic cancer 2 Begum Dariya1, Afroz Alam1, Ganji Purnachandra Nagaraju 1 2

Department of Bioscience and Biotechnology, Banasthali University, Vanasthali, India; Department of Hematology and Medical Oncology, Winship Cancer Institute, Emory University, Atlanta, GA, United States

Abstract Pancreatic cancer (PC) is the fourth leading cause of cancer-related deaths and will become the second most prevalent one by 2030 owing to its aggressive nature. It has the worst prognosis and most limited efficiency of commonly available therapies. The incidence and mortality rate are high in developed countries. Variabilities in geography and gender are proportional to the increase in risk factors. Risk factors include an unhealthy lifestyle, and modifiable factors that cause PC are known but insufficient; therefore, a better understanding of the etiology and epidemiology of PC is essential for its prevention. Germ line mutations, hereditary disorders (familial adenomatous polyposis, familial atypical multiple mole melanoma syndrome, and hereditary nonpolyposis colorectal cancer), inherited disorders (BRCA2 mutations), and gene polymorphism are factors that control the detoxification of carcinogens from the environment and alter risk factors. Inherent resistance developed against chemotherapeutic drugs results from the occurrence of dense stroma developed, including extracellular matrix and nonneoplastic cells (fibroblasts, immune, and invasive and vascular cells), which significantly impairs the drug delivery mechanism. Hence, an increased understanding of cellular pathways including Notch, Wnt, Hedgehog, and Kristen rat sarcoma (Kras) involved in impairing DNA repair processes, cancer cell metabolism, and metastasis can provide new therapeutic strategies for developing novel therapeutic regimens. Clinically, the use of markers including diagnostic and prognostic markers and somatic altered genes such as Kras and TP53 has been investigated, although these tests remain inconsistent owing to their inappropriate clinical performance. Thus, elucidating crucial molecular mechanisms involved in the biology of PC is necessary and will enable future research aimed at developing novel and effective biomarkers for the early diagnosis and therapy of PC.

Keywords: DNA repair; Epidemiology; Kras; Notch; Pancreatic cancer; Risk factors; Wnt.

G.P. Nagaraju, S. Ahmad (eds.) Theranostic Approach for Pancreatic Cancer ISBN 978-0-12-819457-7 https://doi.org/10.1016/B978-0-12-819457-7.00001-3

Copyright © 2019 Elsevier Inc. All rights reserved.

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Theranostic Approach for Pancreatic Cancer

List of abbreviations APC Adenomatous polyposis coli ARK AMPK related kinase BRCA Breast cancer type 1 CA19-9 Carbohydrate antigen CDCP1 CUB domain contained protein 1 CDH1 Epithelial or E-cadherin CDKN2A Cyclin dependent kinase inhibitor 2A DHSA Dihydrosanguinarine DNMT DNA methyltransferase DPC4 Deleted in PC ECM Extracellular matrix protein EMT Epithelial mesenchymal transition FAMMM Familial atypical multiple mole melanoma syndrome FOXC1 Forkhead box C1 GNAS Guanine nucleotide binding protein GSK3b Glycogen synthase kinase 3b HDAC Histone deacetylase HES Hair enhance of split family HP Hereditary pancreatitis IGF Insulin growth factor IPMN Intraductal papillary mucinous neoplasma LEF Lymphoid enhancing factor LITAF Lipopolysaccharide induced tumor necrosis-a factor LRP5 Lipoprotein receptor related protein MCN Mucinous cystic neoplasm MGMT Methylguanine methyl transferase MiRNA MicroRNA MMP Matrix metalloproteinases MUC Mucin NICD Notch intracellular domain NOX NAD(P)H oxidase PCSC Pancreatic cancer stem cells PDAC Pancreatic ductal adenocarcinoma PDX Patient-derived xenografts PSC Pancreatic stellate cells RARb Retinoic acid receptor b RB Retinoblastoma SEER Surveillance, Epidemiology and End Results SHH Sonic Hedgehog SIRT Sirtuin SPARC Secreted protein acidic and rich in cysteine TCF T-cell factor TGF-b Transforming growth factor-b

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TME Tumor microenvironment THOR TERT hypermethylated oncologic region UPSM UltraepH sensitive micelles

Introduction Pancreatic cancer (PC) is a lethal aggressive disease with high mortality rates. It represents the fourth most frequent cause of cancer-related deaths. It will soon be ranked as the second most malignant cancer worldwide, with an overall survival rate of 26% for 1 year, 8.5% for 5 years in advanced stages of the disease, and 22% for early-stage detection with surgical resection of the tumor. Statistical analysis for PC globally estimates 1,000,000 new cases with 65,000 deaths annually [1]. According to reports from the National Cancer Institute Surveillance, Epidemiology and End Result Program, estimated new cases in 2018 were 55,440, with 44,330 deaths. The diagnosis of PC in later stages, its high inherent resistance against conventional chemotherapy, the lack of biomarkers, and insufficient treatment options result in its poor prognosis. Clinical and epidemiological studies demonstrated that early detection of neoplastic precursors and early diagnosis are effective means to regulate cancer-related mortality. Hence, the detection of pancreatic precursor lesions including mucinous cystic neoplasm (MCN), macroscopic intraductal papillary mucinous neoplasma (IPMN), and microscopic pancreatic intraepithelial neoplasia, could reduce the incidence and mortality rate of PC patients [2,3] and has been successfully tested in individuals with a strong family history of PC by imaging and surgical techniques [4e6]. PC progresses in a multistep fashion involving the transformation of healthy cells into malignant ones. This mass of malignant cells is a heterogeneous complex of tumor cells consisting of endothelial cells, immune cells, and stromal and hematopoietic cells driven by genetic alterations [7]. The most common risk factors identified include the onset of diabetes mellitus (DM), hereditary and recent pancreatitis, smoking, obesity, age, and hereditary PC, which are responsible for developing 50%e60% of PCs, whereas 5%e10% is due to genetic mutations in certain genes such as Kristen rat sarcoma (Kras). In addition to genetic mutations, epigenetic aberrations of oncogenes and silencing of tumor suppressor genes such as p16, TP53, and cyclin-dependent kinase inhibitor 2A (CDKN2A) are risk hallmarks of PC.

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Theranostic Approach for Pancreatic Cancer

The most common form of PC is pancreatic ductal adenocarcinoma (PDAC), which represents about 90% of PC cases. Novel therapeutic routes targeting epigenetics, modulators, and regulators of oncogenes and tumor suppressors are emerging, although molecular and pathological insights into the fatal cancer entity remain essential to improve early detection of the disease, survival time, and quality of life. This chapter highlights the epidemiology, biology, and histopathology of PC, providing an entity of tumor for therapeutic approaches to develop the survival rate and provide a better prognosis.

Epidemiology of pancreatic cancer The asymptomatic nature of PC restricts its diagnoses to only its advanced stage with a poor prognosis. Thus, it is essential to improve methods to detect precursors at earlier stages. Furthermore, knowing and understanding the disease epidemiology could be important for primary prevention, allowing the elucidation and identification of etiology risk factors including environmental and genetical factors associated with PC. The epidemiology of PC generally measures the accurate difference of incidence and mortality rates, in which the incidence rate is the new cases and the mortality rate is the number of deaths occurring annually in a specified group. In 1999, Parkin et al. first obtained a data bank for a incidence and mortality rate from 23 areas around the world, comparing registries of cancer. They arrived at the conclusion that PC ranked ninth as the most common cause of cancer-related mortality, with 168,000 calculated deaths and the 13th cause of death in both sexes [8]. GLOBOCAN is Windowsbased software that uses International Agency for Research on Cancer data to estimate the incidence and mortality rate by country. In the 2012, GLOBOCAN estimated that there were 338,000 new cases and 331,000 deaths, and thus that PC ranked seventh for mortality in both sexes [9]. It was further estimated that PC ranked 14th for new cases in 2018, with very low variations seen in males and females, but that it ranked seventh for cancer-related morality worldwide and caused 330,000 deaths per year [9]. The incidence and mortality rate are always identical because of the high fatality of disease. The incidence and mortality rates of PC calculated were higher in developed countries; they ranked second in France, fourth in the United States (North America was 7.4 per 100,000), sixth in Europe

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(6.8 per 100,000), and lower in countries such as Africa (2 per 100,000) and Asia (3.2 per 100,000). However, it was more commonly noticed among men than women and varied geographically and according to gender. According to an estimation of the global incidence rate, about 178,000 men and 160,000 women were diagnosed with PC in the 2012. However, the PC mortality rate has been increasing in both genders in countries such as the United States, Japan, China, and Europe [10e13]. The high mortality rate was attributed to diagnosis of the fatal disease in its later stages. As revealed from Surveillance, Epidemiology. and End Results (SEER) data, only 10% of patients are diagnosed at early localized stages and 52% are diagnosed only at stage 4, with the disease having metastasized to the other organs of the body. Hence, a survival rate of only 5 years is estimated for PC despite novel technologies, although the rate varies owing to data quality worldwide [14]. The incidence and mortality rate correlate with an increase in age and represents predominantly a disease of elder populations diagnosed after 55 years of age [10,11,15], owing to the accumulation of DNA damage over time resulting from exposure to risk factors as well to certain biological processes. The incidence rate of PC in patients aged 55e59 years was 10.4 per 100,000 and 24.0 for age 65e69 years [16]. The occurrence of PC also varies with race, which could be because of differences at the molecular level. K-ras mutations to valine leading to PC are more frequently seen among the black race of the United States than the Caucasian race [17]; similarly, differential expression in K-ras and P53 showed a racial difference among Chinese and Japanese patients [18,19]. However, this estimation is not clear. The Cancer Statistics Review as well as SEER revealed that the incidence rate of PC has increased over the years [11]. Quante et al. from Germany also reported that the incidence rate of PC surpassed CRC and that PC might rank second for cancer-related mortalities by 2030 [20]. The low survival rate noted in PC results from high serum albumin levels, modalities in therapy, health care system differences, and the size of the tumor, in addition to the advanced-stage diagnosis. The increase in the incidence rate is always proportional to the increase in risk factors and detecting the risk factors is not simple.

Etiology of pancreatic cancer Exploring and identifying the critical risk factors in the group is essential because the screening techniques of PC could detect the disease only at the

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advanced stage. The risk factors are determined to be 40% responsible for the development of PC. They can be differentiated as unmodifiable hereditary risk factors (10%) and modifiable environmental risk factors. As discussed, black people are at higher risk for developing PC compared with white, Asian, and Hispanic people. In the case of gender, men are more highly prone to PC than are women. However, increased age makes both genders at higher risk for PC.

Hereditary, genetic, and mutation factors Many other inherited genetic disorders constitute risk factors for PC. The risk for PC is about 62%e67% in patients with first-degree relatives with a medical history of PC, as estimated from metaanalyses and pooled analyses [21,22]. People with hereditary nonpolyposis colorectal cancer, also called Lynch syndrome, with a microsatellite instability in the mismatch repair genes PMS2, MSH2, MSH6, EPCAM, and MLH1 at chromosomes 2 and are prone to early-onset colorectal cancer (CRC) and PC [23]. Familial adenomatous polyposis is another syndrome characterized by the onset of polyps in the tract of gastrointestine, which later become malignant; they develop owing to a mutation in the adenomatous polyposis coli (APC) gene affected at chromosome 5q12-21 [24]. Its occurrence is uncertain and is misdiagnosed as ampulla. Hereditary breast and ovarian cancer syndrome also causes 17%e19% of hereditary PC. It is generally caused by a mutation in the genes breast cancer type 1 (BRCA1) and especially BRCA2, with chromosome 13 affected [25]. PeutzeJeghers syndrome, otherwise called hamartomatous polyposis syndrome, results from a germinal mutation in STK11/LKB1 at chromosome 19p; it causes PC as well gastrointestinal neoplasia [26]. Patients with familial atypical multiple mole melanoma syndrome (FAMMM) are at increased risk for PC. This is because of the gene mutation in p16INK4a at chromosome 9p21, which dysregulates the normal cellular cycle, in addition to a mutation in CDKN2A and a family history of familial melanoma [27]. When detected, it is characterized as malignant melanoma in a first- or second-degree relative, who are highly prone for the occurrence of PC (a 13- to 20-fold increased risk). Hereditary pancreatitis (HP) is a disease caused by a mutation in the PRSS1 gene, resulting in recurrent acute pancreatitis from childhood leading to dysregulation and chronic inflammation in the pancreas; it causes an increased

Biology, pathophysiology, and epidemiology of pancreatic cancer

Table 1.1 Heritable diseases and PC. Mutation/ Syndrome disorder

Hereditary nonpolyposis colorectal cancer

Familial adenomatous polyposis Hereditary breast and ovarian cancer PeutzeJeghers syndrome

Familial atypical multiple mole melanoma syndrome Hereditary pancreatitis Cystic fibrosis

Estimated risk

Chromosome

Gene

Microsatellite instability/ mismatch repair mutation Early onset of polyps

2,3

MSH2, MSH6, EPCAM, MLH1, PMS2 APC

Common inherited mutation Sporadic and inherited disease/germ line mutation P16 Leiden mutation

13

BRCA1, BRCA2

17%e19%

19p

STK11/ LKB1

10%e30%

9p21

P16INK4a CDKN2A

Acute pancreatitis and chronic inflammation Acute pancreatitis

7q35

PRSS1, SPINK1, CFTR

13- to 20-fold Increased risk 40%

7q31

CFTR

5q12-21

7

1%e6%

2%

40%

risk for PC [28]. The lifetime risk estimate owing to this disease is 40%. Cystic fibrosis is disorder similar to HP; it is caused by a mutation in the cystic fibrosis transmembrane gene and results in recurrent acute pancreatitis, and thus the developing onset of PC [29]. Chronic pancreatitis is again an increased risk for PC, with an estimated risk of 4% [30]. Chronic inflammation of the pancreas provokes the production of interleukin (IL)-6, IL-8, tumor necrosis factor-a, transforming growth factor-b (TGF-b), and inducing cellular proliferation [17]. Hereditary diseases and their estimated risks for causing PC are listed in Table 1.1. DM is detected in almost 80% of PC; however the relation is still unclear. The onset of DM types 1 and 2 can

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double the risk of PC, and DM type 2 is often detected with higher levels of insulin growth factor-1 (IGF-1) and hyperinsulinemia. From studies on the PC burden, it was estimated from a population in Italy that 9.7% of cases of PC result from DM [31e34]. Researchers observed that hyperinsulinemia and hyperglycemia create an interaction between tumorassociated macrophages and pancreatic stellate cells (PSCs), inducing fibrosis, inhibiting apoptosis, and promoting the proliferation of cells [35,36]. Furthermore, certain bacteria called Helicobacter pylori cause inflammation in the stomach; they are also at increased risk for stomach cancer and PC. However, the estimated risk was high in the stomach compared with the pancreas (4%e25%). Hepatitis virus was also studied as a risk for PC, because it is commonly detected in PC patients.

Environmental factors Although the incidence rate increases with an increase in risk factors, not all risk factors are easy to detect. However, few unmodifiable hereditary and modifiable environmental factors are detected as associated risk factors for the cause of PC. Environmental factors include alcohol consumption, smoking, DM, and chronic pancreatitis. Smoking produces carcinogens from tobacco, which makes its way to the pancreas through the bloodstream from the upper aerodigestive tract. In a few cases, the consumed tobacco is also directly refluxed into the ducts of the pancreas, released from the duodenum. As published in various reports, the risk for PC is twofold higher in patients habituated to smoking and was estimated at 20%e35% [37]. Furthermore, the prevalence of PC is significantly higher in males because of temporal trends in cigarette smoking. Tobacco intake mutates Kras and p53 genes and induces chronic inflammation, leading to pathological differences in the mechanism promoting cytokine induction and the activation of growth factors, which finally results in cellular transformation. Consumption of alcohol causes the production of metabolites that promote chronic inflammation and genetic instability. The relative risk calculated was 1.22e1.36 for developing PC [38]. Besides smoking, there are certain risk factors including physical inactivity, obesity, and dietary factors that also influence PC mortality. Developing countries are also high in mortality rate in the same way as developed countries because of their adaptations to the lifestyle of developed countries. Epidemiological data showed that dietary food including red meat and a high caloric food intake cause obesity and lead to higher risk for PC. Furthermore, a few studies demonstrated that the

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estimated relative risk for PC was 1.12 for every 5-kg/m2 increase in body mass index (BMI). Adiposopathy is a disorder observed in obese people; it is also called chronic adipose disease. It causes a hormonal imbalance with high levels of leptin and low levels of adiponectin observed owing to proinflammatory cytokines produced by macrophages, which indicates a relationship between PC and high BMI. However, regular physical activity and the consumption of low-calorie foods including green vegetables, fruit, tofu, and fish in Japan reduced the risk to a certain extent. Furthermore, chemicals are risk factors for PC, because exposure to certain chemicals including aspirin, pesticides, petrochemicals, and benzenes may cause PC.

Histopathophysiology of pancreatic cancer The clinical, anatomical, and molecular pathology represent a bridge between research and medicine. Tumors in the pancreas are mostly exocrine and include adenosquamous carcinoma, colloid carcinoma, acinar cell carcinoma, signet ring cell carcinoma, pancreatoblastoma, cystadenocarcinoma, and hepatoid carcinoma. The malignant tumor that commonly occurs in pancreas is ductal adenocarcinoma; it is characterized as a glandular structure and is also called PC. Clinically, the carcinoma arises in the head of the pancreas and rarely in the body and tail of the organ. It has nonspecific symptoms and varies with the region. PC that occurs in the head (75%) of the pancreas is commonly seen in patients with pancreatitis and obstructive jaundice. This cancer causes blockage in the common bile duct, resulting in jaundice with symptoms including nausea, vomiting, itching, dark urine, and light-colored stool. Carcinoma in the body (15%) and tail (10%) of the pancreas are diagnosed later and have a worse prognosis, with symptoms including abdominal pain that exudes toward the sides of the back [39]. It is also reported that the intensity of pain is associated with immune and inflammatory cells, leading to perinuclear invasion. The progression of PC is a stepwise process involving the activation and deactivation of oncogenes and tumor suppressor genes, respectively, with deregulation of the cell cycle. A better understanding of genetics and their molecular biology by the scientific community may be achieved by determining the precursor lesions of the cancer and their classification. This may pave the way for further research studies that could lead to early diagnosis. The first transgenic mouse model developed in 2003 described low-grade precursor lesions in metastatic cancer and improved knowledge

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about cellular interactions and molecular mechanisms involved in initiating and developing PC, which were essential in the field of drug testing [40]. On the basis of biology, morphology, and clinical behavior, pancreatic neoplasia is differentiated into three neoplasias with distinct precursor lesions. Intraductal papillary mucinous neoplasm: These are cystic neoplasms that differ in their potential to developing malignancy, according to their origin and histological subtypes. They arise in the head of the pancreas and develop into chief pancreatic ducts. Their main branches are mucin (MUC)producing neoplastic cells. Dilation of the branches and duct irregularly filled with mucin are observed on sectioning the tissue. Their size was observed to be several centimeters in length of papillary formation from microscopic projections to visible papillae. Mutations observed in IPMN lesion are Kras (mutation in codon 12 or 13) and guanine nucleotide binding protein (GNAS1), detected in the early stages, whereas Kras mutations arise as lowgrade gastric type IPMN and GNAS1 as high-grade lesions of intestinal type developing into invasive carcinoma of a colloid type [41]. Furthermore, mutations in TP53 are also detected in the later stages of IPMN, leading to invasive cancer, in addition to CDKN2A and SMAD4. Mucinous cystic neoplasm: These cysts exist as a separate neoplasms consisting of thick MUC and hemorrhagic material with no communication with the native pancreatic ductal system. They are composed of epithelial cells that are unilocular or multilocular, ranging from millimeters to several centimeters. They are associated with ovarian-type stroma and can be differentiated into benign, borderline, and malignant components with low (cytological atypia and no mitosis), moderate (papillary projection), and high-grade (complex branching and loss of polarity) dysplasia, respectively. Genetic alterations including point mutations in the Kras gene at codon 12 are detected early and mutations in tumor suppressor genes including SMAD4 are also observed in their later stages. Moreover, mutations in TP53 and CDKN2 are associated with invasive disease. It was suggested that IPMN and MCN lesions express highemolecular weight glycoprotein MUC2. Pancreatic intraepithelial neoplasia: Longnecker and Klimstra proposed that pancreatic intraepithelial neoplasia (PanIN) lesions are wellcharacterized premalignant precursors of PC described as noninvasive lesions. They initiate from pancreatic ducts at a small caliber, measuring less than 5 mm in diameter, differentiated histologically into PanIN-1 (PanIN-1A/PanIN-1B), PanIN-2, and PanIN-3 lesions that differ in

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their architectural and cytoplasmic atypia. These lesions are reported to develop PC with their expression in the early stage. Exocrine progenitor cells, insulin-producing cells, and adult acinar cells can also be targeted for these lesions [42]. PanIN-1 (PanIN-1A/PanIN-1B) are lesions originating from the epithelium and appear as flat, elongated, columnar papillary cells with uniform nuclei producing MUC. They are low-grade lesions with activating Kras mutations initiating from them. In addition, Kras are even detected in PanIN-2 lesions that have more numerous papillary cells with abnormalities in the nucleus, including nuclear atypia, loss of polarity in the nucleus, pseudostratification, and hyperchromasia in the nucleus. The lesion shows inactivation of tumor suppressor genes including CDKN2A/ p16INK4A, which encodes proteins CDK4/6 and p16, which are generally involved in arresting cell cycle at G1 phase. Moreover, owing to the inactivation of these proteins, apoptosis is inhibited in PC [43e45]. PanIN-3 lesions, otherwise called “carcinoma in situ” with epithelial cells, appear as clusters with high rates of mitosis and nuclear polymorphism that finally separate and enter into the noninvasive lumen. Various mutations in SMAD4, TP53, BRCA2, and DPC4 promote the progression from PanIN-3 to adenocarcinoma. In addition to the hypothesis that involves PanIN, IPMN, and MCN mediating the progression from the centro-acinar-acinar compartment into the carcinoma that carries on through the metaplasiaedysplasia sequence, an alternative model involves tumor progression through the atypical flat lesion/acinar ductal carcinogenesis model, not including PanIN, that is also considered precursor lesions for PC [46,47]. Morphologically, they appear to be flat to cuboidal epithelial cells with an enlarged nucleus and prominent nucleoli with a high mitotic rate. They have altered expression in p16/ CDKN2A owing to hypermethylation or genetic deletion. However further characterization is essential to determine the molecular views and genetic points, because this could be a novel biomarker for detecting PC at an early stage. The tissue diagnosis and pathological staging of PC as done by the American Joint Committee on Cancer Tumor-Node-Metastasis Classification assesses the resectability of the tumor enhanced through multidetector computed tomography (CT). In addition, there a few other cases are essential that are classified for nonresectable PC based on their histopathological and cytological specimens for biopsy examination. Furthermore, microscopic marginal resection factors including pancreatic, biliary, and retroperitoneal margins are beneficial for overcoming poor prognosis in PC.

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Advanced techniques used to determine the prognosis of PC include contrast-enhanced multislice CT scan for noninvasive staging, endoscopic ultrasonography-guided fine-needle aspiration for diagnosing tissue, endoscopic retrograde cholangiopancreatography for brushing cytology done at the preoperative stage during endoscopic stenting, and laparoscopy done to determine metastatic and vascular involvement.

Markers for diagnosis and prognosis PC is a heterogeneous and asymptomatic disease composed of complex neoplastic cells that are detectable through the expression of immunohistochemical markers. Current research findings focus on investigating and developing biomarkers related to the disease stage. They also develop targeted therapeutic approaches associated with these disease-related biomarkers. Techniques widely used to assess biomarkers in the field of translational research include chromogenic in situ hybridization, immunohistochemistry, microarray, and mutation analysis using serum, urine, and pancreatic juice, of which immunohistochemistry is a widely accepted technique [48,49]. Carbohydrate antigen (CA19-9) is a Lewis antigen isolated from MUC1, a tumor-associated protein. Elevated levels of CA19-9 are detected in the diagnosis of benign, chronic pancreatitis, liver cirrhosis, and obstructive jaundice. High levels are correlated with poor prognosis whereas low levels of CA19-9 marker are detected in poorly differentiated and early stages of PC. Furthermore, a-enolase is also upregulated in PC and complements CA19.9 levels as a diagnostic marker in the resectable and advanced PC stage [50]. In addition to these markers, the mesothelin gene, osteopontin, macrophage inhibitor cytokine 1, and matrix metalloproteinase-1 (MMP) inhibitors are detected as markers, although they are not superior to CA19-9 [51e54]. Certain oncogenic proteins can also function as biomarkers, including sirtuin (SIRT)-3 and 7, insulin-like growth factore2 messenger ribonucleic acid protein, and zinc/iron-regulated transporter related protein-3. Other markers are composed of vimentin (encoded by the VIM gene in mesenchymal cells), the CD99 gene (cell surface transmembrane glycoprotein), CDCP1 (CUB domain contained protein 1), K homology domain containing protein, and phosphorylated extracellular signalregulated kinases. Furthermore MUC1, MUC5AC, and MUC4 are membrane MUC proteins detected as tumor markers for PC diagnosis.

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Precursor lesions in PC accompanied by genetic alterations are also considered to be biomarkers and include mutant Kras, which can also be detected as an early diagnosis marker, identified at its nonresectable PC stage [55,56]. Similarly, osteopontin is a protein upregulated with the expression of proinflammatory cytokines, which are detected as diagnostic markers for detection at early stages of various cancers including lung, gastric, and PC. MicroRNA (miRNA) has also been investigated for effectiveness as a marker that regulates the proliferation and apoptosis of PC cells. It includes miR-221, miR-222, miR-155, and miR-21, which can segregate benign and malignant pancreatic tissues [57e59]. Moreover, p21, SMAD4, and Bcl-2 are also considered to be tissue-based markers for PC diagnosis [60,61]. miR-196a-2 expression indicates poor survival. It has been investigated as a prognostic marker with Kras and TP53 and is recommended for clinical progression in PC patients [60]. The carcinoembryonic antigen is a glycoprotein detected as a biological marker in the prognosis of PC. Its level correlates with the size, differentiation of the tumor, and metastasis to the liver and lymphatic system. S100A6 are highly expressed in PanIN and IPMN lesions that influence progression, metastasis, and invasion in PC. Thus, markers that focus on the early diagnosis could help clinicians to select specific therapeutic regimens suitable for the cancer stage. However, future research into potent markers is required for improved diagnosis and management of PC.

Biology of pancreatic cancer The biology of PC can be easily understood from studies involving genetic and epigenetic alterations and molecular overexpression. PC is a genetic disease that develops mostly from epithelial cells that line the pancreatic ducts, also known as pancreatic exocrine cancer or pancreatic adenocarcinoma. These cells produce digestive enzymes. Rarely, PC can also develop from the endocrine cells of pancreas called islets of Langerhans, which produce hormones including insulin and glucagon released into the bloodstream, involved in controlling blood sugar levels in the body. This type of cancer is called pancreatic endocrine cancer. In their review Alvin et al. [62] described PC in evolutionary terms, classifying PC into three stages including the initiation of normal cells driven by gene mutation resulting from environmental exposure and clonal expansion, in which mutated cells continue dividing; the creation of a clonal population; and finally, introduction into a foreign microenvironment, in which tumor cells

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Theranostic Approach for Pancreatic Cancer

break the basement membrane and pass into the surrounding stroma. Multistep PC starts from pancreatic intraepithelial neoplasia (PanIN-IA, IB, II, and III) driven by gene mutation and finally develops into invasive neoplastic lesions. PanIN are the histological precursors of PC characterized by enlarged nuclei, crowding of the nucleus, polarity loss, hyperchromatin, and pseudostratification [63]. The progression originates from dysplastic epithelium cells including PanIN-IA and B, develops into dysplasia cells including PanIN-II and -III, and finally evolves into invasive carcinoma characterized by mutations in oncogenes such as Kras and tumor suppressor genes such as CDKN2A, TP53, and SMAD4. In one way, PC results from epigenetics modulations and the inhibition of digestive enzymes that start in childhood and eventually become chronic in adults. A statistical analysis of PC revealed that initiation of the disease is associated with cell division and time. This was followed by a scientific debate confirming that extrinsic factors including radiation and carcinogens are highly responsible for cancer, compared with intrinsic factors involved in DNA replication (familial mutation) [64e66]. Wu et al. [67] agreed that mutation causes PC, 50% of which results from intrinsic factors. The genome sequencing of 593 familial PC patients determined that familial PC is polygenic, and the risk is related to a relative of the family with one or more variant germ lines. Germ line variants generally associated with increased PC risk are BRCA1, BRCA2, and ataxia telangiectasia mutated, which are involved in DNA repair mechanisms [62,68]. Mutations in caretaker genes including DNA repair genes and gatekeeper genes, including tumor suppressor genes, perhaps increase cell division and further drive gene mutation, leading to somatic and genetic alterations [69]. The genetic alterations in these genes result in the instability of molecular mechanisms including cell growth, cell division, apoptosis, and migration.

Epigenetics in pancreatic cancer The epigenome is driven by histone acetylation and DNA methylation, but its aberrant activity leads to alterations in gene expression with no change in the sequence of DNA. Epigenetics have a crucial role in regulating the initiation of carcinogenesis in the pancreas in addition to Kras mutations. Thus, the growth and progression of tumors are caused not only by genetic alteration but also by epigenetic modifications. The normal mechanism involved is the common gene expression of various oncogenes and tumor suppression genes. However, the altered epigenome

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leads to overexpression of genes that trigger tumor development. These epigenetic modifications generally include DNA methylation and histone modifications.

DNA methylation DNA methylation occurs when a methyl group is added at the 50 cytosine position, mainly in a CpG dinucleotide catalyzed by DNA methyltransferase (DNMT). Hypermethylation mediates the silencing of genes at their promoter level, including tumor suppressor genes involved in DNA repair, cell cycle regulation, and apoptosis [70e72]. DNMT controls the progression of cancer, and thus is a potential therapeutic approach. It includes DNMT1 (which maintains DNA methylation after replication) and DNMT3a and DNMT3b (which initiate DNA methylation). Moreover, DNMT1 has been detected in various cancers including 40% of PCs [73]. Hypermethylation through DNMT1 could be inhibited by blocking the process through RNA interference in the newly synthesized DNA, causing tumor suppressor gene expression [74]. Hypermethylated genes identified in PC include hMLH1, MUC2, GATA-4 and -5, secreted protein acidic and rich in cysteine (SPARC), E-cadherin, CXCR4, CDKN1C, BNIP3, and ppENK (neuropeptide transmitter gene) [75e78]. Furthermore, MingZhou et al. [79] tested 14 tumor suppressor genes for methylation, including APC, E-cadherin, TIMP3, glutathione-S-transferase, BRCA1, p16, p73, p14, and p15, death-associated protein kinase, hMLH, retinoic acid receptoreb-2 (RARb), thrombospondin, and MGMT, and determined that APC (50%), p16 (35%), p73 (33%), and p15(35%), RARb (35%), and BRCA1 (46%) are frequently methylated, and at least one gene is mutated in 94% of neoplasias and two to more mutated genes are in 69% of tumors. Ramon et al. [80] determined that TERT hypermethylated oncologic region (THOR) can be used as an early disease biomarker in PC diagnosis; it is also correlated with recurrence of the disease. Telomerase reactivation results in the expression of the TERT gene. Furthermore, its expression is positively correlated with methylation levels of THOR. Similarly, lipopolysaccharide-induced tumor necrosis factor-a factor (LITAF), a tumor suppressor gene regulated by p53, is downregulated in PC and serves as a potential biomarker for early diagnosis. Yuan et al. [81] suggested that hypermethylation at the promoter region of LITAF results in PC pathogenesis. However, demethylation at the promoter region promotes inhibition, survival, and the cell cycle of PC cells. Hong et al. [82]

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Theranostic Approach for Pancreatic Cancer

determined a significant interaction among DNMT1, miR-148a, and PC progression and suggested that the aberrant activity or upregulation of DNMT1 causes the hypermethylation of miR-148a. However, further demethylation or restoration reactivates tumor suppressor genes including RASSF1A, p16, and ppENK. Thus, miR-148a can also considered a potential therapeutic strategy targeting DNMT1 in PC therapy.

Histone deacetylase Histone deacetylase (HDAC) is another epigenetic modification that has a critical role in gene expression. HDAC catalyzes acetyl group removal from the histone, resulting in the formation of a complex compact made from DNAehistone by stimulating the condensation of chromatin, blocking transcription [83,84]. HDACs are classified into two groups: classical HDAC (zinc-dependent metalloproteins), which includes classes I (HDAC1, 2, 3, and 8), IIa (HDAC4, 5, 7, and 9), IIb (HDAC6 and 10), and IV (HDAC11); and sirtuins (class III, nicotinamide adenine dinucleotideedependent enzymes). Classical HDAC promotes the proliferation and progression of cancer cells as well as resistance and angiogenesis. Meiying et al. [85] analyzed HDAC protein expression from the human protein atlas and determined that HDAC1, HDAC3, HDAC6, and HDAC9 are more highly expressed in PC tissue than in healthy control tissue. They also observed that silencing the HDAC1 gene upregulates the expression of E-cadherin and downregulates the expression of vimentin, which suggests that the overexpression of HDAC1 promotes malignant potentiality in PC cells. Moreover, epigenetic modification at the promoter region of the E-cadherin/CDH1 gene is mediated in the presence of Snail by binding with its C2H2-type zinc finger domain to the E-box region of CDH1, recruiting corepressor proteins including HDAC and polycomb repressive complex [86,87], and thus deacetylating the promoter region, and downregulating E-cadherin expression. Therefore, HDAC1 is involved in the proliferation and metastasis of cancer cells and the inhibition of apoptosis. To inhibit the activity of HDAC and attenuate epithelial mesenchymal transition (EMT), the HDAC inhibitor CUDC-101 was combined with gemcitabine, which led to EMT inhibition in PC through the Snail signaling pathway. Obesity is considered a modifiable risk factor. It correlates with elevated levels of leptin in patients with PC. Leptin upregulates the expression of miR21, which in turn upregulates HDAC3 expression, leading to

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combined cross-talk in the progression of cancer [88]. Pathway studio platform data analysis also showed that leptin is involved in maintaining a cross-talk mechanism among PSCS, miR21, classical HDAC, and miR200a, -b, and -c. Jin et al. [89] explored a novel inhibitor of HDAC, AR-42, which can inhibit PC progression by cell-cycle arrest at G2/M phase, activating various pathways involved in apoptosis. They determined that AR-42 lowers the expression of mutant p53 by upregulating negative regulators of p53 including miR-33, miR-30d, and miR-125b in PC cells. Clinical investigation targeting HDAC is ongoing. Available HDAC inhibitors approved by the Food and Drug Administration are Vorinostat (suberoylanilide hydroxamic acid), Romidepsin, and Belinostat [90e93].

Genetic alterations Oncogenes and tumor suppressor genes in pancreatic cancer Whole-genome sequencing analysis is used to explore the mutational landscape of PC. A total of 119 somatic chromosomal variants have been reported on average per patient. Variations included were tandem duplications, inversions, intrachromosomal deletions, duplications, rearrangements, and fold back inversions. Like chromosomal instabilities and mutations in PC, these genetic instabilities modify proto-oncogenes and tumor suppressor genes. It was reported that 90% of PC is characterized by aneuploidy owing to the presence of chromosomal instability. Protooncogenes are the genes that encode signal transducers, growth factors, transcription factors, and apoptotic inhibitors involved in molecular mechanisms. Thus, mutated proto-oncogenes eventually develop into heterozygous oncogenes. Mutation types include point mutations, gene rearrangements, deletions, and amplifications causing the products of oncogenes to remain active and resulting in uncontrolled cell growth. Mutated proto-oncogenes in PC are Kras, CTNNB1 (b-catenin), and AKT. Tumor suppressor genes, including TP53, SMAD4, APC, and TP16, affected by mutations lose their inhibiting functionality. Tumor suppressor genes are recessive only if both alleles are affected [94]. Hung et al. [95] identified both Kras and TP53 primary mutated tumor cells from same tumor clones with a difference in the allele frequency. Kras mutation resulted from the amplification of oncogenic Kras allele, whereas TP53 mutation was caused by the deletion of wild-type TP53 allele [96]. Hypermethylation is another mechanism involved in deactivating the

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Theranostic Approach for Pancreatic Cancer

Table 1.2 Prevalent mutated genes in pancreatic cancer. Location of Mutation and Signaling Gene chromosome frequency pathway

Kras

12p

Point/58%

PIK3CA

14q

Heterozygous mutation/5% Heterozygous mutation/24% Intragenic mutation/ loss of second allele and missense mutation/37% Intragenic mutation, chromosomal instability, hypermethylation/ 29% Intragenic mutation, and loss of heterozygous and homozygous deletion/22% Hypermethylation/ 16%

CTNNB1 P53

17q

P16

9p

TGF/ SMAD4

APC

18q

5q

Ras/Raf/ MAPK PTEN/ PI3K/AKT Wnt Cell cycle control and apoptosis

Type

Proto oncogene Proto oncogene Protooncogene Tumor suppressor

Cell cycle control

Tumor suppressor

SMAD4/ Deleted in PC

Tumor suppressor

Wnt

Tumor suppressor

function of tumor suppressor genes [97]. A list of genes and their signaling pathways with an estimated mutated frequency is provided in Table 1.2.

Kristen rat sarcoma The oncogenic Kras mutation is the key initiator and critical determinant for pathogenesis in the early stages of PC within the epithelium of tumor cells and is identified by its occurrence in PanIN lesions, with a prevalence rate ranging between 88% and 100% [98,99]. It drives dedifferentiated mature pancreatic cells into duct-like cells, resulting in PDAC. Kras encodes the guanosine triphosphate (GTP) binding protein of the Ras family, located on chromosome 12p. Downstream of Ras is controlled by the guanine nucleotide exchange factor; hence, GTPase activating factor causes its activation only if it binds to GTP, and its inactivation when

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bound to guanosine diphosphate (hydrolyzed form). However, the Kras gene behaves aberrantly because of point mutations at the G12 or G13 codon; it is liable to develop ductal metaplasia and PanIN from acinar cells. This results in impairment in its protein intrinsic GTPase activities, including cell survival, differentiation, and remodeling of the cytoskeleton by locking it in its active form (GTP-bound), which finally results in the development of PC [100,101]. This in turn leads to the mutation or amplification of its downstream genes, including BRAF, as found in 3%e4% of patients, which causes the aberrant activation of signaling pathways involved in differentiation and survival [102,103]. Hence, activated Ras affects various other signaling pathways including the phosphatidylinositol 3-kinase, rapidly accelerated fibrosarcoma (Raf), extracellular signal-regulated kinase (ERK), ERK kinase, and mitogenactivated protein kinase (MAPK) cascades, involved in promoting cellcycle progression and inhibiting apoptosis (Fig. 1.1). Hingorani et al. [40] developed a mouse model mimicking PC progression in humans with the endogenous expression of mutated KrasG12D and detected the most common mutation, showing modified exon 1 with the transition of aspartic acid from glycine, which is observed in human PC. The allele remains inactive and is activated with age or time when it is crossed with pdx1-Cre (Cre recombinase) mice with PanIN lesions. Guang et al. [104] determined that hyperinsulinemia, which they created by feeding KrasG12D with high-calorie fat food, develops PanIN lesions in KrasLSLG12D-pdx1Cre mice, indicating that Kras mutations are essential in developing cytological atypia in PC. Insulin also induced migration and invasion in human pancreatic nestin-expressing (HPNE) cells as well in the KrasG12D variant. Furthermore, Kong et al. [105] used ultraepH sensitive micelles (UPSM) that had a high pH buffer with chloroquine and observed that UPSM inhibits lysosomal catabolism/acidification which is in turn essential for the supply of nutrients and amino acid to mediate the biological process in a PC microenvironment. Mutant Kras is also responsible for lysosomal acidification, which inhibits lysosomal acidification and can reduce PC cell viability promoted by Kras mutants, driving it under starved conditions. Thus, this could represent a promising therapeutic strategy against Kras-mutant PC/PDAC. This strategy could enhance cytotoxicity using pH-triggered rapid drug release from UPSM loaded with a triptolide prodrug with no side effects in nonepH sensitive micelles. PC is also associated with mutations in P53, P16, and SMAD4, which are correlated with metastatic PC [106].

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Figure 1.1 Representation of signaling cascade of pancreatic cancer. The ligands (epidermal growth factor receptor [EGFR], fibroblast growth factor receptor [FGFR], hepatocyte growth factor [HGF], insulin growth factor [IGF], and vascular endothelial growth factor [VEGF]) bind at their respective receptors, activating various signaling pathways including PI3K and Ras. The transforming growth factor (TGF) pathway includes the phosphorylation of SMAD4, resulting in the transcription of genes in the nucleus. The signaling cascades Hedgehog, Notch, and Wnt are involved in embryonic development that translocates Gli, Notch intracellular domain (NICD), and b-catenin respectively, to the nucleus, binding to transcription factors for gene expression. APC, adenomatous polyposis coli; GSK3b, glycogen synthase kinase3b; LRP, lipoprotein receptor related protein; MAPK, mitogen-activated protein kinase; mTOR, mechanistic target of rapamycin; NF-kb, nuclear factor-kb; Raf, rapidly accelerated fibrosarcoma; TCF-LEF, T-cell factorelymphoid enhancing factor.

p53 Tumor suppressor p53 (TP53) is a latent transcription factor also called the “the guardian protein of the cell”. Up to 50%e70% of cases of PC are associated with the inactivation of TP53 [100,107]. Activated p53 located on chromosome 17 encodes for proteins that have a major role in crucial cellular mechanisms including progression of the cell cycle and regulation of G1/S checkpoints, DNA damage repair by inducing the cell cycle at G2/M phase, and apoptosis [108,109]. These genes are inactivated owing to somatic mutations, and 66% of identified mutations result from missense mutations that affect the DNA binding domain with accumulations in the

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nucleus of tumor cells and the loss of checkpoints in the cell cycle [110,111]. TP53 mutation also has a role in developing PC. However, the correlation between TP53 and other cellular mutations in PC remains unclear. This was evident in mouse models produced by Wagner et al. [112], who reported that the development of PC lesions resulted from the overexpression of TGF-a and overactivation of Ras, and determined that the lesion progressed rapidly when the mouse model was crossed with null p53 mice, whereas lesion progression required more time when it was induced with p53. TP53 mutation was also seen in invasive carcinoma, which suggests that this mutation occurs during the mid to late stages of PC. To support this, Hingorani et al. [113] combined an endogenous mutant of Trp53R172H with KrasG12D, which led to the development of metastatic PC from preinvasion lesions. Amit et al. [114] characterized the role of RET as a targetable driver of PC, for which they developed triplet mutant transgenic mice with KrasG12D, Pdx-1-Cre, and P53R172H evaluated using KaplaneMeier analysis. They reported alterations in the signaling of Ras and RET. For instance, RET was upregulated with activation of the MAPK pathway and involved in invasive PC development along with inactivation of the P53 background. Hence, hyperactive rearranged during trasfection (RET) can be targeted for therapy [115]. Wu et al. [116] revealed that dihydrosanguinarine (DHSA), a crude compound of Chelidonium majus L., can induce cell-cycle arrest at G0/G1 and G2/M phase through the activity of p53 proteins, induce apoptosis, and inhibit invasiveness through Ras/Raf/mitogen-activated protein kinase kinase/ ERK pathways. Furthermore, they determined that DHSA inhibits the growth of PC cells bidirectionally using both mutant p53/Ras and wildtype p53/Ras protein in separate cell lines (PANC-1 and SW1990), which suggests a novel strategy for treating PC cells.

p16 Another tumor suppressor gene encoded by the CDKN2A gene is located on chromosome 9; its inactivation approximately causes 95% of PC [107]. p16 inhibits the progression of the cell cycle at the G1/S checkpoint arbitrated by cyclin-dependent kinase proteins including CDK4 and CDK6 of retinoblastoma protein in healthy cells. However, inactivated p16 genes with loss of function are facilitated by chromosomal instability (hypermethylation), fold back inversions including the accumulation of structural rearrangements, loss of heterozygous and homozygous deletions, intragenic

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mutation, and 50 -CpG island promoter methylations that result in an increase in retinoblastoma phosphorylation leading to the loss of G1/S checkpoints, causing uncontrolled cell growth and proliferation [117,118]. The coexistence of p16 as well as Kras mutations is observed in PanIN lesions, because the mutation of Kras leads to mutations in p16, although this occurs at later stages of the disease [119]. It was also evident from the report of Bardeesy et al. [120] that PC developed rapidly in cells affected by KrasG12D and p16 mutations compared with KrasG12D alone. Huai et al. [121] also unequivocally determined that NAD(P)H oxidase4 (NOX4) has a major role in reprogramming glycolysis via NOX4-dependent replenishment of NADþ levels induced through either the upregulation of p22phox (catalytic subunit of NOX) expression by nuclear factor-kb (NF-kb)-activated oncogenic Kras or the loss of p16 expression through the RB-E2F pathway, both of which induce the expression of NOX4. Thus, targeting NOX4 could be a potential therapeutic strategy for controlling the growth of PC.

Transforming growth factor-beSMAD4 The TGF-b pathway (Fig. 1.1) functions because of SMAD, but it differs in the dependency or independency of SMAD. It is the only pathway with tumor suppression activity in healthy cells and is implicated in tumorigenesis owing to mutations in the SMAD gene, commonly SMAD4. It was found that PC locus 4 genes (DPC4), also called SMAD4 or MADH4, are located on chromosome 18q21 and their inactivation causes 55% of PC. Inactivation of SMAD4 results from intragenic mutation with a loss of heterozygous and homozygous deletion or mutation in one allele, with a second allele loss [122] observed in early lesions of PC and most commonly in late lesions. This leads to aberrant signaling of TGF-b involved in cell growth, differentiation, invasion, angiogenesis, and EMT [107]. Adrian et al. [123] created a mouse model crossing KrasG12D and Tgfbr1 haploinsufficient mice to determine the role of altered TGF in developing PC and compared it with a KrasG12D, controlled Tgfbr1 mouse model. The later model had high frequency levels of PC lesions with additional Kras mutations, whereas the risk for PC was lower with the individual mutation (TGFBR1 only). Like all cancers, PC is a multistep process involving the accumulation of mutated oncogenes and nonfunctional tumor suppressor genes forming a complex, heterogenous, and unstable disease. It initiates from the

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intraepithelial neoplasia lesion of pancreas with a progressive altered pathway throughout tumorigenesis including early genetic alterations, shortening of telomerase, Kras mutation, and loss of p16 function followed by additional pathways characterized by late genetic alterations including loss of p53, SMAD4, and BRCA2, function leading to invasive carcinoma. Mutated Kras, which is an initiator of PC, can obstruct the signaling pathways of TGF-b through SMAD inhibition and inhibit p53, which is required for transactivation of TGF-b in association with SMAD4 [124]. TGF-b can also interact in a cross-network with other pathways including Ras/Ras/ERK [125]. Hence, the complex signaling network of tumorigenesis driven by mutated genes disrupts the systematic biology of the cell. In addition to these genes, certain other growth factors including epidermal growth factor receptor (EGFR) and vascular endothelial growth factor promote cell proliferation, survival, and angiogenesis and are overexpressed in developing tumor stroma, also known as the tumor microenvironment (TME), resulting in poor prognosis.

Molecular level expression Molecular mechanisms involved in various stages, including the initiation, development, and progression of PC, are complex and were studied in the 1970s and 1980s. In the later stages of PC, molecular-level alterations include overexpression of growth factors such as epidermal growth factor (EGF), gastrin, nerve growth factors, proangiogenic factors such as plateletderived growth factor, fibroblast growth factor (FGF), and vascular endothelial growth factor (VEGF), invasive factors such as metalloproteinases, tissue plasmogen activators, and increased telomerase activity. The microenvironment of the tumor is essential for invasion and metastasis involved in continuous interaction between tumor cells and PSCs. Information related to molecular transformation from the lesions of precursors to invasive carcinoma and to metastasis is crucial for the development of novel diagnostic strategies for detecting premalignant lesions and of targeted therapies.

Tumor microenvironment The TME is a characteristic feature of PC and is a heterogeneous fibrous tissue responsible for communicating with surrounding cells through external signals known as desmoplastic reactions. Its cellular and acellular components include the extracellular matrix (ECM), PSCs, cancer stem

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cells, endothelial cells, fibroblast inactive form, nerve fibers, and the hypoxic environment characterized by hypovascularization. The desmoplastic reaction of PC makes it more aggressive, resulting in the spread of disease and resistance against chemoradiation or radiation therapies, which affects therapy effectiveness owing to cross-talk between the TME and the heterogenous stroma. However, the introduction of genetically engineered modified animals in an attempt to investigate the TME is helpful for determining the pathogenesis and resistance developed by cancer cells. The TME is not static and constantly progresses from preneoplastic PanIN lesions to invasive pancreatic adenocarcinoma. The cells and proteins involved have a crucial role in tumor progression and metastasis. PC stellate cells are involved in stroma formation, and it was reported that stromal and epithelial cells are in continuous interaction, secreting various cytokines and growth factors. In addition to Kras (secondary effect) and CDKN2 (no direct effect), TGF-b is highly effective in the stroma and is sensitive to ligands secreted from the tumor cells, as demonstrated by an experiment on a transgenic mouse model showing overexpression of TGF-b with increased collagen and stellate cells [126]. It was also found that the signaling pathway of TGF-b, namely its paracrine activity with ligands secreted from pancreatic tumor cells, can activate tumor stroma [127,128].

Extracellular matrix The pathological PSCs secrete ECM proteins and cytokines at a high concentration, leading to a desmoplastic reaction. The ECM is filled with polysaccharides, glycoproteins, laminin, proteoglycans, and fibronectin, which differ in their physical and chemical properties, resulting from the activity of stromal and epithelial cells; they are meant to separate the compartments. Aberrant reactions in the ECM are caused by cross-talk between mutated cells and the microenvironment of the tumor, resulting in the activation of fibroblasts and the recruitment of inflammatory cells, promoting progression and angiogenesis of PC cells. Collagen, fibronectin, and laminin form a mesh for the basement membrane. Furthermore, fibronectin forms 90% of the tumor environment of PC. The ECM also has a vital role in the progression and differentiation of PC. It is controlled by PSCs, epithelial and immune cells, and endothelial and fibroblast cells through MMP enzymes imbedded in the ECM. The expression of MMP proteins, especially MMP-2, MMP-9, and MMP-1, is highly observed in PC cells; they were found to interact with proteins of ECM, remodeling

Biology, pathophysiology, and epidemiology of pancreatic cancer

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these proteins [129,130]. This suggests that the ECM is fueled by the production of collagens and MMP, which shape the microenvironment [131,132]. During remodeling, certain MMPs including MMP-2, MMP-7, MMP-12, and MMP-23, are upregulated and MMP-11, MMP-19, and MMP-24 are downregulated. The process includes the cleavage of collagen by MMP, resulting in the production of proline catabolized in the presence of proline oxidase (POX). POX is a downstream target of p53 and functions in autophagy signaling, although it is involved in the progression and differentiation of tumor cells when p53 acts aberrantly [133,134]. Kamphorst et al. [135] reported that the presence of glucose, serine, creatine phosphate, and glutamine responsible for metabolism are low in the ECM compared with healthy cells, although proline compensates for the metabolic challenge in the ECM. Ran et al. [136] used bioinformatics to determine the expression of messenger RNA (mRNA) during interaction between PC cells and PSCs using gene expression omnibus. They also used other techniques including proteineprotein interaction network analysis and principal component analysis to analyze differentially expressed genes involved in the cross-talk of tumor. They validated almost 15 genes from the specimens of human PC including S100Z, SARIB, LAMB3, AKAP12, JUND, ZBTB33, LSM4, CLDN1, KLRC3, CP, FKBP1A, STYX, YWHAZ, MTMR3, and PRKAR1A.

Pancreatic stellate cells The three different normal cells identified in the TME are stromal cells, fibroblasts, and immune cells. Stromal cells are also called PSCs. They were first identified in 1982 as star-shaped lipid-containing cells in healthy pancreatic cells in the inactive form, which resemble stellate cells in liver [137] and were further isolated from rats and humans in 1998 [138,139]. Although their origin is unclear, PSCs are believed to be of neuroectodermal and mesenchymal origin. As an inactive form in healthy parenchymal cells, PSCs along with vitamin A and droplets of fats in the cytoplasm function to develop immunity, stimulate the secretion of amylase in pancreas, and develop phagocytosis [140]. However, under certain pathological conditions including acute or chronic inflammatory conditions, PSCs are activated by the loss of lipid droplets, resulting in the expression of desmin, fibroblast features, and a-smooth muscle actin [141]. Thus, aberrantly activated PSCs develop ECM proteins including collagen, laminin, and fibronectin inducing desmoplastic reactions of the pancreas,

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Theranostic Approach for Pancreatic Cancer

resulting in tumor progression and metastasis. Fibroblastic cell formation caused by PSCs contributes to angiogenesis in tumor cells. Fluorescent in situ hybridization also revealed that PSCs impede developing intravasation and extravasation of cancer cells from blood cells metastasizing to other sites of the body [142]. They also promote EMT, which increases mesenchymal protein expression including transcription factors, N-cadherin, and vimentin and decreases epithelial marker expression including E-cadherin. Shang et al. [143] suggested that PC tissue possesses activated PSCs. These stellate cells promote viability and invasiveness and decrease apoptosis of PC cells induced by chemotherapy drugs such as gemcitabine, leading to chemoresistance. Moreover, PSCs can be blocked by the inhibitor SB535334 of TGF-b, which indicates that the inhibitor sensitizes tumor cells to gemcitabine. TGF-b is a group of cytokines that control the proliferation, differentiation, adhesion, migration, and EMT of cells. In addition to TGF-b, other pathways are involved in the active functionality of PSCs, including hepatocyte growth factor (HGF), EGF, and FGF. Apte et al. [144] revealed in their work that PSCs have a vital role in the activity of TGF-b, Rho-associated kinase pathway, and metalloproteinases. They produce ECM and are the chief sources of collagen growth in the matrix. They have critical roles in the interaction between cells in the TME and promote the activation of PSCs. They may represent a novel therapeutic target in the therapy of PC, although understanding the pathophysiology of PC remains challenging. For instance, Tanaka et al. [145] measured the thickness of human PC, ranging from blood vessel walls to the tumor, and then developed a three-dimensional model of fibrosis using PSCs. They determined the presence of collagen fibers and fibronectin fibril remodeled by PSCs and found that SPARC contributes to remodeling the ECM present in PSCs. SPARC, a secreted protein, is acidic and rich in cysteine secreted from fibroblasts; it is involved in the proliferation and migration of tumor cells and is highly expressed in resected PC patients, correlating with poor progression [146,147]. In addition, other signaling cascades have vital roles in the development of the TME, including Kras, which is prevalent in the epithelial compartment of the tumor, and TGF-b, which is essential for both the epithelial and stromal compartments [148]. Overexpression of TGF-b in a transgenic mouse model also showed collagen expression in the initial stages followed by the expression of fibronectin, which mediates fibrosis, resulting in PC progression and metastasis. Fibrogenesis is a process caused by various factors including chronic pancreatitis, injury, and cell death. These injuries

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promote the secretion of various cytokines and growth factors, which activate PSCs and contribute to myofibroblast development, which in turn are responsible for protein depositions in ECM. The hypoxia condition in TME requires a high oxygen demand for the influx of certain chemokines and cytokines, leading to physiochemical and molecular alterations in the TME, including increased production of reactive oxygen species, a reduced pH level, and activation of transcription factors such as hypoxia inducible factor (HIF). HIF has a vital role during tissue injury, maintaining the circulation of angiogenic cells and contracting the wound. However, its overexpression correlates with the development of desmoplasia, which recruits macrophages. Thus, HIF-1a coupled with c-mesenchymal epithelial transition factor (c-MET) and hedgehog signaling contributes to the emergence of drug resistance. Li et al. [149] demonstrated that hyperglycemic or diabetic patients had a high level of expression of HIF-1a and MMP-9 associated with high invasion and migration capacity, as well as poor prognosis in PC patients compared with euglycemic patients. Zhang et al. [150] also demonstrated the importance of hypoxia in the development of chemoresistance, because they observed that gemcitabine can enhance the stemness of the tumor by activating the AKT/ Notch1 signaling pathway, synergistically supplemented by the hypoxia environment. Abdalla et al. [151] reported a novel therapeutic regimen that targets genes involved in hypoxia including heme oxygenase (HO-1), which is upregulated during PC development and induces tumor progression. They used an HO-1 inhibitor and tin and zinc protoporphyrin to inhibit HO-1 and sensitize tumor cells to gemcitabine.

Pancreatic cancer stem cells PC stem cells (PCSCs) are self-renewing, immortal cells that inhabit a hypoxic niche composed of various cells including endothelial and immune cells, cytokines, and growth factors maintained by the activity of almost 20 different transcription factors such as HIF and NF-kB. They acquire glycolysis metabolism from oxidative phosphorylation, which has a vital role in the development of resistance owing to the high autophagic flux against anticancer drugs. The high expression of multidrug resistance, apoptotic proteins, and ABC transporters is responsible for the emergence of resistance against radiotherapy and chemotherapy [152]. Furthermore, chemoresistance developed by PCSCs results from the activation of certain mechanisms including drug efflux, owing to the low expression of

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Theranostic Approach for Pancreatic Cancer

equilibrative nucleoside transporters and concentrative nucleoside transporters and the overexpression of proteasomes and aldehyde dehydrogenase (ALDH). In addition, genetic alterations caused by mutations at the target level of the drug contribute to chemoresistance [153,154]. The aberrant pathways involved in developing resistance and self-renewal include the EMT, Notch, Wnt, and Sonic Hedgehog. Studies revealed that PCSCs are derived from the bone marrow, genetic mutation, and stem cells or progenitors, although their origin is still unclear. They can be identified by a number of markers including CD133, CD22, CD44, c-Met, ALDH1, and epithelium-specific antigen [154]. Begum et al. [155] determined that cancer-associated fibroblasts are involved in progression, metastasis, and resistance against drugs and that they enhance the capacity for self-renewal and frequency of cancer stems by producing type I collagen through the integrinefocal adhesion kinase signaling pathway. Moreover, Biondani et al. [156] reported that PCSCs develop symbiotic cross-talk with parenchymal cells of PC and thus are involved in initiation and metastasis. Daur et al. [157] demonstrated that glucose regulatory protein 78 (GRP78), the master regulator of unfolded protein response involved in proliferation, invasion, and metastasis, is also responsible for developing stemness in PC. Downregulating GRP78 dysregulates certain pathways including fatty acid metabolism and decreases clonogenicity and self-renewal properties. C-Met is a PCSC marker and a receptor tyrosine kinase that has a crucial role in cell survival and growth, promoting angiogenesis and metastasis by activating various pathways including Ras-MAPK, Wnt/glycogen synthase kinase 3 (GSK-3b)/b-catenin, and PI3K/AKT, NF-kB. Thus, targeting c-Met could be a therapeutic strategy for controlling the self-renewal of PCSCs.

Epithelial mesenchymal transition PC invasion and metastasis are a complex process involving PCSCs as well as EMT, causing dissemination and stemness. EMT has a crucial role in embryonic development, which downregulates E-cadherin, claudin-1, claudin-7, and occludin to ensure epithelial cellecell contact while upregulating Slug, ZEB1, ZEB2, Snail, twist, and vimentin to acquire mesenchymal features. These phenomena can be simply defined as a phenotypic transition from an epithelial (constant and competent for colonization) to a mesenchymal (metastable) state regulated by various signaling pathways (TGF-b, Wnt, and Notch), growth factors (EGF and HGF), transcription

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factors (NF-kB), transcription regulators (miRNA), and cytokines [158e160]. However, because of the aberrant action of EMT, the expression of certain proteins is los, whereas the functionality of the mesenchymal vimentin cytoskeleton leads to chemoresistance and invasiveness. Epigenetic alterations involved in EMT include X-chromosome inactivation, DNA methylation, and histone modifications and can regulate EMT, affecting miRNA-Snail-ZEB networks [161e163]. Otsurur et al. [164] determined a relation among leucine-rich glycoprotein (LRG), EMT, and metastasis, in which LRG enhances TGF-b/SMAD, which in turn promotes EMT. They observed that E-cadherin expression is decreased owing to the exposure of TGF-b, with an increase in invasion in LRG overexpressing PDAC cells, which indicates that LRG enhances EMT induced by TGF. Furthermore, adenosine 50 monophosphateactivated protein kinaseerelated kinase 5 (ARK5) is a protein kinase associated with developing resistance against chemotherapy drugs by lowering the sensitivity of PC cells against these drugs (gemcitabine). Hence, suppression of ARK5 enhances sensitivity against gemcitabine, upregulating the expression of E-cadherin and downregulating vimentin, inhibiting EMT under hypoxic and normoxic conditions [165]. FOXO3a is a transcription factor inhibited in various cancer cells; its inhibition also drives EMT and metastasis. Li et al. [166] determined that SPRY2, a regulator protein of the receptor tyrosine kinase signaling pathway, also induces EMT in FOXO3 knockout mice, inducing EMT by activating the b-catenin/T-cell factor (TCF4) pathway. N-Myc downstream-regulated gene-1 (NDRG-1) is a multifaceted protein that has a crucial role in inhibiting signaling pathways of oncogenes and EMT. Menezes et al. [167] determined that NDRG-1 upregulates the expression of E-cadherin, regulating its repressors including SNAIL, SLUG, and ZEB1.They also identified that the aberrant activity of NF-kB is responsible for EMT. They found that NDRG-1 inhibits the expression of NF-kB by suppressing pathways involved in Ikka and Ikkb expression and decreased the nuclear localization of NF-kB p65, which indicates that NF-kB is a target of NDGR1 and that TGF-b has a crucial role in upregulating Snail, Slug, and ZEB1. They also determined that the anticancer agent di-2-pyridylketone thiosemicarbazone can upregulate NRDG protein. Hence, the pathways involved in EMT development can be targeted to enhance the sensitivity of PC cells against anticancer drugs, improving common treatment strategies.

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Epidermal growth factor EGFR belongs to the family of cytoplasmic tyrosine kinase receptors that harbor an extracellular N-terminal region that binds to ligands such as EGF and TGF-a, leading to intracellular kinase functions and dimerization. This results in the activation of certain domains resulting in autophosphorylation, which regulates proliferation, differentiation, migration, angiogenesis, and apoptosis. In its active state, EGFR partakes in various downstream signaling pathways such as RAS, RAF, PI3K, AKT, and MAPK (Fig. 1.1). Hence, its aberrant action results in tumor growth and metastasis. EGFR is overexpressed in 90% of PCs and has a crucial role in PC recurrence owing to the development of resistance against chemotherapeutic drugs and EGFR inhibitors [168,169]. The emergence of resistance is associated with aberrant activity in the signal transducer and activator of transcription 3 (STAT3) pathway. The overexpression of ErbB-2 was observed in subsets of human PC tissues and PC lesions [170e172]. EGF and TGF-a were also found to be overexpressed in PC lesions. As previously discussed, high glucose or DM, which are risk factors for PC, induced the expression of EGF, resulting in the progression of PC cells by activating EGFR, ERK, and AKT pathways. Li et al. [173] used curcumin to attenuate cancer induced by hyperglycemia and observed that high levels of glucose were suppressed by curcumin by inhibiting the EGF/EGFR signaling pathways and downregulating E-cadherin and urokinase-type plasminogen activator protein levels. Overexpression of miR-21 along with Kras and EGFR is observed in the early progression of precursor lesions of PC [174]. Furthermore, Qiuvan et al. [175] determined a positive feedback loop between EGFR and miR-21, in which EGF stimulated the overexpression of miR-21, which in turn induced PC proliferation and inhibited apoptosis through the downstream pathways of EGF, including MAPK/ERK and PI3K/AKT. Moreover, overexpression of miR-21 downregulated the expression of its target protein, Spry2, which is involved in PC pathogenesis. Hence, EGFR represents a molecular target for PC therapy [176].

Fibroblast growth factor Fibroblast growth factors belong to the family of growth factors that have a crucial role in biological functions including organogenesis and carcinogenesis. In humans, 22 genes code for FGF and four for FGFR [177,178].

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FGFR belongs to the family of tyrosine kinases that bind with FGF, forming a complex with cofactors such as heparineheparin sulfate cofactor proteoglycans, resulting in downstream signaling pathways including activation of MAPK, AKT, STAT, and phosphoinositide phospholipase C-g involved in the proliferation, migration, and survival of cells during embryonic development (Fig. 1.1). FGFR-2 is the key growth factor involved in tumor progression. The aberrant activity of FGFR-2 is noted in various cancers including gastric (overexpression and amplification), breast, and endometrium (single nucleotide polymorphism). In PC, overexpression of FGF1, FGF2, FGF5, and FGF7 is observed with the receptor FGFR-2IIIb isoform obtained from alternative splicing of the FGFR-2 extracellular domain along with FGFR-2IIIc [179]. FGFR-IIIb and FGFR-IIIc bind with FGF, exerting both autocrine and paracrine effects. FGFR-IIIb is expressed mostly in epithelial cells, whereas FGFR-IIIc is mostly seen in EMT. However, its expression is inversely proportional, in which the decreased expression of FGFR-IIIb increases FGFR-IIIc expression. FGF10 is another ligand that mediates the morphogenesis of mesenchymal cells to epithelial cells by activating FGFR-2b and is involved in pancreatic carcinogenesis [180]. Pathologically, altered FGF signaling pathways stimulate tumor progression with increased expression of VEGF-A and venous invasion. It is also involved in maintaining close interaction between both tumor and stromal cells. Yoko et al. [181] observed that 45% of PC cell lines harbored overexpressed FGFR-2 gene amplification and its inhibition reduced the growth and migration of PC cells in vitro. FGF-10 has a crucial role in embryonic development. However, because of their aberrant activity, they are highly expressed in the stromal cells of PC. This growth factor promotes the progression of PC by maintaining cross-talk interaction, inducing the expression of type 1 MMPs as well as TGF-b1, which leads to EMT. On the other hand, it maintains a positive feedback loop with transcription factor SOX-9, contributing to PC initiation and progression owing to inflammation [180]. Furthermore, Ishiwata [182] showed that FGFR-IIIc suppression and EMT regulation by epithelial splicing regulatory protein-1 are effective in treating PC in immune-deficient mice. Moreover, FGFR-2 is considered to be a novel target for therapy because of its prominent role in carcinogenesis. Zhonghai et al. [183] also reported that AZD4547, an inhibitor of FGFR that targets FGFR 1eamplified PC in a patient-derived xenograft (PDX) model, was effective in PC therapy.

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Insulin growth factor and insulin The signaling pathway of insulin and IGF is dysregulated in PC and contributes to tumor progression. Insulin and IGF develop from different organs including the pancreas (islets of Langerhans) and liver (growth hormone stimulation), respectively, and are closely related. Receptors for these growth factors belong to the receptor tyrosine kinase family including IR-A, IR-B for insulin, and IGF-1R and IGF-2R. Insulin and IGF in its activated state participate in various signaling pathways. They have a major role in regulating the basal growth rate of PC cells. IGF and IGF-1R are abundantly expressed in PC tissue [184,185]. IGF-1R and EGFR are coexpressed and associated with poor survival of PC. IGF-2R is upregulated in PC tissue, as revealed by the overexpression of mRNA and protein levels in the nucleus [186]. Thus, this can be a novel target and a promising approach for PC therapy. However, this strategy is unsuccessful because of the dual role of insulin and IGF-1 in the stroma of PC, together with IGF binding proteins (IGFBPs), which are produced as a response gene of p53 involved in inducing apoptosis independent of IGF-1 signaling. Their alteration results in the overexpression of IGF-1R in PC. Ayse et al. [187] proposed that therapy including targeting stroma cellederived IGFs and IGFBPs could be a novel target for PC therapy affecting both stroma and PC cells. However, IGF-1R inhibition resulted in adverse side effects, whereas targeting Forkhead Box C1, a downstream transcription factor of IGF-1R, inhibits angiogenesis and metastasis in PC [188].

c-Mesenchymal epithelial transition factor/hepatocyte growth factor Hepatocyte growth factor is a tyrosine protein kinase receptor encoded by c-met, a proto-oncogene involved in the development of embryo and wound healing. The mRNA of this protein is observed ay a low level in exocrine pancreas, but its level is upregulated in PC and the epithelial cells of pancreatitis. The aberrant signaling pathway is a major cause for the progression of PC in its early stage and is also responsible for invasion and angiogenesis owing to the hyperactivation of oncogenes. HGF acts as a mediator in the interaction of tumor and stroma of PC in enhancing angiogenesis, metastasis, and chemoresistance. Furthermore, C-met can induce chemoresistance in mutant Kras against gemcitabine because of the increased endogenous expression of c-met potentially owing to gene amplification [189]. Tomioka et al. [190] found a fragment called kringle 4

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protein (NK4) in HGF, which has a dual role as an angiogenesis inhibitor and antagonist. Hence, they administered a recombinant form of NK4 to PC mice to inhibit growth, invasion, and metastasis. They observed suppression of ascites accumulation, peritoneal dissemination, and metastasis of cancer cells into the peritoneal wall, with improved survival time. Thus, the activation of various other signaling pathways including Wnt, JAK, PI3K, and STAT could be a remarkable approach to targeting HGF-mediated metastasis and angiogenesis for the therapy of PC.

Hedgehog signaling pathway Hedgehog signaling pathway (HH) is a key regulator during embryonic development mediating differentiation and proliferation; it is aberrantly activated in precursor lesions in PC. The pathway is activated by three ligands including Desert (DHH), Sonic (SHH), and Indian Hedgehog (IHH) that bind to receptors Ptch-1 and Ptch-2 (patched) with a smoothened (Smo) signal transducer and transcription factors (Gli1, Gli2, and Gli3) carried as canonical and noncanonical pathways. Summing the flow of the pathway (Fig. 1.1), ligand binding to the receptor induces the release of Smo and promotes the translocation of Gli to the nucleus, leading to transcription of target genes of Hedgehog including Gli1 and Ptch. The Ptch receptor is a tumor suppressor gene that inhibits the activity of Smo, encoded by the Smo gene, a proto-oncogene in the absence of ligand [191,192]. The mutation causes activation in Smo and deactivation in Ptch. Furthermore, the aberrantly activated HH signaling pathway is engaged in activating PCSCs. Cells in the exocrine pancreas are affected when in contact with pathogens, leading to activation of the quiescent HH pathway. Hence, PCSC secretes components of ECM, which participate in the repair mechanism [193,194]. Furthermore, the activated PCSCs assist in enhancing the migration of PC cells, resulting in metastasis along with axons and nerve of malignant cells [195]. Studies have also shown IHH ligand can enhance PCSCs, leading to migration by promoting the activation of type 1 MMPs [196]. The SHH proteins are secreted vigorously by PC cells under hypoxia conditions by overexpression of HIF-1 secretion in a dependent manner, resulting in the development of fibroblasts developing a stroma-rich microenvironment [197]. Furthermore, the SHH pathway is responsible for the development of resistance against gemcitabine, in which SHH is activated owing to the activation of HO-1, which is considerably overexpressed in PC and leads to resistance against chemotherapy drugs.

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Hence, its inhibition decreases cell proliferation and chemosensitizes PC cells to gemcitabine. This endogenous mechanism could represent a novel therapeutic target for PC therapy [198]. Eberl et al. [199] determined that cooperation between EGFR and the HH signaling pathway promotes tumor cell proliferation; they identified a cooperation response gene involved in HH-EGFR including the expression of SOX2, FGF-19, JUN, CXCR4, and SOX9, which, when synergistically activated through HHEGFR signaling, can initiate tumor progression in PC. This indicates that targeting this pathway could be a strategy for PC therapy.

Wnt signaling pathway The Wnt signaling pathway is a complex cascade including canonical (b cateninedependent) and noncanonical pathways (b-catenineindependent), which has a fundamental role in embryonic development and the maintenance of tissue homeostasis in adults. The intracellular signaling cascades (Fig. 1.1) involved in Wnt ligands and frizzled b-catenin receptor/ lipoprotein receptor-related protein 5 receptor complex are translocated to the nucleus, where they bind with transcriptional factors including TCF/ lymphoid enhancing factor, leading to transcription of target genes including cyclin D1, c-jun, and c-myc [200,201]. The destruction complex in the cytoplasm, including glycogen synthase kinase 3 (GSK3b), AXIN1, casein kinase 1, and APC, inhibits b-catenin by phosphorylating it and making it ready for ubiquitination by b transducin repeat containing protein. However, the dysregulation or aberrant activity of the pathways leads to pathogenesis and fibrosis. Previous reports suggested that mutation in AXIN1, APC, or CTNNB1 are rarely detected, but with increased levels of nuclear and cytoplasmic b-catenin, they lead to the development of pancreatic ductal adenocarcinoma [202e206]. Moreover, the aberrant activity of b-catenin with mutation in Kras develops intraductal tubular tumor, a rare form of pancreatic tumor [207]. PCSCs that contribute as a result of fibrosis are also caused by the aberrant activity of b-catenin. The pathway assisting or maintaining cross-talk activation with other pathways including TGF-b and NF-kB lead to the development of fibrosis and inflammation in pancreas [208,209]. Jinfeng et al. [210] determined that the transcription factor of TCF7L2 is a predicting novel marker for the detection of PC; they also suggested that it potentiates aerobic glycolysis and can be regulated by targeting Egl-9 family hypoxia inducible factor 2 EGLN2 (an oxygen sensing enzyme)/HIF-1a. In addition, an analysis of

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the Cancer Genomic Atlas dataset revealed a positive correlation of these. Furthermore, TCF7L2 has a b-catenin binding domain that participates in positive aerobic glycolysis regulation by suppressing EGLN2 and upregulating HIF-1a. Thus, targeting TCF7L2 could be a promising way to control tumor growth and differentiation. MiRNA-27a also promotes cell proliferation and inhibits apoptosis in PC cell lines through the Wnt signaling cascade, as shown by anti-miRNA, which suppressed serine/ threonine protein kinase (PAK1) protein, which is usually highly expressed in tumor cells and is responsible for cell division and migration. Furthermore, Wnt can activate PAK1 by inducing the expression of Wnt-1 responsive Cdc42 homolog. Thus, targeting the Wnt signaling cascade could be a promising route for PC therapy [211].

Notch signaling pathway The Notch signaling pathway has a critical role in the proliferation, differentiation, and homeostasis of cells. The pathway is also involved in embryonic development and the stem cell niche, decides cell fate, and drives carcinogenesis when it acts aberrantly. Evidence from the report suggests that it contributes to PC progression and is a ligand receptor pathway. The cascade (Fig. 1.1) is initiated when the ligand binds to the receptor among adjacent cells. There are four transmembrane receptors: Notch-1, Notch-2, Notch-3, and Notch-4, and five ligands, Jagged-1, Jagged-2, Delta-like-1, Delta-like-3, Delta-like-4, that are canonical [212]. When the pathway is activated, the cascade is followed by three repeated proteolytic cleavages releasing Notch intracellular domain (NICD) in the presence of various enzymes, including g-secretase enzyme, as coactivators. It then translocates to the nucleus and activates various Notch target genes including MMP-9, VEGF, p53, p27, p21, hair enhance of split family, Hey family, cox-2, c-myc, ERK, mTOR, NF-kB, cyclin D1, Bcl-2, and AKT [213]. However, its aberrant activity promotes PanIN lesions in addition to upregulating Jagged-1, Jagged-2, Notch-1, Notch-2, and Notch target genes. Thus, it has a crucial role in PC progression, migration, invasion, metastasis, and angiogenesis. It also cross-talks with various pathways including MEK/ERK, Wnt, Hedgehog, and TGF-b, which may result in PC progression. Ma et al. [214] reported that the expression of Notch-1 is regulated and mediated by EGFR, as shown by its activation, which leads to the upregulation of MMP-9 and VEGF expression and promotes invasion and metastasis. Furthermore,

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EMT-mediated PC progression is mediated through the Notch cascade, as evidenced by the overexpression of Notch genes, including Delta like-4 coupled with the upregulation of ZEB, Snail, and vimentin, which cause the EMT phenotype in PC cells [215,216]. The pathway also has a crucial role in deciding the fate of CSCs that develop resistance against chemotherapy drugs in PC. It was reported that PC harbors increased levels of Notch1, Notch2, and Delta like-4 with the upregulation of transcription factors including octamer-binding transcription factor 4 (self-renewal of embryonic stem cells) and homeobox protein Nanog (maintains pluripotency) [217]. Furthermore, it was reported that PC cells develop resistance against chemotherapy drug fluorouracil (5-FU) by activating the Notch signaling pathway [218]. Herbuzariu et al. [218] determined that adipokine leptin has a pivotal role in PC development, linking it to pandemic obesity, which modulates or maintains a cross-talk relation with the Notch signaling cascade and promotes chemoresistance against chemotherapy drugs such as 5-FU with increased expression of ABC proteins. The activation of Kras and Notch in PanIN lesions was also found to exhibit a strong synergy during tumor development [40,219]. Furthermore, Maria et al. [220] determined that miR-148a controls PC progression targeting Notch signaling components ADAM17 and EP300, leading to their inhibition and overexpression of miR-148a. Hence, targeting the Notch signaling pathway as well other pathways involved in its activation could be a better strategy in controlling PC.

Conclusion Efforts are under way to analyze the epidemiology of PC. Further clinical research in this field may lead to the development of efficient strategies for controlling PC. The survival rate of PC can be managed by exhaustive validation of the registries of cancer patients, regular follow-up, and maintenance of the quality of registration data. Developing clinical strategies for the early detection and prognosis of PC could improve the efficacy of therapies and decrease the risk for PC. Profiling tumor samples from patients is an advisable strategy because it allows a detailed evaluation of gene alterations involved in the developing stroma and immune cells, leading to targeted therapies tailored to the individual patient’s disease progression stage. Furthermore, researchers could focus on the discovery of novel biomarkers for early diagnosis and the development of preclinical models for understanding the complex process of PC development, which

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could inform the progress of novel and effective anticancer therapies. Extensive clinical trials could further support the development of personalized cancer medicine. This scenario can be achieved only with improved understanding of the molecular biology underlying PC. The microenvironment of PC includes a multifaceted network that promotes differentiation, worst prognosis, metastasis, EMT transition, invasion, and resistance against chemotherapy drugs including nab-paclitaxel, gemcitabine, and FOLFIRINOX. Hence, the development of targeted neoadjuvant therapies represents a promising strategy for inhibiting PC.

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