phosphodiesterases signaling pathway for colorectal cancer prevention

Accepted Manuscript Title: Targeting the Paracrine Hormone-Dependent Guanylate cyclase/cGMP/Phosphodiesterases Signaling Pathway for Colorectal Cancer Prevention Authors: Yarla N.S., Gali H., Pathuri G., Smriti S., Madka V., Panneerselvam J., Kumar G., Farooqui M, Rao C.V. PII: DOI: Reference:

S1044-579X(18)30064-6 https://doi.org/10.1016/j.semcancer.2018.08.011 YSCBI 1508

To appear in:

Seminars in Cancer Biology

Received date: Revised date: Accepted date:

14-6-2018 30-8-2018 31-8-2018

Please cite this article as: Yarla NS, Gali H, Pathuri G, Smriti S, Madka V, Panneerselvam J, Kumar G, Farooqui M, Rao CV, Targeting the Paracrine Hormone-Dependent Guanylate cyclase/cGMP/Phosphodiesterases Signaling Pathway for Colorectal Cancer Prevention, Seminars in Cancer Biology (2018), https://doi.org/10.1016/j.semcancer.2018.08.011 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Targeting the Paracrine Hormone-Dependent Guanylate cyclase/cGMP/Phosphodiesterases Signaling Pathway for Colorectal Cancer Prevention Yarla N.S.1, Gali H.2, Pathuri G.1, Smriti S. 1 Madka V. 1, Panneerselvam J. 1, Kumar G. 1,3,

1

SC RI PT

Farooqui M1, Rao C.V.1,3* Center for Cancer Prevention and Drug Development, Department of Medicine, Hematology-

Oncology Section, Stephenson Cancer Center, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, USA

Department of Pharmaceutical Sciences, College of Pharmacy, and Stephenson Oklahoma Cancer

U

2

VA Medical Center, Oklahoma City, Oklahoma, USA.

A

3

N

Center, The University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, USA

M

Corresponding author at: Center for Cancer Prevention and Drug Development, Department of

D

Medicine, Hematology-Oncology Section, Stephenson Cancer Center, University of Oklahoma

TE

Health Sciences Center, Oklahoma City, Oklahoma, USA.

EP

Email addresses: [email protected] (C.V. Rao)*;

A

CC

Phone: +1-405-271-3224; Fax; 1-405-271-3225

1

Abstract: Colorectal cancer (CRC) is one of the leading causes of cancer related-deaths. The risk of development of CRC is complex and multifactorial, and includes disruption of homeostasis of the intestinal epithelial layer mediated though dysregulations of tumor suppressing/promoting

SC RI PT

signaling pathways. Guanylate cyclase 2C (GUCY2C), a membrane-bound guanylate cyclase receptor, is present in the apical membranes of intestinal epithelial cells and maintains homeostasis. GUCY2C is activated upon binding of paracrine hormones (guanylin and uroguanylin) that lead to formation of cyclic GMP from GTP and activation of downstream signaling pathways that are associated with normal homeostasis. Dysregulation/suppression of the GUCY2C-mediated signaling promotes CRC tumorigenesis. High-calorie diet-induced obesity is associated with deficiency of guanylin expression and silencing of GUCY2C-signaling in colon

U

epithelial cells, leading to tumorigenesis. Thus, GUCY2C agonists, such as linaclotide, exhibit

N

considerable role in preventing CRC tumorigenesis. However, phosphodiesterases (PDEs) are

A

elevated in intestinal epithelial cells during CRC tumorigenesis and block GUCY2C-mediated signaling by degrading cyclic GMP to 5`-GMP. PDE5-specific inhibitors, such as sildenafil, show

M

considerable anti-tumorigenic potential against CRC by amplifying the GUCY2C/cGMP signaling pathway, but cannot achieve complete anti-tumorigenic effects. Hence, dual targeting the elevation

D

of cGMP by providing paracrine hormone stimuli to GUCY2C and by inhibition of PDEs may be

TE

a better strategy for CRC prevention than alone. This review delineates the involvement of the GUCY2C/cGMP/PDEs signaling pathway in the homeostasis of intestinal epithelial cells. Further,

EP

the events are associated with dysregulation of this pathway during CRC tumorigenesis are also discussed. In addition, current updates on targeting the GUCY2C/cGMP/PDEs pathway with

CC

GUCY2C agonists and PDEs inhibitors for CRC prevention and treatment are described in detail.

A

Abbreviations ACF

Aberrant Crypt Foci

2

Keywords: Colorectal tumorigenesis; guanylate cyclase; GUCY2C agonists; PDEs inhibitors;

A

CC

EP

TE

D

M

A

N

U

SC RI PT

Linaclotide; Sildenafil; Chemoprevention

3

1. Introduction Colorectal cancer (CRC) cancer is one of the leading causes of cancer-related deaths in the US and world-wide [1, 2, 3]. An estimated 140,250 cases of CRC will be diagnosed in the US in 2018 [3].

SC RI PT

Identification of novel biomarkers/targets is needed for early diagnosis and to aid in the development of novel chemopreventive and therapeutic agents for CRC. Several research groups have worked to identify novel biomarkers/targets in CRC. A significant progress has been made in understanding the underlying histopathologic and molecular processes in the transition from

U

normal epithelium to an invasive adenocarcinoma.

The adenomatous polyposis coli (APC) gene is mutated in more than 80% of patients with CRC

A

N

[4]. However, APC mutation and activation of the wnt/β-catenin cascade is insufficient for CRC

M

initiation and progression. Disruption of the homeostasis of intestinal epithelial cells is associated with tumorigenesis and progression, which results from dysregulation of several signaling

D

processes, including the guanylate cyclase (GUCY2C)/cyclic GMP/ Phosphodiesterases (PDEs)

TE

pathway.

EP

GUCY2C, a membrane-bound guanylate cyclase receptor, is present in the apical membranes of intestinal epithelial cells from the duodenum to the distal rectum [5, 6]. It is a key regulator of

CC

secretions of intestinal fluid and electrolytes, and the pathophysiology of enterotoxigenic diarrhea [5, 6]. Paracrine hormones, such as guanylin (found throughout the intestine) and uroguanylin

A

(present selectively in small intestine), and the enterotoxins of diarrheagenic bacteria are cognate ligands, structurally peptides, that, when bound to GUCY2C, lead to the formation of cyclic GMP (cGMP) from GTP [5, 6]. Further, the cGMP-dependent protein kinases, also called as protein kinase G (PKG), are activated as a result of formation of cGMP, and transduce the signals from GUCY2C though activation of downstream signaling mediators [7]. 4

PDEs, a group of metallophosphohydrolases, hydrolyze the 3′,5′-cyclic phosphate group on cyclic nucleotides to a 5′-monophosphate, thereby blocking cyclic AMP (cAMP) or cGMP signaling [8, 9]. There are different types of PDE isozymes, with different substrate specificities. PDE5 (specific

SC RI PT

to cGMP) and PDE10 (specific to both cGMP and cAMP) are more associated with GUCY2Cmediated signaling than are other PDEs [9]. PDEs block GUCY2C-associated second messenger signaling by degrading cyclic GMP, while inhibition of PDEs activity prevents cGMP degradation and leads to amplification of GUCY2C-dependent cGMP signaling [10, 11]. Accumulating evidence suggests that the paracrine hormone-mediated GUCY2C/cGMP/PDEs signaling pathway

U

plays a key role in homeostasis of the intestinal crypt-villus axis, and its dysregulation is associated

N

with intestinal tumorigenesis [5, 6, 11, 12, 13, 14, 15,16].

A

The present article reviews the role of the GUCY2C/cGMP/PDEs signaling pathway in

M

homeostasis of the intestinal crypt and villus axis, and how dysregulation of homeostasis via this

D

pathway is associated with CRC tumorigenesis. In addition, recent advances in the development

TE

of the modulators of the GUCY2C/cGMP/PDEs pathway for CRC prevention are also discussed. 2. Role of the GUCY2C/cGMP/PDEs pathway in homeostasis of intestinal crypt-villus axis

EP

and colorectal tumorigenesis

CC

In the intestine, villi projecting into the lumen and flank-like crypts embedded in the mesenchyme are called the intestinal crypt-villus axis [17]. The luminal surface of the crypt-villus axis is

A

covered with a single epithelial layer that provides a physical barrier between systemic and mucosal compartments. In the colon, tubular crypts in the mesenchyme possess proliferating zones that contain various types of cells, including stem cells. Intestinal stem cells can self-renew, and have the potential to give rise to all differentiated cell types (multipotency), including enterocytes, goblet cells, entero-endocrine cells, and paneth cells, in the bottom of the crypt [17]. The 5

proliferation, migration, differentiation, and apoptosis of epithelial cells of the crypt-villus axis occur via a highly regulated process, and several signaling pathways are involved in maintenance of homeostasis [12, 17]. Disruption of homeostasis in the crypt-villus axis leads to tumorigenesis

SC RI PT

in the colorectum [12, 17]. The GUCY2C-paracrine hormone axis is considered a key regulator of homeostatic processes, including cell proliferation, migration, differentiation and apoptosis, as well as functions that are vital to the integrity of genome [5, 6, 14, 15, 16]. Suppression of GUCY2C is associated with altering genomic integrity (loss of APC heterozygosity and DNA damage) in colorectum that leads

U

to tumorigenesis [15]. GUCY2C agonist suppressed proliferation and increased goblet cell density

N

in non-neoplastic intestinal epithelium of APCMin/+ mice [9]. In GUCY2C knockout mice, there

M

A

was increased cell proliferation, crypt hyperplasia, and migration (Table 1). Wilson et al. [18] reported that guanylin was found in colorectal mucosa specimens from healthy

D

individuals (n=30), but absent from patients with colorectal tumors (n=54). Silencing of guanylin

TE

and uroguanylin, the endogenous hormones for GUCY2C, resulted in CRC tumorigenesis. GUCY2C/cGMP signaling maintains integrity and homeostasis of intestinal epithelial cells and

EP

preserves APC function, opposing neoplasia (Table 1) [19]. Disruption of the intestinal epithelial

CC

barrier resulted in inflammation and the generation of reactive oxygen species that cause DNA damage; the genotoxic effects led to intestinal tumorigenesis [20, 21]. Silencing of GUCY2C is

A

associated with intestinal epithelial barrier disruption and hyperpermeability, as well as elevated levels of oxidative DNA damage in circulating leukocytes. Conversely, protecting barrier integrity and restricting permeability was observed after activating GUCY2C with an agonist [20, 21]. Obesity is one of the risk factors for CRC, which is associated with deficiency of the guanylin– GUCY2C signaling [22, 23, 24, 25]. Lin et al. [22] found that high-calorie diet-induced obesity is 6

associated with deficiency of guanylin expression and blockade of GUCY2C-signaling in colon epithelial cells, leading to tumorigenesis. Mechanistic studies revealed that high-calorie dietinduced endoplasmic reticulum (ER) stress, which suppressed expression of guanylin during colon

SC RI PT

tumorigenesis [22, 26]. Chemical chaperone taurodeoxycholic acid (oral dosage of 150 mg/kg body weight for 12 days), an FDA-approved drug for the treatment of cholestatic liver diseases, blocked ER stress and rescued expression of guanylin in colons of C57BL/6 mice fed with highfat diet. PKR-like ER-localized eIF2α kinase (PERK) is involved in ER stress-induced unfolded protein response, and its inhibition allowed guanylin expression in intestinal epithelial cells [22,

U

27]. Therefore, ER stress inhibitors and/or GUCY2C agonists, such as linaclotide, can be

N

suggested as novel chemopreventive agents for obesity-induced CRC [22, 23, 24, 25].

A

PDEs block GUCY2C-associated second messenger signaling by degrading cyclic nucleotides,

M

whereas inhibition of PDEs activity prevents cyclic nucleotide degradation (Fig. 1). Several

D

researchers have described the pathophysiological roles of various PDEs in numerous tumor cell

TE

types, including CRC cells [9, 28, 29, 30]. Accumulating evidence suggests that PDEs are involved in colon tumorigenesis and progression.

EP

Recently, Lehrer et al. [28] collected data from The Cancer Genome Atlas (TCGA) to analyze the

CC

survival of patients with colon cancer associated with mutations of the PDE5A gene. They found that 96.77% of patients with colon cancer do not have the PDE5 mutation [28]. Patients without

A

mutation/elevation of PDE5 had significantly poorer survival than did patients with PDE5 mutation [28]. Zhu et al. [29] demonstrated that suppression of cGMP-specific PDE5 led to inhibition of proliferation and pro-apoptotic induction in colon cancer cells. In a clinical study, Li et al. [30] found that PDE10 expression levels are high in colon tumors from patients with stage I – IV disease compared with normal colonic mucosa from healthy individuals. The researchers also 7

reported that PDE10 expression levels were elevated in intestinal tumors of APCMin/+ mice compared with levels in normal intestinal mucosa from wild-type mice. Inhibition of PDE5 is associated with suppression of proliferation and enhancement of goblet cell density in non-

SC RI PT

neoplastic epithelium of intestine from APCMin/+ mice (Table 1) [9]. The cGMP-dependent PK maintains homeostasis by reducing crypt hyperplasia, decreasing epithelial apoptosis, and increasing numbers of differentiated goblet and enteroendocrine cells in the colons of mice [31]. 3. The GUCY2C/cGMP/PDEs signaling cross-talk with other tumor suppressive/oncogenic

U

signaling during tumorigenesis of CRC

Tumorigenesis of CRC is a complex process, involving various signaling pathways, including

A

N

APC, wnt, β-catenin, p53, K-RAS, EGFR, and VEGF, and many other signaling molecules [32,

M

33]. Several studies suggested that the GUCY2C/cGMP/PKG signaling pathway maintains homeostasis of the intestinal epithelial barrier and exhibits tumor-suppressive effects in association

D

with several oncogenic and tumor suppressor mediators. Upon paracrine hormonal stimuli,

TE

GUCY2C acts on GTP and converts it to cGMP, which in turn activates PKG. Activated PKG can directly inhibit β-catenin activity through phosphorylation and ubiquitin-dependent proteasomal

EP

degradation, thereby blocking TCF transcriptional activity and expression of other downstream

CC

gene products, cyclin D1 and survivin (Fig. 1) [7, 34, 35, 36]. Thus, the GUCY2C/cGMP/PKG signaling pathway exerts preventive effects against CRC under APC-mutated conditions.

A

However, overexpression of PDEs is associated with degradation of cGMP to 5`-GMP and inhibition of PKG and β-catenin/TCF transcriptional activity in colon epithelial cells (Fig. 1) [30]. Kwon et al. [34] reported that PKG blocked expression of beta-catenin and TCF activity at the transcriptional level in colon cancer cells.

8

Activation of GUCY2C/cGMP/PKGII signaling is associated with dephosphorylation of AKT1, which maintains homeostasis in intestinal epithelial cells and suppresses intestinal tumorigenesis [21, 37, 38]. knockout of GUCY2C in mice is associated with reduced PTEN expression and

SC RI PT

activity, as well as decreased expression of tumor suppressors p27 and p21 [37, 39]. Silencing of phosphatase and tensin homolog (PTEN), a tumor suppressor, is associated with blockade of GUCY2C/cGMP-mediated effects and inactivation of AKT and its downstream signaling in colon cancer cells. Therefore, it is clear that GUCY2C/cGMP/PKGII mediated anti-tumorigenic effects against colon cancer via activation of tumor suppressor PTEN and inactivation of oncogenic AKT

U

[21, 37, 38]. Overexpression of PDE5 disrupts homeostasis of intestinal epithelial cells by

N

promoting proliferation and apoptosis, while inhibiting differentiation mediated through the

A

cGMP/PKG2/dual specificity protein phosphatase 10/ c-Jun N-terminal kinase pathway [40, 41].

M

4. Modulators of the GUCY2C/cGMP/PDEs signaling pathways for CRC prevention and

D

therapy (pre-clinical and clinical development)

TE

Several modulators of the GUCY2C/cGMP/PDEs signaling pathways, including GUCY2C agonists and PDEs inhibitors, have been developed and explored as chemopreventive agents for

EP

CRC. Some of these agents are at various stages of their preclinical and clinical development

CC

(Table 1, Figures 1&2). Exisulind, a sulindac derivative with PDE inhibition activity, suppressed tumorigenesis in familial adenomatous polypsis (FAP) in patients. Due to adverse effects,

A

however, this agent was not approved by the FDA [42]. However, linaclotide, plecanatide, and dolcanatide (some of the GUCY2C agonists), and sildenafil and vardenafil (PDEs inhibitors) have been reported to possess prominent chemopreventive potential against CRC [43]. 4.1. GUCY2C agonists/modulators for CRC prevention

9

Linaclotide (a peptide containing 14 amino acids) (Fig. 2A) is an FDA-approved oral GUCY2C agonist formulated for treatment of chronic idiopathic constipation under the trade name of Linzess [9]. Linaclotide has been explored as chemopreventive agent for colon cancer by several

SC RI PT

investigators. This agent inhibited tumorigenesis in APCMin/+ mice, with up to 67% inhibition of the number of polyps [9]. Further, Weinberg et al. [44] aimed to evaluate the efficacy of oral linaclotide to induce a pharmacodynamic response in the colorectal epithelial cells of humans. Oral administration of linaclotide (0.87 mg per day) to healthy volunteers with a colon preparation using polyethylene glycol solution (Movi Prep) resulted in activation of GUCY2C and elevation of

U

cGMP in epithelial cells of the colon and rectum, and maintained homeostasis by inhibiting

N

proliferation of epithelial cells. However, these effects were absent from epithelial cells of the

A

colorectum without colonoscopy preparation [44, 45]. Further investigations are required to

M

improve the delivery of linaclotide to the intestinal epithelial layer.

D

Plecanatide (a peptide containing 16 amino acids) is an analog of human uroguanylin, a GUCY2C

TE

agonist, and an FDA-approved drug (brand name Trulance) for the treatment of chronic idiopathic constipation and irritable bowel syndrome with constipation [46]. Wen-Chi et al. [47] reported that

EP

oral treatment of APCMin/+ FCCC mice with plecanatide showed a significant reduction in the formation of inflammation-mediated polypoid, flat, and indeterminate dysplasias. The researchers

CC

concluded that oral treatment with plecanatide inhibited inflammation-driven CRC carcinogenesis

A

via activation of the GUCY2C-signaling pathway, resulting in suppression of the Wnt/β-catenin pathway and pro-inflammatory cytokines [47]. Dolcanatide, a GUCY2C analog, has been in clinical trials for ulcerative colitis [43, 48]. Dolcanatide has an additional advantage over other peptide-based GUCY2C analogs in that it exhibits resistance to digestive proteases in the intestine [43, 49]. However, detailed investigations 10

are required to demonstrate the chemopreventive efficacy of dolcanatide against CRC tumorigenesis. Previously, radiolabeled-conjugates of GUCY2C agonists have been synthesized and further

SC RI PT

explored for diagnostic/therapeutic purposes in colon tumors [50, 51]. Moreover, several research groups are developing GUCY2C-based immunotherapeutic strategies against CRC. AntiGUCY2C-monoclonal antibody-drug conjugate TAK-264 was developed to target against GUCY2C-expressing gastrointestinal malignancies, including CRC [52, 53]. Almhanna et al. [53] published results of a phase I study (ClinicalTrials.gov identifier: NCT01577758) on TAK-264

U

(MLN0264) in patients with advanced gastrointestinal malignancies, including metastatic CRC.

4.2.

M

A

targeted at CRC metastases without autoimmunity.

N

Magee et al. [54, 55] developed GUCY2C-directed chimeric antigen receptor (CAR)-T cells

PDEs inhibitors for CRC prevention

D

Researchers have used in vitro and in vivo methods to explore several PDE5 and 10 inhibitors as

TE

chemopreventive agents. At the clinical level, sulindac is one of the most effective chemopreventive agents against CRC among all NSAIDs. However, long-term use of sulindac for

EP

cancer prevention is not recommended, due to its adverse effects. Sulindac derivatives exert

CC

anticancer properties in colon tumor cells by modulating the cGMP/PDE/PKG/wnt/β-catenin pathway, but not through a mechanism involving cyclooxygenase inhibition [30, 35, 56, 57].

A

PDE10 and PDE5 dual inhibitor (sulindac derivative ADT-094) exhibits better cancer preventive potency than that of PDE 5 inhibitors [30]. Exisulind, a sulindac derivative, suppressed tumorigenesis in patients with FAP. However, this agent was not approved by the FDA due to adverse effects. Nevertheless, the studies on exisulind for FAP highlighted cGMP/PDE as a target for colon cancer prevention [42]. Later, several investigators began developing sulindac 11

derivatives that target the GUCY2C/cGMP/PDEs/PKGII signaling pathway for CRC prevention [30, 35, 56, 57]. Sildenafil, an FDA-approved drug for the treatment of erectile dysfunction (ED) (Fig. 2B), is an

SC RI PT

inhibitor of PDE5, which has been validated as a CRC chemoprevention target in mice [10, 13]. Sharman et al. [9] reported that sildenafil inhibited tumorigenesis in APCMin/+ mice, with inhibition of the number of polyps up to 50%. This drug prevents azoxymethane/dextran sulfate sodium (AOM/DSS) inflammation-driven CRC in mice by elevating the cGMP [10, 58].

U

Vardenafil (Fig. 2C), an FDA-approved drug for the treatment of ED, is another PDE5 inhibitor

N

that has been explored as a chemopreventive agent. This drug increased cGMP levels in colonic

A

mucosa of wild-type mice and suppressed proliferation and apoptosis, but increased secretory cell

M

lineage differentiation, in the colonic epithelium [40]. Vardenafil activates cGMP-mediated signaling in the colonic epithelium. Increased PKG2 activity modulates homeostasis by inhibiting

D

proliferation and apoptosis, while promoting differentiation. Tadalafil, another FDA-approved

TE

drug for the treatment of ED and a PDE inhibitor, selectively inhibited the growth of colon cancer cells that expressed high levels of PDE5 compared with normal colonocytes [59, 60]. Abadi et al.

EP

[60] synthesized PDE5-inhibitory novel tadalafil analogs with improved colon cancer cell growth

CC

inhibition properties.

5. Concluding remarks and future perspectives

A

CRC tumorigenesis is a complex process, and many of the underlying histopathologic and molecular processes involved in the transition from normal epithelium to an invasive adenocarcinoma are unknown. Disruption of intestinal homeostasis is associated with tumorigenesis, which results from dysregulation of several signaling processes, including the

12

GUCY2C/cGMP/PDEs pathway. The molecular link between the GUCY2C pathway and obesityinduced tumorigenesis of CRC is also well established. The GUCY2C/cGMP/PDEs/PKG pathway regulates several tumor-suppressive (APC, PTEN, p27, and p21) and oncogenic (AKT) mediators.

SC RI PT

Genetic and pharmacologic modulation of mediators of the GUCY2C/cGMP/PDEs pathway is associated with either chemoprevention or tumorigenesis of CRC. Hence, mediators of this pathway (GUCY2C, cGMP, PDEs and PKG) have been considered as biomarkers and/or preventive targets for CRC.

Several NSAIDs have been explored as chemopreventive agents for CRC. NSAIDs modulate

U

several signaling pathways, including the cGMP/PDEs/PKG signaling pathway. For instance,

N

exisulind, a sulindac derivative, prevents tumorigenesis in patients with FAP. But, exisulind was

A

not FDA-approved for clinical use due to adverse effects. However, these studies on exisulind for

M

FAP highlighted the cGMP/PDE pathway as a target for CRC prevention. Further, various sulindac

D

derivatives, which modulate the cGMP/PDE/PKG/wnt/β-catenin pathway in CRC, have been

TE

synthesized and are being evaluated for their preventive capabilities and toxicity effects. GUCY2C agonists (linaclotide, plecanatide, and dolcanatide) and PDE5 inhibitors (sildenafil,

EP

vardenafil, and tadalafil) have been explored pre-clinically and clinically as chemopreventive

CC

agents against CRC. GUCY2C agonists can prevent colon tumorigenesis, but activated PDEs reduce the GUCY2C/cGMP signaling-mediated tumor-suppressive effects by degrading cGMP.

A

Hence, dual targeting GUCY2C/cGMP towards elevation and PDEs toward inhibition may be a better strategy for CRC prevention. In this scenario, combinational use of GUCY2C agonists, like linaclotide, and PDEs inhibitors, like sildenafil, could be a better chemopreventive strategy against CRC than either agent alone. High-calorie diet induces ER stress, which suppresses expression of guanylin during obesity13

induced CRC tumorigenesis. Hence, exploration of a combination of GUCY2C agonists and PDEs inhibitors, along with inhibitors of ER stress (taurodeoxycholic acid and/or GSK2606414), may be a potential preventive strategy for obesity-induced CRC tumorigenesis.

SC RI PT

Overall studies suggest that the GUCY2C/cGMP/PDEs/PKG signaling pathway can be a promising target for prevention of CRC. However, further studies are required to elaborate the molecular mechanism(s) that are associated with the GUCY2C/cGMP/PDE/PKG signaling pathway in CRC tumorigenesis and progression, and to develop novel drugs for CRC prevention

U

by targeting this pathway.

N

Conflict of Interest: The authors declare that there are no conflicts of interest.

A

Acknowledgement: Dr. C.V. Rao acknowledges R01CA 213987, N01-CN-55003-47, and VA

Azoxymethane

D

help.AOM

M

Merit Award for financial support. We would like to acknowledge Kathy J. Kyler for editorial

Adenomatous Polyposis Coli

CAR

Chimeric Antigen Receptor

CRC

Colorectal Cancers

EP

TE

APC

CC

COX Cyclooxygenases

A

FAP GI

Familial Adenomatous Polyposis Gastrointestinal

GUCY2C Guanylate cyclase 2C NSAIDs Non-Steroidal Anti-inflammatory Drugs

14

PDEs

Phosphodiesterases

A

CC

EP

TE

D

M

A

N

U

SC RI PT

PTEN Phosphatase and tensin homolog

15

References [1]

Siegel RL, Miller KD, Fedewa SA, Ahnen DJ, Meester RGS, Barzi A, Jemal A. Colorectal cancer statistics, 2017. CA Cancer J Clin. 2017;67(3):177-193.

[2]

Cronin KA, Lake AJ, Scott S, Sherman RL, Noone AM, Howlader N, Henley SJ, Anderson

SC RI PT

RN, Firth AU, Ma J, Kohler BA, Jemal A. Cancer. 2018 May 22. Annual Report to the Nation on the Status of Cancer, part I: National cancer statistics. [3]

American Cancer Society. Cancer Facts & Figures 2018. Atlanta: American Cancer Society; 2018, pp.13.

Lawrence N. K., William F. D. APC and its modifiers in colon cancer. Adv Exp Med Biol.

U

[4]

Kuhn M. Molecular Physiology of Membrane Guanylyl Cyclase Receptors. Physiol Rev.

A

[5]

N

2009;656:85–106.

[6]

M

2016; 96(2):751-804.

Crystal L. Kraft, Jeffrey A. Rappaport, Adam E. Snook, Amanda M. Pattison, John P.

D

Lynch, Scott A. Waldman1. GUCY2C maintains intestinal LGR5+ stem cells by opposing

Browning DD, Kwon IK, Wang R. cGMP-dependent protein kinases as potential targets

EP

[7]

TE

ER stress. Oncotarget. 2017; 8(61): 102923–102933.

for colon cancer prevention and treatment. Future Med Chem. 2010;2(1):65-80. doi:

CC

10.4155/fmc.09.142. [8]

Dousa TP. Cyclic-3',5'-nucleotide phosphodiesterase isozymes in cell biology and

A

pathophysiology of the kidney. Kidney Int. 1999;55(1):29-62.

[9]

Sharman SK, Islam BN, Hou Y, Singh N, Berger FG, Sridhar S, Yoo W, Browning DD. Cyclic-GMP-Elevating Agents Suppress Polyposis in ApcMin mice by Targeting the Preneoplastic Epithelium. Cancer Prev Res (Phila). 2018;11(2):81-92.

16

[10]

Islam BN, Sharman SK, Hou Y, Bridges AE, Singh N, Kim S, Kolhe R, Trillo-Tinoco J, Rodriguez PC, Berger FG, Sridhar S, Browning DD. Sildenafil Suppresses InflammationDriven Colorectal Cancer in Mice. Cancer Prev Res (Phila) 2017; 10(7):377-388. Nalli AD, Bhattacharya S, Wang H, Kendig DM, Grider JR, Murthy KS. Augmentation

SC RI PT

[11]

of cGMP/PKG pathway and colonic motility by hydrogen sulfide. Am J Physiol Gastrointest Liver Physiol. 2017;313(4):G330-G341. [12]

Li P, Waldman SA. Corruption of homeostatic mechanisms in the guanylyl cyclase C signaling

pathway

underlying

colorectal

tumorigenesis.

Piazza GA. Validation of PDE5 as a Chemoprevention Target. Cancer Prevention

A

Research 2017; 10(7):373.

Li P, Lin JE, Chervoneva I, Schulz S, Waldman SA, Pitari GM. Homeostatic control of

M

[14]

Ther.

N

[13]

Biol

U

2010;10(3):211-8.

Cancer

the crypt-villus axis by the bacterial enterotoxin receptor guanylyl cyclase C restricts the

Li P, Schulz S, Bombonati A, Palazzo JP, Hyslop TM, Xu Y, Baran AA, Siracusa LD,

TE

[15]

D

proliferating compartment in intestine. Am J Pathol. 2007; 171(6):1847-58.

EP

Pitari GM, Waldman SA. Guanylyl cyclase C suppresses intestinal tumorigenesis by restricting proliferation and maintaining genomic integrity. Gastroenterology. 2007;

CC

133(2):599-607.

A

[16]

[17]

Pitari GM, Di Guglielmo MD, Park J, Schulz S, Waldman SA. Guanylyl cyclase C agonists regulate progression through the cell cycle of human colon carcinoma cells. Proc Natl Acad Sci U S A. 2001; 98(14):7846-51. Shahid U, Intestinal Stem Cells. Curr Gastroenterol Rep. 2010; 12(5): 340–348.

17

[18]

Wilson C, Lin JE, Li P, Snook AE, Gong J, Sato T, Liu C, Girondo MA, Rui H, Hyslop T, Waldman SA. The paracrine hormone for the GUCY2C tumor suppressor, guanylin, is universally

lost

in

colorectal

cancer.

Cancer

Epidemiol

Biomarkers

Prev.

[19]

SC RI PT

2014;23(11):2328-37. doi: 10.1158/1055-9965.EPI-14-0440. Shailubhai K1, Yu HH, Karunanandaa K, Wang JY, Eber SL, Wang Y, Joo NS, Kim HD, Miedema BW, Abbas SZ, Boddupalli SS, Currie MG, Forte LR. Uroguanylin treatment suppresses polyp formation in the Apc(Min/+) mouse and induces apoptosis in human colon adenocarcinoma cells via cyclic GMP. Cancer Res. 2000;60(18):5151-7.

Ullman TA, Itzkowitz SH. Intestinal inflammation and cancer. Gastroenterology

U

[20]

Lin JE, Snook AE, Li P, Stoecker BA, Kim GW, Magee MS, et al. GUCY2C Opposes

A

[21]

N

2011;140: 1807–1816.

M

Systemic Genotoxic Tumorigenesis by Regulating AKT-Dependent Intestinal Barrier Integrity. PLoS ONE 2012;7(2): e31686. Lin JE, Colon-Gonzalez F, Blomain E, Kim GW, Aing A, Stoecker B, Rock J, Snook AE,

D

[22]

TE

Zhan T, Hyslop TM, Tomczak M, Blumberg RS, Waldman SA. Obesity-Induced

EP

Colorectal Cancer Is Driven by Caloric Silencing of the Guanylin-GUCY2C Paracrine Signaling Axis. Cancer Res. 2016;76(2):339-46. doi: 10.1158/0008-5472.CAN-15-1467-

CC

T.

A

[23]

[24]

Blomain ES, Merlino DJ, Pattison AM, Snook AE, Waldman SA. Guanylyl Cyclase C Hormone Axis at the Intersection of Obesity and Colorectal Cancer. Mol Pharmacol. 2016;90(3):199-204. doi: 10.1124/mol.115.103192. Jessica A, Silence of the GUCY2C tumor suppressor. Science Translational Medicine. 2016;8:325,325ec22.

18

[25]

Kim GW, Lin JE, Waldman SA. GUCY2C: at the intersection of obesity and cancer. Trends Endocrinol Metab. 2013;24(4):165-73.

[26]

Kim GW, Lin JE, Snook AE, Aing AS, Merlino DJ, Li P, Waldman SA. Calorie-induced

2016; 23;6:e211. doi: 10.1038/nutd.2016.18. [27]

Blomain, ES, Waldman SA. Does obesity promote the development of colorectal cancer? Expert Rev Anticancer Ther. 2016; 16(5): 465–467.

[28]

SC RI PT

ER stress suppresses uroguanylin satiety signaling in diet-induced obesity. Nutr Diabetes.

Lehrer S, Rheinstein PH, Rosenzweig K. Mutations of the PDE5A gene confer a survival

U

advantage in patients with colon cancer. Cancer Prev Res (Phila). 2018. pii:

Zhu B, Vemavarapu L, Thompson WJ, Strada SJ. Suppression of cyclic GMP-specific

A

[29]

N

canprevres.0105.2018. doi: 10.1158/1940-6207.CAPR-18-0105.

Biochem. 2005;94(2):336-50.

Li N, Lee K, Xi Y, Zhu B, Gary BD, Ramírez-Alcántara V, Gurpinar E, Canzoneri JC,

D

[30]

M

phosphodiesterase 5 promotes apoptosis and inhibits growth in HT29 cells. J Cell

TE

Fajardo A, Sigler S, Piazza JT, Chen X, Andrews J, Thomas M, Lu W, Li Y, Laan DJ,

EP

Moyer MP, Russo S, Eberhardt BT, Yet L, Keeton AB, Grizzle WE, Piazza GA. Phosphodiesterase 10A: a novel target for selective inhibition of colon tumor cell growth

CC

and β-catenin-dependent TCF transcriptional activity. Oncogene. 2015;34(12):1499-509. doi: 10.1038/onc.2014.94.

A

[31]

Wang R, Kwon IK, Thangaraju M, Singh N, Liu K, Jay P, Hofmann F, Ganapathy V, Browning DD. Type 2 cGMP-dependent protein kinase regulates proliferation and differentiation in the colonic mucosa. Am J Physiol Gastrointest Liver Physiol. 2012;303(2):G209-19.

19

[32]

Janakiram NB, Rao CV. Molecular markers and targets for colorectal cancer prevention. Acta Pharmacol Sin. 2008;29(1):1-20.

[33]

Mahasneh A, Al-Shaheri F, Jamal E. Molecular biomarkers for an early diagnosis,

SC RI PT

effective treatment and prognosis of colorectal cancer: Current updates. Exp Mol Pathol. 2017;102(3):475-483. doi: 10.1016/j.yexmp.2017.05.005. [34]

Kwon IK, Wang R, Thangaraju M, Shuang H, Liu K, Dashwood R, et al. PKG inhibits TCF signaling in colon cancer cells by blocking beta-catenin expression and activating FOXO4. Oncogene 2010;29:3423–34.

Thompson WJ, Piazza GA, Li H, Liu L, Fetter J, Zhu B, Sperl G, Ahnen D, Pamukcu R.

U

[35]

N

Exisulind induction of apoptosis involves guanosine 3',5'-cyclic monophosphate

[36]

M

Cancer Res. 2000;60(13):3338-42.

A

phosphodiesterase inhibition, protein kinase G activation, and attenuated beta-catenin.

Liu L, Li H, Underwood T, Lloyd M, David M, Sperl G, et al. Cyclic GMP-dependent

D

protein kinase activation and induction by exisulind and CP461 in colon tumor cells. J

Lin JE, Li P, Snook AE, Schulz S, Dasgupta A, Hyslop TM, Gibbons AV, Marszlowicz

EP

[37]

TE

Pharmacol Exp Ther 2001;299:583–92.

G, Pitari GM, Waldman SA. The hormone receptor GUCY2C suppresses intestinal tumor

CC

formation by inhibiting AKT signaling. Gastroenterology 2010; 138: 241-254.

A

[38]

Rui Wang, In-Kiu Kwon, Muhan Hu, Darren D. Browning. Regulation of AKT-FoxO signaling by PKG2 inhibits colon cancer cell growth. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl): Abstract nr 2583. doi:10.1158/1538-7445.AM2013-2583.

20

[39]

Basu N, Saha S, Khan I, Ramachandra SG, Visweswariah SS. Intestinal cell proliferation and senescence are regulated by receptor guanylyl cyclase C and p21. J Biol Chem 2014; 289: 581-593. Wang R, Kwon IK, Singh N, Islam B, Liu K, Sridhar S, Hofmann F, Browning DD. Type

SC RI PT

[40]

2 cGMP-dependent protein kinase regulates homeostasis by blocking c-Jun N-terminal kinase in the colon epithelium. Cell Death Differ. 2014;21(3):427-37. doi: 10.1038/cdd.2013.163. [41]

Soh JW1, Mao Y, Kim MG, Pamukcu R, Li H, Piazza GA, Thompson WJ, Weinstein IB.

U

Cyclic GMP mediates apoptosis induced by sulindac derivatives via activation of c-Jun

Arber N, Kuwada S, Leshno M, Sjodahl R, Hultcrantz R, Rex D. Sporadic adenomatous

A

[42]

N

NH2-terminal kinase 1. Clin Cancer Res. 2000;6(10):4136-41.

M

polyp regression with exisulind is effective but toxic: a randomised, double blind, placebo controlled, dose-response study. Gut 2006;55:367–73. Shailubhai K, Palejwala V, Arjunan KP, Saykhedkar S, Nefsky B, Foss JA, Comiskey S,

D

[43]

TE

Jacob GS, Plevy SE. Plecanatide and dolcanatide, novel guanylate cyclase-C agonists,

EP

ameliorate gastrointestinal inflammation in experimental models of murine colitis. World J Gastrointest Pharmacol Ther. 2015;6(4):213-22. doi: 10.4292/wjgpt.v6.i4.213. Weinberg DS, Lin JE, Foster NR, Della'Zanna G, Umar A, Seisler D, Kraft WK,

CC

[44]

A

Kastenberg DM, Katz LC, Limburg PJ, Waldman SA. Bioactivity of Oral Linaclotide in

[45]

Human Colorectum for Cancer Chemoprevention. Cancer Prev Res (Phila). 2017;10(6):345-354. doi: 10.1158/1940-6207.CAPR-16-0286. https://clinicaltrials.gov/ct2/show/study/NCT01950403

21

[46]

FDA approves Trulance for Chronic Idiopathic Constipation". FDA.gov. U.S. Food and Drug Administration. Retrieved 20 January 2017.

[47]

Wen-Chi, LC, Shet Masih, Anusha Thadi, Viren Patwa, Apoorva Joshi, Harry S Cooper,

SC RI PT

Vaseem A Palejwala, Margie L Clapper, and Kunwar Shailubhai. Plecanatide-mediated activation of guanylate cyclase-C suppresses inflammation-induced colorectal carcinogenesis in Apc+/Min-FCCC mice. World J Gastrointest Pharmacol Ther. 2017;8(1): 47–59. [48]

Shailubhai K, Jacob GS, inventors; Synergy Pharmaceuticals Inc (New York, NY, US),

U

assignee. Agonists of guanylate cyclase useful for the treatment of gastrointestinal

N

disorders, inflammation, cancer and other disorders. United States patent US 7, 879, 802

Zhang G, Arjunan KP, Foss J, Comiskey S, Shailubhai K. SP-333, a proteolysis-resistant

M

[49]

A

B2. 2011.

agonist of guanylate cyclase-C, inhibits activation of NF-kB and suppresses production of

D

inflammatory cytokines to ameliorate DSS induced colitis in mice (Abstract 1555) Am J

Gali H, Sieckman GL, Hoffman TJ, Owen NK, Mazuru DG, Forte LR, Volkert WA.

EP

[50]

TE

Gastroenterol. 2012;107(Supplement 1):S626.

Chemical Synthesis of Escherichia Coli STh Analogues by Regioselective Disulfide Bond Biological Evaluation of an 111In-DOTA−Phe19-STh Analogue for

CC

Formation: 

Specific Targeting of Human Colon Cancers. Bioconjugate Chem., 2002;13 (2):224–231.

A

[51]

Wolfe HR, Mendizabal M, Lleong E, Cuthbertson A, Desai V, Pullan S, Fujii DK, Morrison M, Pither R, Waldman SA. In vivo imaging of human colon cancer xenografts in immunodeficient mice using a guanylyl cyclase C--specific ligand. J Nucl Med. 2002;43(3):392-9.

22

[52]

Gallery M, Zhang J, Bradley DP, Brauer P, Cvet D, Estevam J, Danaee H, Greenfield E, Li P, Manfredi M, Loke HK, Rabino C, Stringer B, Williamson M, Wyant T, Yang J, Zhu Q, Abu-Yousif A, Veiby OP. A monomethyl auristatin E-conjugated antibody to guanylyl

SC RI PT

cyclase C is cytotoxic to target-expressing cells in vitro and in vivo. PLoS One. 2018 Jan 25;13(1):e0191046. doi: 10.1371/journal.pone.0191046. eCollection 2018. [53]

Almhanna K, Kalebic T, Cruz C, Faris JE, Ryan DP, Jung JA, Wyant T, Fasanmade A, Messersmith WA, Rodon J. Phase I study of the investigational anti-guanylyl cyclase antibody-drug conjugate TAK-264 (MLN0264) in adult patients with advanced malignancies.

Clin

Cancer

Res

2016;

22(20):5049-5057.

U

gastrointestinal

Magee MS, Kraft CL, Abraham TS, Baybutt TR, Marszalowicz GP, Li P, Waldman SA,

A

[54]

N

http://dx.doi.org/10.1158/1078-0432.ccr-15-2474

M

Snook AE. GUCY2C-directed CAR-T cells oppose colorectal cancer metastases without autoimmunity. Oncoimmunology. 2016;5(10):e1227897. eCollection 2016. Magee MS, Abraham TS, Baybutt TR, Flickinger JC Jr, Ridge NA, Marszalowicz GP,

D

[55]

TE

Prajapati P, Hersperger AR, Waldman SA, Snook AE. Human GUCY2C-Targeted

EP

Chimeric Antigen Receptor (CAR)-Expressing T Cells Eliminate Colorectal Cancer Metastases. Cancer Immunol Res. 2018;6(5):509-516. doi: 10.1158/2326-6066.CIR-16-

CC

0362.

A

[56]

Whitt JD, Li N, Tinsley HN, Chen X, Zhang W, Li Y, Gary BD, Keeton AB, Xi Y, Abadi AH, Grizzle WE, Piazza GA. A novel sulindac derivative that potently suppresses colon tumor cell growth by inhibiting cGMP phosphodiesterase and β-catenin transcriptional activity. Cancer Prev Res (Phila). 2012;5(6):822-33. doi: 10.1158/1940-6207.CAPR-110559.

23

[57]

Tinsley HN, Gary BD, Thaiparambil J, Li N, Lu W, Li Y, Maxuitenko YY, Keeton AB, Piazza GA. Colon tumor cell growth-inhibitory activity of sulindac sulfide and other nonsteroidal anti-inflammatory drugs is associated with phosphodiesterase 5 inhibition.

[58]

SC RI PT

Cancer Prev Res (Phila). 2010;3(10):1303-13. doi: 10.1158/1940-6207.CAPR-10-0030. Lin S, Wang J, Wang L, Wen J, Guo Y, Qiao W, Zhou J, Xu G, Zhi F. Phosphodiesterase5 inhibition suppresses colonic inflammation-induced tumorigenesis via blocking the recruitment of MDSC. Am. J. Cancer Res. 2017;7(1):41-52. [59]

Li N1, Xi Y, Tinsley HN, Gurpinar E, Gary BD, Zhu B, Li Y, Chen X, Keeton AB, Abadi

U

AH, Moyer MP, Grizzle WE, Chang WC, Clapper ML, Piazza GA. Sulindac selectively

N

inhibits colon tumor cell growth by activating the cGMP/PKG pathway to suppress

A

Wnt/β-catenin signaling. Mol Cancer Ther. 2013;12(9):1848-59. doi: 10.1158/1535-

[60]

M

7163.MCT-13-0048.

Abadi AH, Gary BD, Tinsley HN, Piazza GA, Abdel-Halim M. Synthesis, molecular

D

modeling and biological evaluation of novel tadalafil analogues as phosphodiesterase 5

TE

and colon tumor cell growth inhibitors, new stereochemical perspective. Eur J Med Chem.

[61]

EP

2010;45(4):1278-86.

Steinbrecher KA, Wowk SA, Rudolph JA, Witte DP, Cohen MB. Targeted inactivation

CC

of the mouse guanylin gene results in altered dynamics of colonic epithelial proliferation.

A

Am J Pathol. 2002; 161: 2169-2178.

24

Figure legends

SC RI PT

Fig. 1. GUCY2C/cGMP/PDEs signaling and its associated oncogenic pathways in CRC tumorigenesis and progression. A. Paracrine hormones/GUCY2C agonists bind on GUCY2C that leads to formation of cGMP from GTP. Elevation of cGMP levels activates PKGII which further inhibits β-catenin transcriptional activity and gene products (cyclin D1 and survivin) during APC mutated condition. PDEs (5 and 10) are highly expressed in CRC epithelial barrier during tumorigenesis and degrade cGMP to 5`-GMP and block transduced signaling of paracrine hormone/agonist-mediated GUCY2C. B. PDEs inhibitors allow paracrine hormone/agonistGUCY2C-mediated tumor suppressor effects in CRC epithelial barrier by inhibiting cGMP degradation and activation of PKGII which further inactivates β-catenin-TCF transcriptional activity.

A

CC

EP

TE

D

M

A

N

U

Fig. 2. Chemical structures of some of the GUCY2C agonists and PDE inhibitors with chemopreventive efficacy against colorectal cancer.

25

Table 1: Role of the GUCY2C/cGMP/PDEs signaling pathway in intestinal homeostasis and colorectal tumorigenesis Biomarker/

Experimental type

Observation/Inference

Reference

Target Knockout

Disrupted homeostasis of crypt-villus axis

[61]

SC RI PT

GUCY2C

represented as an increase in proliferative

cells, accelerated cell cycle, and enhanced

depth of crypt, leads to increased risk of tumorigenesis of the colon.

U

Increased DNA damage and several folds [14, 15]

N

(8-fold) increase in colon tumors in

A

APCMin/+ mice.

M

Upregulated expression or activation of [37, 39]

D

proliferating markers (cyclin D1, pRb, β-

A

CC

EP

TE

catenin, and phosphorylated-AKT), and suppressed expression of tumor suppressors (p27 and p21). Increased crypt length (hyperplasia) and

[14, 15]

increases in cell migration. Phase I clinical GUCY2C activation and cGMP elevation in trial

epithelial cells of the colorectum in humans.

(NCT01950403)

upon oral dose of a GUCY2C agonist linaclotide (0.87 mg per day).

26

[44, 45]

Knockout

Dextran sulfate sodium colitis caused >80%

[21]

deaths in GUCY2C−/− (knockout) mice compared with GUCY2C+/+ mice, which

Knock-in

SC RI PT

showed 100% survival. GUCY2A+ mice resisted against disruption of

barrier

and

DSS-induced

[21]

colitis

compared with wild-type mice Knockout

APC

agents

suppressed [9]

polyposis in APCMin/+ mice by regulating

U

(Heterogenous)

cGMP–elevating

guanylin levels

are [9]

decreased and increased proliferation in

M

(Heterogenous)

Uroguanylin and

A

Knockout

N

homeostasis in preneoplastic epithelium

non-polyp mucosal tissues of APCMin/+ mice

TE

D

compared with mucosal tissue of wild-type

Knock-in

A

CC

EP

Guanylin

mice. Increased

expression

intestinal

epithelial

of

guanylin

cells

in [9]

activated

GUCY2C signaling and prevented intestinal tumorigenesis.

Clinical study

Guanylin was found in colorectal mucosa [18] specimens from healthy individuals (n=30), but was absent from patients with colorectal tumors (n=54).

27

Uroguanylin

APCMin/+

Suppressed polyp formation in APCMin/+

Knockout

mice.

[19]

(Heterogenous) Knockout

Caused crypt hyperplasia, as well as

[31]

SC RI PT

PKG2

increased apoptosis of epithelial cells, and

reduced numbers of differentiated goblet

and enteroendocrine cells in the colons of mice.

data 96.77% CRC patients are without PDE5 [28]

analysis

mutation. Patients without mutation of

U

Clinical

N

PDE5

A

PDE5 had significantly poorer survival than

inhibition

Elevated the cGMP levels in colon mucosa of

D

Pharmacological

M

did patients with PDE5 mutation.

wild-type

mice

and

[40]

suppressed

TE

proliferation and apoptosis, but increased

APCMin/+

EP

PDE10

A

CC

Knockout

(Heterogenous)

secretory cell lineage differentiation, in the colon epithelium. PDE10 expression levels were high in [30] intestinal

tumors

of

APCMin/+

mice

compared with levels in mucosa of normal intestine from wild-type mice.

Clinical Study

PDE10 expression levels are higher in colon tumors from patients with stage I - IV than

28

[30]

in normal colonic mucosa from healthy

A

CC

EP

TE

D

M

A

N

U

SC RI PT

individuals.

29

D

TE

EP

CC

A

SC RI PT

U

N

A

M

Figr-1

30

31

D

TE

EP

CC

A

SC RI PT

U

N

A

M

D

TE

EP

CC

A

SC RI PT

U

N

A

M

Figr-2

32