Autophagy is a therapeutic target in anticancer drug resistance

Biochimica et Biophysica Acta 1806 (2010) 220–229

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Biochimica et Biophysica Acta j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / b b a c a n

Review

Autophagy is a therapeutic target in anticancer drug resistance Suning Chen a,b, Sumaiyah K. Rehman c, Wei Zhang d, Aidong Wen a, Libo Yao b,⁎, Jian Zhang a,b,⁎ a

Department of Pharmacy, Xijing Hospital, The Fourth Military Medical University, Xi'an, Shaanxi Province, 710032, People's Republic of China The Department of Biochemistry and Molecular Biology and The State Key Laboratory of Cancer Biology, The Fourth Military Medical University, Xi'an, Shaanxi Province, 710032, People's Republic of China c Department of Molecular and Cellular Oncology, The University of Texas, MD Anderson Cancer Center and the University of Texas Graduate School of Biomedical Sciences at Houston, Houston, TX, 77030, USA d Department of Biopharmaceutics, School of Pharmacy, The Fourth Military Medical University, Xi'an, Shaanxi Province, 710032, People's Republic of China b

a r t i c l e

i n f o

Article history: Received 29 April 2010 Received in revised form 5 July 2010 Accepted 7 July 2010 Available online 13 July 2010 Keywords: Autophagy Cancer Drug resistance

a b s t r a c t Autophagy is a type of cellular catabolic degradation response to nutrient starvation or metabolic stress. The main function of autophagy is to maintain intracellular metabolic homeostasis through degradation of unfolded or aggregated proteins and organelles. Although autophagic regulation is a complicated process, solid evidence demonstrates that the PI3K-Akt-mTOR, LKB1-AMPK-mTOR and p53 are the main upstream regulators of the autophagic pathway. Currently, there is a bulk of data indicating the important function of autophagy in cancer. It is noteworthy that autophagy facilitates the cancer cells' resistance to chemotherapy and radiation treatment. The abrogation of autophagy potentiates the re-sensitization of therapeutic resistant cancer cells to the anticancer treatment via autophagy inhibitors, such as 3-MA, CQ and BA, or knockdown of the autophagy related molecules. In this review, we summarize the accumulation of evidence for autophagy's involvement in mediating resistance of cancer cells to anticancer therapy and suggest that autophagy might be a potential therapeutic target in anticancer drug resistance in the future. © 2010 Elsevier B.V. All rights reserved.

Contents 1. 2.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Molecular regulation mechanism of autophagy in cancer . . . . . . . . . . . . 2.1. PI3K-Akt-mTOR . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. LKB1-AMPK-mTOR . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. p53 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4. Beclin1 and Bcl-2 . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5. Other mechanisms . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Double face of autophagy's function in cancer . . . . . . . . . . . . . . . . . 3.1. Autophagy inhibits tumorigenesis . . . . . . . . . . . . . . . . . . . . 3.2. Autophagy promotes cancer cells survival . . . . . . . . . . . . . . . 4. Autophagy inhibition facilitates anticancer drug resistance . . . . . . . . . . . 4.1. Autophagy confers the anticancer drug resistance . . . . . . . . . . . . 4.2. Autophagy inhibition sensitizes the tumor cells to anticancer therapy . . . 4.3. Autophagy inhibitors as the potential drug to overcome anticancer therapy 5. Concluding remarks. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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1. Introduction ⁎ Corresponding authors. J. Zhang is to be contacted at Department of Pharmacy, Xijing Hospital, The Fourth Military Medical University, Xi'an, Shaanxi Province, 710032, People's Republic of China. Tel./fax: + 86 29 84774513. L. Yao, Tel./fax: + 86 29 84774513. E-mail addresses: [email protected] (L. Yao), [email protected] (J. Zhang). 0304-419X/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.bbcan.2010.07.003

Autophagy, also called macroautophagy, is a kind of cellular catabolic degradation response to nutrient starvation or metabolic stress [1–3]. During the initial stages of autophagic process, cellular proteins, organelles and cytoplasm are sequestered and engulfed by the intracellular double-membrane-bound structures, called

S. Chen et al. / Biochimica et Biophysica Acta 1806 (2010) 220–229

autophagosomes (early autophagic vesicles) [1,2,4,5]. These autophagosomes mature by fusing with lysosomes to form the autolysosomes (late autophagic vesicles) [2,4–8], in which the sequestered proteins and organelles are digested by lysosomal hydrolases and recycled to sustain cellular metabolism [9–13]. Whenever we talked about autophagy, we had to refer to apoptosis (process of programmed cell death), which shared several common regulatory elements with autophagy [14–16]. Autophagy was believed as a non-apoptotic programme of cell death or “type-II” cell death to distinguish with apoptosis [17]. Under most circumstances, autophagy promoted cell survival by adapting cells to the stress conditions, which was functionally paradoxical to apoptosis. However, it is still fundamentally important to clarify whether autophagy is a main strategy for cell survival, or if it also serves as a trigger for cell death [17]. Autophagy is an evolutionarily conserved process from yeast to mammals [3,18,19]. The main function of autophagy is to maintain intracellular metabolic homeostasis. In parallel with the ubiquitin proteasome degradation pathway, the dynamic autophagic process supervises and maintains the protein and organelle quality control to prevent the accumulation of unfolded and aggregated proteins [6– 8,11,13]. In addition to this key function, autophagy was also found to be responsible for other important functions, especially under stressful situation [18,20,21]. Whenever faced with environmental stressors, such as metabolic deprivation DNA damage caused by chemotherapy or radiation and hypoxia, the process of autophagy is activated which leads to cell survival or death [2,18,22–27]. There is an accumulation of evidence that highlights the important function of autophagy in cancer [2,5,22–25,28]. Although it is still controversial about whether autophagy kills cancer cells or sustains their survival under stressful conditions, more and more reports provide data to support that autophagy promotes cancer cell survival after chemotherapy or radiation therapy [22,29,30]. For example, autophagy facilitates resistance chronic myeloid leukemia (CML) to Imatinib mesylate (IM) [31], and also potentiates resistance of HER2 positive breast cancer cells to anti-HER2 monoclonal antibody trastuzumab [32]. Intriguingly, abrogation of autophagy by autophagy inhibitors, such as 3MA, CQ and BA, or by shRNA knockdown of autophagy related molecules, re-sensitizes the resistant cancer cells to the chemotherapy or radiation [30,31]. It is also noteworthy that the autophagic inhibitor hydroxychloroquine (HCQ) has already been applied in a clinical trial. So, it is possible to expect autophagic inhibitors to be the next generation of drugs to overcome anticancer therapeutic resistance. Herein, in this review, we will discuss the state-of-art of the molecular regulation mechanisms of autophagy, autophagy function in cancer, and emphasize the facilitation of autophagic inhibition in anticancer drug resistance.

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constitutive PI3K activation, which are detected in many human cancers [48–53]. As the major downstream of class I PI3K, Akt can activate mTOR and lead to inhibition of autophagy [39,54]. Contrary to class I PI3K, class III PI3K can induce autophagy. So, the class III PI3K inhibitor, such as 3-MA, LY294002 and wortmannin, can repress the autophagy process [55–57]. In addition, the tumor suppressor gene PTEN can activate autophagy through inhibition of Akt activity [58]. Hence, the loss or mutation of PTEN will cause Akt activation and inhibit autophagy. Similar to PTEN, another tumor suppressor ARHI also can induce autophagy through inhibition of the PI3K-Akt signaling pathway [59]. As the downstream target of Akt, the serine/threonine kinase mTOR controls most of the autophagy-related genes, both transcriptionally and translationally. It is speculated that the inhibition of autophagy by mTOR is mainly dependent on p70S6 kinase and the eukaryotic initiation factor 4EBP1 activity [60,61]. Thus, the mTOR inhibitors rapamycin and RAD001 could induce autophagy via inhibition of mTOR activity and demonstrate their intriguing potential in cancer treatment [62–64]. 2.2. LKB1-AMPK-mTOR As the central metabolic sensor, AMP-activated protein kinase (AMPK) regulates lipid, cholesterol and glucose metabolism and is also closely related with cell metabolism and cancer [65,66]. AMPK activates under the energy stress conditions with increased AMP/ATP ratio to repress mTOR and initiate autophagy [67]. So far, the serine/ threonine kinase LKB1 (also known as STK11) is believed to be the major upstream regulator of AMPK, which directly phosphorylates and activates AMPKα at Thr172 site [68,69]. It is noteworthy that AMPK can also be activated by other upstream kinases, such as calcium and calmodulin-dependent protein kinase kinase 2 (CAMKK2), in hypothalamic neurons [70], T cells and endothelial cells [71,72]. It is suggested that these group of cells are more sensitive to response to the cellular calcium concentration, which signifies the link between calcium concentration mediated regulation and autophagy. Currently, Griselda et al. identified Transforming growth factor-β-activating kinase 1 (TAK1) as another new AMPK upregulator [73]. Whenever treated with the tumour necrosis factor-related apoptosis inducing ligand (TRAIL), the untransformed human breast epithelial MCF10A cells generated cytoprotective autophagy via AMPK-mTOR pathway, which was dependent on TAK1 and TAK1-binding subunit 2, but not the LKB1 or CAMKK2. Taken together, it is possible to identify more AMPK upregulators, but it is beyond doubt that the AMPK-mTOR-autophagy pathway already displays the well-recognized contribution to metabolic remodeling, cancer development and anticancer drug resistance.

2. Molecular regulation mechanism of autophagy in cancer 2.3. p53 Autophagy is a complicated regulatory process, which involves a great number of upstream regulating signaling pathways [2,22,28]. Nevertheless, no matter it is normal or cancer cells, the mammalian target of rapamycin (mTOR) serves as the main regulator of autophagy [22,33–43]. In response to nutrient availability, mTOR activation suppresses autophagy and stimulates cell proliferation. However, under nutrient deprivation, hypoxia, genomic instability or other stress conditions, the suppression of mTOR triggers the autophagic cascade and inhibits the cell proliferation [21,22,34,35,44]. Herein, we mainly introduce PI3K-Akt-mTOR, LKB1-AMPK-mTOR, P53, Beclin1 and Bcl-2 pathway in the regulation of autophagy. 2.1. PI3K-Akt-mTOR Increasing evidence shows that the over-active PI3K-Akt pathway promotes tumor cell survival [45–47]. The somatic mutations of PI3K, such as E542K, E545D and E545K, are common mutations that lead to

As a well-known tumor suppressor gene, p53 also involves in genotoxic stress response and DNA damage repair [74–76]. Therefore, it is no surprise that p53 can also regulate autophagy [77–79], though p53's function in the control of autophagy is a little complicated [80,81] mainly due to the intricate cellular microenvironment. On one hand, p53 can activate AMPK and then inhibit mTOR to induce the autophagy process [67,82]. Simultaneously, p53 promotes autophagy through triggering other downstream targets, such as Sestrin2 [83,84], damage-regulated autophagy modulator (DRAM) [78,85], Bcl-2-associated X protein (Bax) and p53-upregulated modulator of apoptosis (PUMA) [86], which contribute to the induction of apoptosis. Under nutrient deprivation or other stressful conditions, p53 activates autophagy in adaptation to the low ATP level, which facilitates the apoptosis course. Therefore, it is concordant with the tumor suppressor function of p53 in the positive regulatory process of autophagy [78].

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On the other hand, in addition to the autophagy inducer, p53 also can repress autophagy [87]. Tasdemir et al. [88,89] showed that depletion of p53 either in vitro by RNA interference or pharmacological inhibition in cell lines or in vivo by genetic deletion in mice could induce autophagy. And the autophagy induced by p53 loss promotes the survival of p53deficient cells to sustain high ATP levels under conditions of hypoxia and nutrient depletion [89]. Therefore, this dual interplay between p53 and regulation of autophagy is still indefinable due to possible key underlying aspects of metabolism and cancer biology. Actually, it is difficult to reconcile both positive and negative function of p53 in autophagy regulation. Taken together, it is important to note that these two distinct effects take place under different stress conditions, which reflects the ability of p53 to promote cell survival during low levels of stress or damage and induce cell death when the damage is more serious [90,91]. 2.4. Beclin1 and Bcl-2 Beclin1 (also called Atg6) is essential for the double-membrane autophagosome formation, which is required during the initial steps of autophagy [92]. The autophagic induction of Beclin1 facilitates the inhibition of tumorigenesis [93]. The allelic loss of Beclin1 is frequently seen in human breast, ovarian and prostate cancers [94]. Beclin1 promotes the initiation of autophagy mainly through binding with other autophagy-related proteins such as Bcl-2, Vps34, p150, UVRAG, Bif1, Atg14L and Rubicon to form huge protein complexes [95–99]. Bcl-2 proto-oncogene, overexpressed in 50–70% of breast cancers, is one of the major causes that induces resistance to chemotherapy and hormone therapy-induced apoptosis. In addition to the wellestablished anti-apoptosis function, Bcl-2 is also well-documented to inhibit autophagy via the physical interaction with Beclin1 [98]. And the autophagy inhibition might be the accumulative effect on the oncogenic properties of Bcl-2 family. Akar et al. [100] reported that RNAi knockdown of Bcl-2 induced the autophagy and apoptosis in MCF-7 breast cancer cells. Simultaneously, Saeki et al. [101] found that Bcl-2 silencing by antisense mRNA induced the autophagy-dependent cell death in human leukemia HL60 cells. The interaction of Beclin1 with Bcl-2 is mainly dependent on its BH3 domain with BH3-receptor domain on Bcl-2 proteins [102]. Therefore, in addition to the direct knockdown of Bcl-2, it is also feasible to abrogate some of their interactions by some BH3 mimics to induce autophagy. As one BH3-mimetic compound, ABT737 could induce autophagy through competitively disrupting the inhibitory interaction between Beclin1 and Bcl-2/Bcl-X [103,104]. At least, silencing Bcl-2 or disrupting the Beclin1 and Bcl-2 interaction to induce autophagic cell death might be used as a novel therapeutic strategy in cancer cells overexpressing Bcl-2. 2.5. Other mechanisms Under hypoxia and nutrient deprivation condition, a great number of unfolded or misfolded proteins can trigger endoplasmic reticulum (ER) stress. There are evidences showing that the accumulation of ER stress could induce autophagy through protein kinase-like endoplasmic reticulum kinase (PERK) and inositol-requiring kinase 1 (IRE1) dependent mechanisms in the heart [105,106]. Although there is no documented report of ER stress inducing autophagy currently in cancer cells, it is worthwhile to exploit the adaptive mechanism and biological function for therapeutic benefit of cancer treatment. Recently, Criollo et al. [107] reported that IKK (IκB kinase) stimulated autophagy in an NF-κB-independent manner. They found that the constitutively active IKK subunits stimulated autophagy through activating multiple signals, including the phosphorylation of AMPK and JNK1 in starvation-induced condition. These results indicate that IKK has a pivotal role in the stimulation of autophagy by physiological and pharmacological stimuli.

3. Double face of autophagy's function in cancer Although the regulatory mechanism of autophagy is partially manifested, however, the function of autophagy in cancer is still in an undetermined debate. Whether autophagy is a death-induced mechanism or a protective effort for cellular survival is still a controversy [1,2]. There are a number of evidences to support the double-faced function of autophagy in cancer: tumor suppression and also promotion. 3.1. Autophagy inhibits tumorigenesis Distinct from apoptosis, autophagy is termed the type II programmed cell death (PCD) [108]. The basal level of autophagy in normal tissues provides a very important homeostatic and housekeeping function. Autophagy and ubiquitin proteasome degradation system monitor and clean the aggregated unfolded or misfolded proteins, and damaged cellular organelles to maintain intracellular homeostasis [6–8,11,13]. Therefore, abrogation of the dynamic autophagy pathway will result in disease or tumorigenesis. The autophagy related genes knockout mice with Atg5−/− or Atg7−/− developed the progressive deficits in motor function and behavioral defects [9,10]. The major reason is due to the loss of functional autophagy to induce the accumulation of cytoplasmic inclusion bodies. These results suggest that the functional clearance of aggregated cytosolic proteins through basal autophagy is crucial for preventing the accumulation of abnormal proteins. The tumor suppressor role of autophagy was firstly documented through genetic approaches of Beclin1. In 1999, Liang et al. [93] reported Beclin1 as a potential tumor suppressor gene, the expression of which is frequently decreased in human breast epithelial carcinoma cell lines and tissue compared to the higher level in normal tissue. These findings suggested that decreased expression of autophagy proteins might contribute to the initiation or progression of breast cancer. This notion was further confirmed by others Yue et al. discovered that Beclin1−/− mice died early in embryogenesis and Beclin1+/− mutant mice suffered from a high incidence of spontaneous tumors [109]. Also, the heterozygous disruption of Beclin1 increases the spontaneous malignancies frequency and accelerates the development of hepatitis B virusinduced premalignant lesions [110]. These data provided the direct proof of the function of Beclin 1 as a tumor suppressor. Actually, the tumor suppressor function is not only subjected to Beclin1. Alteration of other autophagic genes has also been reported in other tumor types. Marino et al. [111] found that Atg4C knockout mice show an increased susceptibility to the development of fibrosarcomas induced by chemical carcinogens. Deletion of Atg5 conferred natural killer (NK) cells malignancy [112]. Bax-interacting factor-1 (Bif-1) protein, a positive regulator of apoptosis and autophagy, plays a critical role in tumorigenesis. Loss of Bif-1 suppresses programmed cell death and promotes colon adenocarcinomas [113]. Another important autophagy-related molecule, Ultraviolet radiation resistance-associated gene (UVRAG) could bind with Beclin1 and induce autophagy [114]. The frame shift mutations of UVRAG gene exon 8 resulted in decreased autophagy activity and induced the colorectal and gastric carcinomas occurrence [114,115]. All these data support the tumor suppressive function of autophagy. The inhibition of tumorigenesis by autophagy is a common effect, and not limited to one unique autophagic molecule. However, the detailed mechanism by which autophagy functions as a tumor suppressor has not been fully elucidated. Nonetheless, it is believed that the failure of DNA repair to maintain genome integrity from genotoxic stress might be the major reason [2,4]. 3.2. Autophagy promotes cancer cells survival Although many research results display the tumor suppressive function of autophagy, there is growing evidence that supports the

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autophagic role in tumor survival. During progression, cancer cells require plenty of nutrients and oxygen to sustain the rapid cell proliferation. Unfortunately, many tumors undergo the hypoxia and metabolic stress during the progression and invasion, especially solid tumors with their poor vascularization. But how do these cells survive under the metabolic stress conditions? Part of the answer is that autophagy can maintain and promote autophagic tumor cells’ survival. Metabolic stress can robustly induce autophagy in cancer cells with defective apoptosis [116]. Thus, autophagy inhibition by constitutively active Akt or allelic disruption of Beclin1 confers sensitivity to metabolic stress by inhibiting the autophagy-dependent survival pathway in vitro and in vivo. Karantza-Wadsworth [23] found that defective autophagy induced by the allelic loss of Beclin1 sensitized mammary epithelial cells to metabolic stress and accelerated lumen formation in mammary acini. In the growth factordependent Bax/Bak-deficient cells, autophagy can prevent the cell death following growth factor withdrawal [117]. Cancer cells can initiate, proliferate and propagate in many kinds of serious stress conditions, such as glucose starvation, hypoxia, growth-factor withdrawal and even the cytotoxic cellular damage of chemotherapy or radiation [2,4,91,117]. In general, autophagy activation follows the rule of the “Survival of the Fittest” for the cancerous cells. Autophagy facilitates the removal of damaged proteins and organelles, and functions as a back-up energy reserve to provide extra energy to sustain the tumor cells' survival against anticancer therapy. Thus, autophagy may function in tumor promotion by mitigating and limiting metabolic stress to protect the genome’s integrity and confer resistance to anticancer therapy. On the contrary, defective autophagy facilitates DNA damage and genomic instability which ultimately confers apoptosis or death upon cancer cells.

4. Autophagy inhibition facilitates anticancer drug resistance 4.1. Autophagy confers the anticancer drug resistance Adjuvant cancer treatment, such as chemotherapy or radiotherapy, etc., is very important to prevent or postpone the cancer’s relapse and prolong the patients' survival. However, one of the most daunting clinical problems is the frequent relapse after the treatment even with longer dormancy [2,118]. Most of the therapeutic failures are due to the

Table 1 Known sensitizing function of autophagy inhibition in breast cancer. Cancer cell types

Inhibition targets of autophagy

Treatment

Ref.

Breast cancer cells lines with HER2 overexpression SKBR3 Breast cancer cells lines with ER overexpression T47D and MCF-7 Breast cancer cells lines with ER overexpression T47D and MCF-7 Breast cancer cells lines MCF7 and MDA-MB-231 Breast cancer cells lines MCF-7/LCC9 Breast cancer cell lines MCF-7/MCF-7. Beclin1 Breast cancer cell lines MDA-MB-231

Knockdown of LC3

anti-HER2 monoclonal antibody Trastuzumab

[32]

Pharmacological inhibitor 3-MA

Estrogen receptor antagonist Tamoxifen

[119–121]

Knockdown of Beclin1 Knockdown of Atg5, Beclin1 and Atg7

Estrogen receptor antagonist Tamoxifen

[122]

Knockdown of LC3B, ATF4 and HDAC6

Inhibitor of the 26 S proteasome Bortezomib

[123]

Knockdown of Beclin 1 Estrogen receptor antagonist Faslodex (FAS) Knockdown of Beclin 1 Estrogen receptor antagonist Faslodex (FAS) and Tamoxifen Knockdown of Beclin 1 Atg3 Atg4b, Atg4c, Atg5 and atg12

γ-radiation

[124]

[121]

[30]

intrinsic or acquired resistance towards the therapy. Presumably, autophagy is one of the most important mechanisms to enable cancer cells' survival and eventual recurrence after long-term cytotoxic treatment. Especially during the acquired resistance process, therapeutic drugs can kill most of the cancer cells. But a few remaining cells tolerate the dramatically harsh and stressful conditions (Fig. 1) due to the cell’s protective mechanism of autophagy. The recovery of this group of cells results in the tumor to relapse once growth conditions are

Table 2 Known sensitizing function of autophagy inhibition in colorectal cancer. Cancer cell types

Inhibition targets of autophagy

Colon cancer cell lines HT29 and HCT116

Vorinostat Pharmacological inhibitors chloroquine (CQ), Knockdown of Atg7 Pharmacological TFT and 5-FU inhibitors 3-MA

[125]

Pharmacological inhibitors 3-MA Knockdown of Beclin1 and UVRAG Pharmacological inhibitors PepA, Knockdown of Atg7 Pharmacological inhibitors 3-MA

5-FU

[56]

UVRAG

[114]

Colorectal cancer cell lines WiDR, Lovo92 and Colo320 Colorectal cancer cell lines colon26 and HT29 Colorectal cancer cell lines HCT116 Colorectal cancer cell lines SW480, DLD-1, WiDr, SW620 and LoVo Colon cancer cell lines HT-29 Colon cancer cell lines HCT116

Fig. 1. Autophagy confers the anticancer therapy resistance. Cancer cells can tolerate the metabolic stress caused by anticancer therapy, including chemotherapy, radiation, tyrosine kinase inhibitors, target therapy antibody, etc. The induced autophagy facilitates to reserve the energy and promote the cancer cells survival, which finally confer the anticancer therapy resistance.

223

Colorectal cancer cell lines DLD1 and HT29 Rectum carcinoma cell lines SW707

Pharmacological nhibitors 3-MA, E64D and PepA Knockdown of atg5, atg7, Beclin1 and UVRAG Knockdown of Beclin1 Knockdown of Beclin 1 Atg3 Atg4b, Atg4c, Atg5 and atg12

Treatment

Ref.

[126]

Amino acid and [127] glucose deprivation Cyclooxygenase inhibition with Sulindac sulfide TRAIL

[128]

Hypoxia and Bortezomib γ-radiation

[130]

[129]

[30]

224

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Table 3 Known sensitizing function of autophagy inhibition in glioblastoma and lung cancer. Cancer cell types

Inhibition targets of autophagy

Treatment

Ref.

CD133+

Pharmacological inhibitor 3-MA and BA Knockdown of Beclin 1 and Atg5 Pharmacological inhibitor 3-MA and BA

γ-radiation

[26,131]

Glioma stem cells Glioma cell lines U87-MG and U373-MG Glioma cell lines LN229, LZN308, U87-MG and U373-MG Astrocytoma, Glioblastoma cells Glioma cell lines

U251, SF188 and U373 Glioma cell lines A172, T98G, U87-MG and U373-MG Lung cancer cell lines A549

Pharmacological inhibitors 3-MA and BA Knockdown of Atg12-Atg5

Cancer cell types

Inhibition targets of autophagy

Pharmacological inhibitors BA and CQ, Knockdown of Atg5 and Atg7 K562, BaF3/E255K cells and Pharmacological BaF3/T315I CML cells inhibitors CQ CML cells Pharmacological inhibitors CQ Chronic myeloid leukemia (CML) cells K562, BV173 and 32D-p210BCR/ABL

N-(4-hydroxyphenyl) [132] retinamide (4-HRP) induced autophagy tyrosine kinase [133] inhibitor (TKI) Imatinib

AGT inhibitor O6-benzylguanine (BG) Pharmacological inhibitors 3-MA and oligomycin Knockdown of Beclin1

Temozolomide (TMZ)

[134]

Temozolomide (TMZ) and Etoposide

[135]

Pharmacological inhibitors 3-MA and BA

Temozolomide (TMZ) [136]

Knockdown of Beclin 1 Atg3 Atg4b, Atg4c, Atg5 and atg12

γ-radiation

[30]

favourable. Since autophagy confers anticancer drug resistance mainly through allowing the residual or metastasized tumor cells to tolerate the cytotoxic stress, the potent inhibition of autophagy in resistant cancer cells might be an efficient approach for cancer eradication. 4.2. Autophagy inhibition sensitizes the tumor cells to anticancer therapy It is remarkable to notice that a great number of studies show that inhibition of autophagy confers cancer cells sensitive to a wide spectrum of therapies. Currently, there are several potential strategies to inhibit autophagy, including autophagy inhibitors and shRNA approach, to re-sensitize the resistant cancer cells to anticancer therapy. The accumulated data show that autophagy is a shared mechanism to induce the tumor cells resistant to different cancer therapies, such as chemotherapy, radiotherapy and targeted therapy

Table 4 Known sensitizing function of autophagy inhibition in cervical and prostate cancer. Cancer cell types

Inhibition targets of autophagy Treatment

Cervical cancer cell lines HeLa

Knockdown of Beclin1

Cervical cancer cell lines Hela

Pharmacological inhibitors 3-MA, HCQ, bafilomycin A1 and monensin; shRNA Knockdown of Atg5, Atg6, Beclin1, Atg10, and Atg12 Inhibition of Bif-1 Nutrient [97] (Endophilin B1) deprivation Knockdown of Beclin 1 Atg3 γ-radiation [30] Atg4b, Atg4c, Atg5 and atg12

Cervical cancer cells Hela HTB35 cervical squamous cell carcinoma cells Normal human prostate epithelial cells and DU145 prostate cancer cells Human prostate epithelial cells (PrEC) LNCap prostate cancer cell line

Table 5 Known sensitizing function of autophagy inhibition in leukemia, esophageal squamous carcinoma and pharyngeal cancer cells.

Ref.

[130] Hypoxia and Bortezomib Nutrient [137] depletion

Pharmacological inhibitors 3-MA

TRAIL and FADD

[138]

Myc overexpression abrogating autophagy Pharmacological inhibitors 3-MA Knockdown of Beclin1

Rapamycin [139]

Ba/F3, K562 and LAMA 84 CML cells and primary human CML cells Esophageal squamous carcinoma (ESCC) EC9706 cell line Pharyngeal HTB43 cancer cells

Knockdown of Beclin 1 Atg3 Atg4b, Atg4c, Atg5 and atg12

Ref.

Imatinib

[31,141]

Imatinib and TPA Imatinib and HDAC inhibitor SAHA Imatinib and HDAC inhibitor SAHA Cisplatin (DDP)

[142]

γ-radiation

[30]

[143]

[29]

[55]

All cell lines are human origin. Abbreviation: 3-MA, 3-methyladenine; Baf-A1, bafilomycin A1; CQ, chloroquine, HCQ, hydroxychloroquine; PepA, pepstatin A; UVRAG, ultraviolet radiation resistanceassociated gene; shRNA, short hairpin RNA.

etc. For example, increased autophagy induced the resistance to the HER2 monoclonal antibody Trastuzumab which is a cytostatic agent used for HER2 positive breast cancers and mediates its effects by targeting HER2 [32]. Interestingly, knockdown of LC3 expression via shRNA resulted in resistant cells being re-sensitized to Trastuzumab treatment [32]. Also, inhibition of autophagy using either pharmacological inhibitors or RNAi of essential autophagy genes potentiates cell death induced by Imatinib mesylate in CML cells [31]. Repression of autophagy enhances the therapeutic efficacy of cisplatin and 5-FU in esophageal and colon cancer, respectively [55,56]. In the following section, we summarize the updated research data according to the different tumor classification, such as breast cancer (Table 1), colorectal cancer (Table 2), glioblastoma (Table 3), cervical and prostate cancer (Table 4), leukemia and esophageal squamous carcinoma (Table 5). In each table, we clearly list out the cancer cell types or cell lines, the inhibited targets of autophagy and the treatment regimen according to the references. 4.3. Autophagy inhibitors as the potential drug to overcome anticancer therapy resistance So far, different inhibitors of autophagy which affect different stages of the autophagy pathway have been developed and used in the studies Table 6 Autophagy inhibitors. Compound

Mechanism & target

Effect

Ref.

3-methyladenine (3-MA) Chloroquine (CQ) Bafilomycin A1 (BA) Hydroxychloroquine (HCQ) Wortmannin

Class III PI3K inhibitor Lysosomal pH

Autophagy inhibition

Vacuolar-ATPase

Autophagy inhibition

Lysosomal pH

Autophagy inhibition

[118,126, 144–156] [29,59,125, 143,157–166] [58,136, 167–169] [158,170]

Class III PI3K inhibitor Change Endocytic and Lysosomal pH Class III PI3K inhibitor

Autophagy inhibition

[171–176]

Autophagy inhibition

[171,177,178]

Autophagy inhibition

[179–182]

Monensin Androgen [140] deprivation

Pharmacological inhibitors 3-MA and CQ, Pharmacological inhibitors 3-MA

Treatment

LY294002

Autophagy inhibition

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225

via different mechanisms, which might be used to re-sensitize the resistant cancer cells to anticancer therapy. Since autophagy might be one of the main reasons to induce the cancer cells survival to anticancer therapy, the investigators want to know whether blocking autophagy while giving standard treatment will improve the treatment of cancer. It is noteworthy that some autophagy inhibitors have already been applied in clinical trials (http://clinicaltrials.gov/ct2/results?term=autophagy). Currently, investigators have mainly explored the potential clinical application of the autophagy inhibitor hydroxychloroquine (HCQ), in addition to the standard regimen in the treatment of cancer, such as non-small cell lung cancer (NSCLC), breast cancer, metastatic prostate cancer, colon cancer, refractory multiple myeloma, melanoma and other solid cancer types (Table 7). It is promising to see inhibitors of autophagy becoming the new therapy in anticancer therapy.

5. Concluding remarks Fig. 2. Autophagy inhibitors facilitate to re-sensitize the resistant cancer cells to antitumor therapies. Autophagy inhibitors such as Hydroxychloroquine (HCQ), 3-methyladenine (3-MA) and Bafilomycin A1 (BA) can facilitate the re-sensitization of the resistant cancer cells to antitumor therapies via abrogating the autophagic cells survival function. It is promising to expect the autophagy inhibitors to be the next generation drugs to overcome the resistant cancer cells to antitumor therapies.

of autophagy in tumorigenesis and cancer therapy. As shown in Table 1 to Table 5, much evidence demonstrates that the autophagy inhibitors facilitate the re-sensitization of resistant tumor cells to anticancer treatment. Herein, the current available autophagy inhibitors are summarized in Table 6. Different autophagy inhibitors block the autophagy effect through different mechanisms. For example, 3-MA, LY294002 and Wortmannin can inhibit the PI3K activity [55,56], one of the major upstream regulators of autophagy. Hydroxychloroquine (HCQ) or chloroquine (CQ) can inhibit autophagosome fusion with lysosomes and autophagosome degradation [31]. As shown in Fig. 2, autophagy inhibitors can abrogate the cell survival effect of autophagy

One of the most daunting clinical issues is the frequent tumors progression after standard treatment, mainly due to therapeutic resistance. It is urgent to elucidate the mechanisms that induce anticancer drug resistance. As we discussed in this review, although the controversy about the pro- or anticancer effect of autophagy is still heated, the in vitro and in vivo data fully support that autophagy can facilitate the tumor cells' survival to anticancer treatment [2,4,21,55,56,183]. The data suggest that cancer cells may take advantage of autophagy to survive despite of treatment with anticancer therapy regimens, such as chemotherapy or radiation. So, it is possible to inhibit autophagy through shRNA knockdown or inhibitors to promote the conversion from autophagic survival to apoptotic process. It is noteworthy that the autophagy inhibitors such as Hydroxychloroquine (HCQ), 3methyladenine (3-MA) and Bafilomycin A1 (BA) can effectively sensitize the cancer cells to therapeutic agents. Therefore, in the near future, it is promising to expect autophagy inhibitors, such as HCQ, in clinical trials, to be the next generation drugs that re-sensitize the resistant cancer cells to anticancer therapies.

Table 7 Clinical trials of autophagy inhibitors in cancer therapy. Cancer types

Treatment regimen

Sponsor

Starting date

Estimated Primary Completion Date

Clinical Trials. gov ID

Study phase

Non-Small Cell Lung Cancer (NSCLC) Metastatic Breast Cancer Ductal Carcinoma in Situ (breast) Refractory Solid Tumors Metastatic Prostate Cancer Metastatic Colorectal Cancer Advanced Non-Small Cell Lung Cancer (NSCLC) Relapsed or Refractory Multiple Myeloma Stage III or IV Resectable Melanoma Small Cell Lung Cancer Advanced Solid Tumors

Hydroxychloroquine, Carboplatin, Paclitaxel and Bevacizumb Ixabepilone and Hydroxychloroquine Tamoxifen and Chloroquine

University of Medicine and Dentistry New Jersey Cancer Institute of New Jersey

June 2008

June 2012

NCT00933803

Phase I/II

February 2009

August 2011

NCT00765765

Phase I/II

Inova Health Care Services

December 2009

December 2011

NCT01023477

Phase I/II

Hydroxychloroquine and Temsirolimus Docetaxel and Hydroxychloroquine Hydroxychloroquine, Capecitabine, Oxaliplatin, and Bevacizumab Hydroxychloroquine, Carboplatin, Paclitaxel, and Bevacizumab Hydroxychloroquine and Bortezomib Hydroxychloroquine

University of Pennsylvania

October 2008

May 2012

NCT00909831

Phase I

Cancer Institute of New Jersey

December 2008

October 2011

NCT00786682

Phase II

Cancer Institute of New Jersey

May 2009

July 2011

NCT01006369

Phase II

Cancer Institute of New Jersey

June 2008

August 2011

NCT00728845

Phase I/II

University of Pennsylvania

November 2007

November 2009

NCT00568880

Phase I/II

Cancer Institute of New Jersey

March 2009

March 2010

NCT00962845

Phase I

Chloroquine and A-CQ 100 Hydroxychloroquine and Vorinostat

June 2010 November 2009

December 2015 November 2010

NCT00969306 NCT01023737

Phase I/II Phase I

Breast Cancer

Ritonavir

October 2009

October 2013

NCT01009437

Phase I/II

Renal Cell Carcinoma Pancreatic Cancer

Hydroxychloroquine Hydroxychloroquine and Gemcitabine

Maastricht Radiation Oncology University of Texas Health Science Center at San Antonio Masonic Cancer Center, University of Minnesota University of Pittsburgh University of Pittsburgh

July 2010 June 2010

July 2012 June 2012

NCT01144169 NCT01128296

Phase I Phase I/II

226

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