The role of ncRNA in the co-regulation of autophagy and exosome pathways during cancer progression

Since its discovery a few decades ago, autophagy has been recognized as a crucial signaling pathway, linked to the recycling of cellular components in nutrient stress. Autophagy is a two-way sword, playing a dual role in tumorigenesis. In this catabolic process, dysfunctional organelles, biomolecules, and misfolded proteins are sequestered in the autophagosome and sent to the lysosome for degradation. Alongside, there are cellular messengers called exosomes, which are released from cells and are known to communicate and regulate metabolism in recipient cells. Multivesicular bodies (MVB) act as the intricate link between autophagy and exosome pathways. The continuous crosstalk between the two pathways is coordinated and regulated by multiple players among which ncRNA is the emerging candidates. The exosomes carry varied cargo of which non-coding RNA exerts an immediate regulatory effect on recipient cells. ncRNA is known to exhibit dual behavior in both promoting and inhibiting tumor growth. There is increasing evidence for the involvement of ncRNAs' in the regulation of different hallmarks of cancer. Different ncRNAs are involved in the co-regulation of autophagy and exosome pathways and therefore represent a superior therapeutic approach to target cancer chemoresistance. Here, we will discuss the ncRNA involved in regulating autophagy, and exosomes pathways and its relevance in cancer therapeutics.     

Autophagy and tumorigenesis

Autophagy, or cellular self-digestion, is activated in cancer cells in response to multiple stresses and has been demonstrated to promote tumor cell survival and drug resistance. Nonetheless, genetic evidence supports that autophagy functions as a tumor suppressor mechanism. Hence, the precise role of autophagy during cancer progression and treatment is both tissue and context dependent. Here, we discuss our current understanding of the biological functions of autophagy during cancer development, overview how autophagy is regulated by cancer-associated signaling pathways, and review how autophagy inhibition is being exploited to improve clinical outcomes.     

Therapeutic implications of GIPC1 silencing in cancer

GIPC1 is a cytoplasmic scaffold protein that interacts with numerous receptor signaling complexes, and emerging evidence suggests that it plays a role in tumorigenesis. GIPC1 is highly expressed in a number of human malignancies, including breast, ovarian, gastric, and pancreatic cancers. Suppression of GIPC1 in human pancreatic cancer cells inhibits in vivo tumor growth in immunodeficient mice. To better understand GIPC1 function, we suppressed its expression in human breast and colorectal cancer cell lines and human mammary epithelial cells (HMECs) and assayed both gene expression and cellular phenotype. Suppression of GIPC1 promotes apoptosis in MCF-7, MDA-MD231, SKBR-3, SW480, and SW620 cells and impairs anchorage-independent colony formation of HMECs. These observations indicate GIPC1 plays an essential role in oncogenic transformation, and its expression is necessary for the survival of human breast and colorectal cancer cells. Additionally, a GIPC1 knock-down gene signature was used to interrogate publically available breast and ovarian cancer microarray datasets. This GIPC1 signature statistically correlates with a number of breast and ovarian cancer phenotypes and clinical outcomes, including patient survival. Taken together, these data indicate that GIPC1 inhibition may represent a new target for therapeutic development for the treatment of human cancers.     

PKC delta and tissue transglutaminase are novel inhibitors of autophagy in pancreatic cancer cells

Apoptosis (type I) and autophagy (type II) are both highly regulated forms of programmed cell death and play crucial roles in physiological processes such as the development, homeostasis and selective, moderate to massive elimination of cells, if needed. Accumulating evidence suggests that cancer cells, including pancreatic cancer cells, in general tend to have reduced autophagy relative to their normal counterparts and premalignant lesions, supporting the contention that defective autophagy provides resistance to metabolic stress such as hypoxia, acidity and chemotherapeutics, promotes tumor cell survival and plays a role in the process of tumorigenesis. However, the mechanisms underlying the reduced capability of undergoing autophagy in pancreatic cancer remain elusive. In a recent study, we demonstrated a novel mechanism for regulation of autophagy in pancreatic ductal carcinoma cells. We found that protein kinase C-delta (PKC delta) constitutively suppresses autophagy through induction of tissue transglutaminase (TG2). Inhibition of PKC delta/TG2 signaling resulted in significant autophagic cell death that was mediated by Beclin 1. Elevated expression of TG2 in pancreatic cancer cells has been implicated in the development of drug resistance, metastatic phenotype and poor patient prognosis. In conclusion, our data suggest a novel role of PKC delta/TG2 in regulation of autophagy, and that TG2 may serve as an excellent therapeutic target in pancreatic cancer cells.     

Novel role of VMP1 as modifier of the pancreatic tumor cell response to chemotherapeutic drugs

We hypothesized that inhibiting molecules that mediate the adaptation response to cellular stress can antagonize the resistance of pancreatic cancer cells to chemotherapeutic drugs. Toward this end, here, we investigated how VMP1, a stress‐induced autophagy‐associated protein, modulate stress responses triggered by chemotherapeutic agents in PDAC. We find that VMP1 is particularly over‐expressed in poorly differentiated human pancreatic cancer. Pharmacological studies show that drugs that work, in part, via the endoplasmic reticulum stress response, induce VMP1 expression. Similarly, VMP1 is induced by known endoplasmic reticulum stress activators. Genetic inactivation of VMP1 using RNAi‐based antagonize the pancreatic cancer stress response to antitumoral agents. Functionally, we find that VMP1 regulates both autophagy and chemotherapeutic resistance even in the presence of chloroquin, ATG5 or Beclin 1 siRNAs, or a Beclin 1‐binding VMP1 mutant. In addition, VMP1 modulates endoplasmic reticulum stress independently of its coupling to the molecular and cellular autophagy machinery. Preclinical studies demonstrate that xenografts expressing an inducible and tractable form of VMP1 show increased resistance to the gemcitabine treatment. These results underscore a novel role for VMP1 as a potential therapeutic target for combinatorial therapies aimed at sensitizing pancreatic cancer cells to chemotherapeutic agents as well as provide novel molecular mechanisms to better understand this phenomenon. J. Cell. Physiol. 228: 1834–1843, 2013. © 2013 Wiley Periodicals, Inc.     

PKCδ and Tissue Transglutaminase are Novel Inhibitors of Autophagy in Pancreatic Cancer Cells

Apoptosis (type I) and autophagy (type II) are both highly regulated forms of programmed cell death and play crucial roles in physiological processes such as the development, homeostasis and selective, moderate to massive elimination of cells, if needed. Accumulating evidence suggests that cancer cells, including pancreatic cancer cells, in general tend to have reduced autophagy relative to their normal counterparts and premalignant lesions, supporting the contention that defective autophagy provides resistance to metabolic stress such as hypoxia, acidity and chemotherapeutics, promotes tumor cell survival and plays a role in the process of tumorigenesis. However, the mechanisms underlying the reduced capability of undergoing autophagy in pancreatic cancer remain elusive. In a recent study, we demonstrated a novel mechanism for regulation of autophagy in pancreatic ductal carcinoma cells. We found that protein kinase C-delta (PKCδ) constitutively suppresses autophagy through induction of tissue transglutaminase (TG2). Inhibition of PKCδ/TG2 signaling resulted in significant autophagic cell death that was mediated by Beclin 1. Elevated expression of TG2 in pancreatic cancer cells has been implicated in the development of drug resistance, metastatic phenotype and poor patient prognosis. In conclusion, our data suggest a novel role of PKCδ/TG2 in regulation of autophagy, and that TG2 may serve as an excellent therapeutic target in pancreatic cancer cells.

Addendum to:

Tissue Transglutaminase Inhibits Autophagy in Pancreatic Cancer Cells

U. Akar, B. Ozpolat, K. Mehta, J. Fok, Y. Kondo and G. Lopez-Berestein

Mol Cancer Res 2007; 5:241-9     

Chemotherapy and autophagy-mediated cell death in pancreatic cancer cells

Autophagy is an evolutionarily preserved degradation process of cytoplasmic cellular constituents and plays important physiological roles in human health and disease. It has been proposed that autophagy plays an important role both in tumor progression and in promotion of cancer cell death, although the molecular mechanisms responsible for this dual action of autophagy in cancer have not been elucidated. Pancreatic ductal adenocarcinoma is one of the most aggressive human malignancies with 2-3% five-year survival rate. Its poor prognosis has been attributed to the lack of specific symptoms and early detection tools, and its relatively refractory to traditional cytotoxic agents and radiotherapy. Experimental evidence pointed at autophagy as a pancreatic cancer cell mechanism to survive under adverse environmental conditions, or as a defective programmed cell death mechanism that favors pancreatic cancer cell resistance to treatment. Here, we consider several phenotypical alterations that have been related to increase or decrease the autophagic process in pancreatic tumor cells. We specially review autophagy as a cell death mechanism in response to chemotherapeutic drugs.     

Autophagy and mesenchymal cell fibrogenesis

Autophagy is a catabolic pathway essential for cellular energy homeostasis that involves the self-degradation of intracellular components in lysosomes. This process has been implicated in the pathophysiology of many human disorders, including infection, cancer, and fibrosis. Autophagy is also recognized as a mediator of survival and proliferation, and multiple pathways induce autophagy under conditions of cellular stress, including nutrient and energy depletion. High autophagic activity has been detected in fibrogenic cells from several tissues; however the role of autophagy in fibrogenesis and mesenchymal cells varies greatly in different tissues and settings, with contributions uncovered to energy metabolism and collagen turnover by fibrogenic cells. Because several chemical modulators of autophagy have already been identified, autophagy regulation constitutes a potential target for antifibrotic therapy. This article is part of a Special Issue entitled: Fibrosis: Translation of basic research to human disease.     

MAPK14/p38α confers irinotecan resistance to TP53-defective cells by inducing survival autophagy

Recently we have shown that the mitogen-activated protein kinase (MAPK) MAPK14/p38α is involved in resistance of colon cancer cells to camptothecin-related drugs. Here we further investigated the cellular mechanisms involved in such drug resistance and showed that, in HCT116 human colorectal adenocarcinoma cells in which TP53 was genetically ablated (HCT116-TP53KO), overexpression of constitutively active MAPK14/p38α decreases cell sensitivity to SN-38 (the active metabolite of irinotecan), inhibits cell proliferation and induces survival-autophagy. Since autophagy is known to facilitate cancer cell resistance to chemotherapy and radiation treatment, we then investigated the relationship between MAPK14/p38α, autophagy and resistance to irinotecan. We demonstrated that induction of autophagy by SN38 is dependent on MAPK14/p38α activation. Finally, we showed that inhibition of MAPK14/p38α or autophagy both sensitizes HCT116-TP53KO cells to drug therapy. Our data proved that the two effects are interrelated, since the role of autophagy in drug resistance required the MAPK14/p38α. Our results highlight the existence of a new mechanism of resistance to camptothecin-related drugs: upon SN38 induction, MAPK14/p38α is activated and triggers survival-promoting autophagy to protect tumor cells against the cytotoxic effects of the drug. Colon cancer cells could thus be sensitized to drug therapy by inhibiting either MAPK14/p38 or autophagy.     

Exosomes derived from imatinib-resistant chronic myeloid leukemia cells mediate a horizontal transfer of drug-resistant trait by delivering miR-365

Chronic myeloid leukemia (CML) is a malignant disorder of hematopoietic stem/progenitor cells. Majority of patients can be effectively treated with tyrosine kinase inhibitors (TKIs) such as imatinib, but a portion of patients will develop drug resistance. Accumulated evidences have identified exosomes in cancer as promoters of tumor progression. Herein, we found that exosomes derived from imatinib resistant CML cells can be internalized into sensitive CML cells and confer drug-resistance traits. We also demonstrated a significant higher level of miR-365 in exosomes derived from drug-resistant CML cells compared with those from sensitive ones using microarray and qRT-PCR. The imatinib sensitive CML cells transfected with pre-miR-365 displayed lower chemosensitivity and apoptosis rate compared with controls. We further confirmed that exosomal transfer of miR-365 induced drug resistance by inhibiting expression of pro-apoptosis protein in sensitive CML cells. In conclusion, our study reveals that exosomes mediate a horizontal transfer of drug-resistant trait in chronic myeloid leukemia cell by delivering miR-365.     

Identification of Double-stranded Genomic DNA Spanning All Chromosomes with Mutated KRAS and p53 DNA in the Serum Exosomes of Patients with Pancreatic Cancer

Exosomes are small vesicles (50–150 nm) of endocytic origin that are released by many different cell types. Exosomes in the tumor microenvironment may play a key role in facilitating cell-cell communication. Exosomes are reported to predominantly contain RNA and proteins. In this study, we investigated whether exosomes from pancreatic cancer cells and serum from patients with pancreatic ductal adenocarcinoma contain genomic DNA. Our results provide evidence that exosomes contain >10-kb fragments of double-stranded genomic DNA. Mutations in KRAS and p53 can be detected using genomic DNA from exosomes derived from pancreatic cancer cell lines and serum from patients with pancreatic cancer. In addition, using whole genome sequencing, we demonstrate that serum exosomes from patients with pancreatic cancer contain genomic DNA spanning all chromosomes. These results indicate that serum-derived exosomes can be used to determine genomic DNA mutations for cancer prediction, treatment, and therapy resistance.     

Tampering with cancer chemoresistance by targeting the TGM2-IL6-autophagy regulatory network

Macroautophagy/autophagy is a well-established process involved in maintaining cellular homeostasis, but its role in cancer is complex and even controversial. Many studies have reported a correlative relationship between increased autophagy and evolving cancer cells under stress conditions such as nutrient or oxygen deprivation; however, there has been a lack of a plausible mechanistic link to properly target the autophagy process in the context of this microenvironment. We recently unveiled a positive regulatory loop involving TGM2 (transglutaminase 2)-NFKB/NF-κB signaling, IL6 and autophagy in cancer using mantle cell lymphoma (MCL) as a model system. These pathways are functionally connected to each other, thereby promoting malignant B cell survival and leading to enhanced lymphoma progression both in mice and in patients. Disruption of this network could provide an opportunity to increase the efficacies of current therapies and to reduce MCL drug resistance.     

Inhibition of autophagy by chloroquine induces apoptosis in primary effusion lymphoma in vitro and in vivo through induction of endoplasmic reticulum stress

Autophagy plays a crucial role in cancer cell survival and the inhibition of autophagy is attracting attention as an emerging strategy for the treatment of cancer. Chloroquine (CQ) is an anti-malarial drug, and is also known as an inhibitor of autophagy. Recently, it has been found that CQ induces cancer cell death through the inhibition of autophagy; however, the underlying mechanism is not entirely understood. In this study, we identified the role of CQ-induced cancer cell death using Primary Effusion Lymphoma (PEL) cells. We found that a CQ treatment induced caspase-dependent apoptosis in vitro. CQ also suppressed PEL cell growth in a PEL xenograft mouse model. We showed that CQ activated endoplasmic reticulum (ER) stress signal pathways and induced CHOP, which is an inducer of apoptosis. CQ-induced cell death was significantly decreased by salbrinal, an ER stress inhibitor, indicating that CQ-induced apoptosis in PEL cells depended on ER stress. We show here for the first time that the inhibition of autophagy induces ER stress-mediated apoptosis in PEL cells. Thus, the inhibition of autophagy is a novel strategy for cancer chemotherapy.     

Protein Kinase A Activation Promotes Cancer Cell Resistance to Glucose Starvation and Anoikis

Cancer cells often rely on glycolysis to obtain energy and support anabolic growth. Several studies showed that glycolytic cells are susceptible to cell death when subjected to low glucose availability or to lack of glucose. However, some cancer cells, including glycolytic ones, can efficiently acquire higher tolerance to glucose depletion, leading to their survival and aggressiveness. Although increased resistance to glucose starvation has been shown to be a consequence of signaling pathways and compensatory metabolic routes activation, the full repertoire of the underlying molecular alterations remain elusive. Using omics and computational analyses, we found that cyclic adenosine monophosphate-Protein Kinase A (cAMP-PKA) axis activation is fundamental for cancer cell resistance to glucose starvation and anoikis. Notably, here we show that such a PKA-dependent survival is mediated by parallel activation of autophagy and glutamine utilization that in concert concur to attenuate the endoplasmic reticulum (ER) stress and to sustain cell anabolism. Indeed, the inhibition of PKA-mediated autophagy or glutamine metabolism increased the level of cell death, suggesting that the induction of autophagy and metabolic rewiring by PKA is important for cancer cellular survival under glucose starvation. Importantly, both processes actively participate to cancer cell survival mediated by suspension-activated PKA as well. In addition we identify also a PKA/Src mechanism capable to protect cancer cells from anoikis. Our results reveal for the first time the role of the versatile PKA in cancer cells survival under chronic glucose starvation and anoikis and may be a novel potential target for cancer treatment.     

MAPK14/p38α confers irinotecan resistance to TP53-defective cells by inducing survival autophagy

Recently we have shown that the mitogen-activated protein kinase (MAPK) MAPK14/p38α is involved in resistance of colon cancer cells to camptothecin-related drugs. Here we further investigated the cellular mechanisms involved in such drug resistance and showed that, in HCT116 human colorectal adenocarcinoma cells in which TP53 was genetically ablated (HCT116-TP53KO), overexpression of constitutively active MAPK14/p38α decreases cell sensitivity to SN-38 (the active metabolite of irinotecan), inhibits cell proliferation and induces survival-autophagy. Since autophagy is known to facilitate cancer cell resistance to chemotherapy and radiation treatment, we then investigated the relationship between MAPK14/p38α, autophagy and resistance to irinotecan. We demonstrated that induction of autophagy by SN38 is dependent on MAPK14/p38α activation. Finally, we showed that inhibition of MAPK14/p38α or autophagy both sensitizes HCT116-TP53KO cells to drug therapy. Our data proved that the two effects are interrelated, since the role of autophagy in drug resistance required the MAPK14/p38α. Our results highlight the existence of a new mechanism of resistance to camptothecin-related drugs: upon SN38 induction, MAPK14/p38α is activated and triggers survival-promoting autophagy to protect tumor cells against the cytotoxic effects of the drug. Colon cancer cells could thus be sensitized to drug therapy by inhibiting either MAPK14/p38 or autophagy.     

Examination of the specificity of tumor cell derived exosomes with tumor cells in vitro

Small endogenous vesicles called exosomes are beginning to be explored as drug delivery vehicles. The in vivo targets of exosomes are poorly understood; however, they are believed to be important in cell-to-cell communication and may play a prominent role in cancer metastasis. We aimed to elucidate whether cancer derived exosomes can be used as drug delivery vehicles that innately target tumors over normal tissue. Our in vitro results suggest that while there is some specificity towards cancer cells over “immortalized” cells, it is unclear if the difference is sufficient to achieve precise in vivo targeting. Additionally, we found that exosomes associate with their cellular targets to a significantly greater extent (> 10-fold) than liposomes of a similar size. Studies on the association of liposomes mimicking the unique lipid content of exosomes revealed that the lipid composition contributes significantly to cellular adherence/internalization. Cleavage of exosome surface proteins yielded exosomes exhibiting reduced association with their cellular targets, demonstrating the importance of proteins in binding/internalization. Furthermore, although acidic conditions are known to augment the metastatic potential of tumors, we found that cells cultured at low pH released exosomes with significantly less potential for cellular association than cells cultured at physiological pH.     

Autophagy in pancreatic cancer: An emerging mechanism of cell death

Pancreatic cancer, the fourth leading cause of cancer-related death in the United States, is resistant to current chemotherapies. Therefore, identification of different pathways of cell death is important to develop novel therapeutics. Our previous study has shown that triptolide, a diterpene triepoxide, inhibits the growth of pancreatic cancer cells in vitro and prevents tumor growth in vivo. However, the mechanism by which triptolide kills pancreatic cancer cells was not known, hence, this study aimed at elucidating it. Our study reveals that triptolide kills diverse types of pancreatic cancer cells by two different pathways; it induces caspase-dependent apoptotic death in some cell lines and death via a caspase-independent autophagic pathway in the other cell lines tested. Triptolide-induced autophagy requires autophagy-specific genes, atg5 or beclin 1 and its inhibition results in cell death via the apoptotic pathway, whereas inhibition of both autophagy and apoptosis rescues triptolide-mediated cell death. Our study shows for the first time that induction of autophagy by triptolide has a pro-death role in pancreatic cancer cells. Since triptolide kills diverse pancreatic cancer cells by different mechanisms, it makes an attractive chemotherapeutic agent for future use against a broad spectrum of pancreatic cancers.Key words: pancreatic cancer, triptolide, apoptosis, caspase-3Pancreatic adenocarcinoma is one of the most lethal human malignancies. It is the fourth leading cause of cancer-related death in the United States. The five-year survival rate for pancreatic cancer is estimated to be <5% due to its aggressive growth, metastasis and resistance to radiation and most systemic chemotherapies. Hence, efforts are ongoing to understand the pathobiology of pancreatic cancer to develop innovative and effective therapies against it. A promising candidate for future therapeutic use against pancreatic cancer is a diterpene triepoxide, triptolide. Our previous studies show that triptolide inhibits the growth of pancreatic cancer cells in vitro and prevents tumor growth in vivo. Since the mechanism by which triptolide kills pancreatic cancer cells was not known, we decided to elucidate it.The K-ras, p53, p16 and DPC4 genes are the most frequently altered genes in pancreatic adenocarcinoma. In this study we have used diverse pancreatic cancer cell lines, MiaPaCa-2, Capan-1, S2-013 and S2-VP10 cells, which have mutations in all the above-mentioned genes and BxPC-3 and Hs766T cells, which have mutations in the p53, p16 and DPC4 genes, but have a wild-type K-ras gene. The treatment of all the cell lines with triptolide results in a significant time- and dose-dependent decrease in cell viability, independent of cell cycle arrest. After treatment with triptolide, only MiaPaCa-2, Capan-1 and BxPC-3 cells show an increase in the apoptosis parameters: cytochrome c release from mitochondria into the cytosol, caspase-3 activation and phosphatidylserine externalization. In contrast to this, S2-013, S2-VP10 and Hs766T cells show an induction of autophagy: an increase in LC3-II levels (by immunoblotting and immufluorescence), increase in acridine orange-positive cells, inhibition of the PtdIns3K/Akt/mTOR pathway and induction of the ERK1/2 pathway. Also, none of the cell lines tested show necrosis as evidenced by the absence of the release of lactate dehydrogenase. These results indicate that triptolide induces apoptosis in MiaPaCa-2, Capan-1 and BxPC-3 cells, whereas it induces autophagy in S2-013, S2-VP10 and Hs766T cells.Since the role of autophagy in cancer was controversial we investigated whether triptolide-induced autophagy has a prosurvival or a pro-death role. As autophagy-associated cell death is independent of caspase-3, we tested the effect of triptolide on pancreatic cancer cells in the absence of caspase-3. Treatment of cells with triptolide post-caspase-3 knockdown shows a significant rescue of cell viability only in MiaPaCa-2, but not S2-013 or S2-VP10 cells. This indicates that in contrast to MiaPaCa-2, triptolide-mediated cell death in S2-013 and S2-VP10 cells is independent of caspase-3. Next, we tested the role of autophagy in triptolide-mediated cell death in pancreatic cancer cells. In spite of a knockdown of autophagy-specific genes (atg5 and beclin 1), treatment of S2-013 and S2-VP10 cells with triptolide show a significant decline in cell viability, which is comparable to the cells treated with triptolide in the presence of autophagy genes. Subsequently we show that death in the absence of autophagy-specific genes is due to the utilization of an alternate cell death pathway, apoptosis. Furthermore, in the absence of both autophagy-specific and apoptosis-specific genes, triptolide-mediated cell death is rescued in S2-013 and S2-VP10 cells. Thus, these results confirm that triptolide-induced autophagy has a pro-death role in S2-013 and S2-VP10 cells and that these cells do not have a defect in the apoptotic machinery; however, they respond to triptolide by activating the autophagic pathway instead of the apoptotic pathway. Our studies also reveal the presence of a crosstalk between the two cell death pathways, apoptosis and autophagy, in pancreatic cancer cells.In conclusion, our study shows for the first time that triptolide induces autophagy in pancreatic cancer cells. It sheds light on the fundamental question as to whether autophagy is protective or causes cell death, proving convincingly that induction of autophagy causes cell death of some pancreatic cancer cells. Although a basal level of autophagy is necessary to maintain cellular homeostasis, its prosurvival role can be switched into a cell death mechanism if the amplitude of autophagy increases above a threshold level which is incompatible with viability, as seen in S2-013, S2-VP10 and Hs766T cells after triptolide treatment. Furthermore, there exists a crosstalk between apoptosis and autophagy in S2-013 and S2-VP10 cells; either both pathways function independently to kill the cells, with autophagy being the preferred pathway or autophagy antagonizes apoptosis and hence apoptosis is seen only after inhibiting autophagy. Although there is no direct correlation between the selection of cell death pathway in response to triptolide and the genotype of the cell lines, the choice of autophagic cell death pathway could depend on the metastatic potential of the cells; S2-013, S2-VP10 and Hs766T cell lines being more metastatic than the others, which merits further investigation. In conclusion, the ability of triptolide to induce cell death in diverse pancreatic cancer cells by either mechanism makes it an attractive chemotherapeutic agent against a broad spectrum of pancreatic cancers.     

Aurora kinase A inhibition-induced autophagy triggers drug resistance in breast cancer cells

We have previously shown that elevated expression of mitotic kinase aurora kinase A (AURKA) in cancer cells promotes the development of metastatic phenotypes and is associated clinically with adverse prognosis. Here, we first revealed a clinically positive correlation between AURKA and autophagy-associated protein SQSTM1 in breast cancer and further demonstrated that AURKA regulated SQSTM1 through autophagy. Indeed, depletion by siRNA or chemical inhibition of AURKA by the small molecule VX-680 increased both the level of microtubule-associated protein 1 light chain 3-II (LC3-II) and the number of autophagosomes, along with decreased SQSTM1. Conversely, overexpression of AURKA inhibited autophagy, as assessed by decreased LC3-II and increased SQSTM1 either upon nutrient deprivation or normal conditions. In addition, phosphorylated forms of both RPS6KB1 and mechanistic target of rapamycin (MTOR) were elevated by overexpression of AURKA whereas they were suppressed by depletion or inhibition of AURKA. Moreover, inhibition of MTOR by PP242, an inhibitor of MTOR complex1/2, abrogated the changes in both LC3-II and SQSTM1 in AURKA-overexpressing BT-549 cells, suggesting that AURKA-suppressed autophagy might be associated with MTOR activation. Lastly, repression of autophagy by depletion of either LC3 or ATG5, sensitized breast cancer cells to VX-680-induced apoptosis. Similar findings were observed in cells treated with the autophagy inhibitors chloroquine (CQ) and bafilomycin A₁ (BAF). Our data thus revealed a novel role of AURKA as a negative regulator of autophagy, showing that AURKA inhibition induced autophagy, which may represent a novel mechanism of drug resistance in apoptosis-aimed therapy for breast cancer.