MicroRNA-520g Confers Drug Resistance by Regulating p21 Expression in Colorectal Cancer

Development of drug resistance is one of the major causes of colorectal cancer recurrence, yet mechanistic understanding and therapeutic options remain limited. Here, we show that expression of microRNA (miR)-520g is correlated with drug resistance of colon cancer cells. Ectopic expression of miR-520g conferred resistance to 5-fluorouracil (5-FU)- or oxaliplatin-induced apoptosis in vitro and reduced the effectiveness of 5-FU in the inhibition of tumor growth in a mouse xenograft model in vivo. Further studies indicated that miR-520g mediated drug resistance through down-regulation of p21 expression. Moreover, p53 suppressed miR-520g expression, and deletion of p53 up-regulated miR-520g expression. Inhibition of miR-520g in p53^−/− cells increased their sensitivity to 5-FU treatment. Importantly, studies of patient samples indicated that expression of miR-520g correlated with chemoresistance in colorectal cancer. These findings indicate that the p53/miR-520g/p21 signaling axis plays an important role in the response of colorectal cancer to chemotherapy. A major implication of our studies is that inhibition of miR-520g or restoration of p21 expression may have considerable therapeutic potential to overcome drug resistance in colorectal cancer patients, especially in those with mutant p53.     

Tissue transglutaminase inhibits autophagy in pancreatic cancer cells

Elevated expression of tissue transglutaminase (TG2) in cancer cells has been implicated in the development of drug resistance and metastatic phenotypes. However, the role and the mechanisms that regulate TG2 expression remain elusive. Here, we provide evidence that protein kinase Cdelta (PKCdelta) regulates TG2 expression, which in turn inhibits autophagy, a type II programmed cell death, in pancreatic cancer cells that are frequently insensitive to standard chemotherapeutic agents. Rottlerin, a PKCdelta-specific inhibitor, and PKCdelta small interfering RNA (siRNA) down-regulated the expression of TG2 mRNA and protein and induced growth inhibition without inducing apoptosis in pancreatic cancer cells. Inhibition of PKCdelta by rottlerin or knockdown of TG2 protein by a TG2-specific siRNA resulted in a marked increase in autophagy shown by presence of autophagic vacuoles in the cytoplasm, formation of the acidic vesicular organelles, membrane association of microtubule-associated protein 1 light chain 3 (LC3) with autophagosomes, and a marked induction of LC3-II protein, important hallmarks of autophagy, and by electron microscopy. Furthermore, inhibition of TG2 by rottlerin or by the siRNA led to accumulation of green fluorescent protein (GFP)-LC3-II in autophagosomes in pancreatic cancer cells transfected with GFP-LC3 (GFP-ATG8) expression vector. Knockdown of Beclin-1, a specific autophagy-promoting protein and the product of Becn1 (ATG6), inhibited rottlerin-induced and TG2 siRNA-induced autophagy, indicating that Beclin-1 is required for this process. These results revealed that PKCdelta plays a critical role in the expression of TG2, which in turn regulates autophagy. In conclusion, these results suggest a novel mechanism of regulation of TG2 and TG2-mediated autophagy in pancreatic cancer cells.     

Anti-Cancer Effect of HIV-1 Viral Protein R on Doxorubicin Resistant Neuroblastoma

Several unique biological features of HIV-1 Vpr make it a potentially powerful agent for anti-cancer therapy. First, Vpr inhibits cell proliferation by induction of cell cycle G2 arrest. Second, it induces apoptosis through multiple mechanisms, which could be significant as it may be able to overcome apoptotic resistance exhibited by many cancerous cells, and, finally, Vpr selectively kills fast growing cells in a p53-independent manner. To demonstrate the potential utility of Vpr as an anti-cancer agent, we carried out proof-of-concept studies in vitro and in vivo. Results of our preliminary studies demonstrated that Vpr induces cell cycle G2 arrest and apoptosis in a variety of cancer types. Moreover, the same Vpr effects could also be detected in some cancer cells that are resistant to anti-cancer drugs such as doxorubicin (DOX). To further illustrate the potential value of Vpr in tumor growth inhibition, we adopted a DOX-resistant neuroblastoma model by injecting SK-N-SH cells into C57BL/6N and C57BL/6J-scid/scid mice. We hypothesized that Vpr is able to block cell proliferation and induce apoptosis regardless of the drug resistance status of the tumors. Indeed, production of Vpr via adenoviral delivery to neuroblastoma cells caused G2 arrest and apoptosis in both drug naïve and DOX-resistant cells. In addition, pre-infection or intratumoral injection of vpr-expressing adenoviral particles into neuroblastoma tumors in SCID mice markedly inhibited tumor growth. Therefore, Vpr could possibly be used as a supplemental viral therapeutic agent for selective inhibition of tumor growth in anti-cancer therapy especially when other therapies stop working.     

NF-κB targeting for overcoming tumor resistance and normal tissues toxicity

Radiotherapy and chemotherapy are two famous modalities in tumor-targeted therapy that lead to systemic and local toxicities for normal tissues. Moreover, several studies have confirmed that exposure of the tumor to radiation or chemotherapy drugs stimulate some signaling pathways in the tumor microenvironment (TME), leading to resistance of cancer cells to apoptosis, as well as promoting angiogenesis and tumor growth. Nuclear factor kappa B (NF-κB) plays a central role in the regulation of inflammatory responses in both normal tissues and tumors via the release of several cytokines, regulation of prostaglandins, reduction/oxidation (redox) reactions, angiogenesis, and cell death. Upregulation of NF-κB in normal tissues causes an appearance of inflammatory reactions and oxidative stress, whereas it regulates angiogenesis and suppresses apoptosis, leading to resistance to subsequent doses of radiation or chemotherapy. Selective inhibition of NF-κB in experimental studies has shown promising results for tumor sensitization via apoptosis induction, inhibition of angiogenesis, and increasing delay of tumor growth. The use of some agents for NF-κB inhibition has been shown to alleviate radiation/chemotherapy toxicities in normal cells/ tissues. In this current review, we explained the pivotal role of NF-κB in both normal tissue toxicity and tumor resistance. We also discussed the promising strategies for overcoming these problems with regard to chemotherapy and radiotherapy.     

失巢凋亡及其在肿瘤侵袭、转移中的调控

作为肿瘤转移的屏障, 细胞与邻近细胞或者细胞外基质(Extracellular matrix, ECM)失去联系后将遭受凋亡, 这种细胞死亡方式称为“失巢凋亡”。正常上皮细胞或不具备转移性质的实体瘤细胞从原位脱落进入血液循环后就会引发失巢凋亡, 失巢凋亡的意义在于防止这些脱落的细胞种植并生长于其他不适宜的地方。而肿瘤细胞, 尤其是一些容易发生远距离转移的恶性肿瘤细胞, 具有极强的抗失巢凋亡特性, 便于转移侵袭。研究发现肿瘤细胞能通过多种方式抵抗失巢凋亡, 比如细胞自分泌生长因子或者由邻近细胞旁分泌, 激活促存活信号通路; 细胞改变整合蛋白的表达模式, 使之能够接收新环境的生存信号; 活性氧(Reactive oxygen species, ROS)通过不依赖配体的方式激活生长因子受体, 从而逃逸凋亡; 上皮间质转化(Epithelial-mesenchymal transition, EMT)激活等。这些方式导致细胞存活信号激活和凋亡途径抑制, 最终使肿瘤细胞抗失巢凋亡, 促进转移。文章综述了当前研究的肿瘤转移的关键机制, 这些策略也将成为肿瘤治疗的重要靶点。     

Dynamic reprogramming of the kinome in response to targeted MEK inhibition in triple-negative breast cancer

Kinase inhibitors have limited success in cancer treatment because tumors circumvent their action. Using a quantitative proteomics approach, we assessed kinome activity in response to MEK inhibition in triple-negative breast cancer (TNBC) cells and genetically engineered mice (GEMMs). MEK inhibition caused acute ERK activity loss, resulting in rapid c-Myc degradation that induced expression and activation of several receptor tyrosine kinases (RTKs). RNAi knockdown of ERK or c-Myc mimicked RTK induction by MEK inhibitors, and prevention of proteasomal c-Myc degradation blocked kinome reprogramming. MEK inhibitor-induced RTK stimulation overcame MEK2 inhibition, but not MEK1 inhibition, reactivating ERK and producing drug resistance. The C3Tag GEMM for TNBC similarly induced RTKs in response to MEK inhibition. The inhibitor-induced RTK profile suggested a kinase inhibitor combination?therapy that produced GEMM tumor apoptosis and regression where single agents were ineffective. This approach defines mechanisms of drug resistance, allowing rational design of combination therapies for cancer.     

The small heat shock protein alpha B-crystallin is a novel inhibitor of TRAIL-induced apoptosis that suppresses the activation of caspase-3

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a member of the tumor necrosis factor alpha family of cytokines that preferentially induces apoptosis in transformed cells, making it a promising cancer therapy. However, many neoplasms are resistant to TRAIL-induced apoptosis by mechanisms that are poorly understood. We demonstrate that the expression of the small heat shock protein alpha B-crystallin (but not other heat shock proteins or apoptosis-regulating proteins) correlates with TRAIL resistance in a panel of human cancer cell lines. Stable expression of wild-type alpha B-crystallin, but not a pseudophosphorylation mutant impaired in its assembly and chaperone function, protects cancer cells from TRAIL-induced caspase-3 activation and apoptosis in vitro. Furthermore, selective inhibition of alpha B-crystallin expression by RNA interference sensitizes cancer cells to TRAIL. In addition, wild-type alpha B-crystallin promotes xenograft tumor growth and inhibits TRAIL-induced apoptosis in vivo in nude mice, whereas a pseudophosphorylation alpha B-crystallin mutant impaired in its anti-apoptotic function inhibits xenograft tumor growth. Collectively, these findings indicate that alpha B-crystallin is a novel regulator of TRAIL-induced apoptosis and tumor growth. Moreover, these results demonstrate that targeted inhibition of alpha B-crystallin promotes TRAIL-induced apoptosis, thereby suggesting a novel strategy to overcome TRAIL resistance in cancer.     

Nano carriers that enable co-delivery of chemotherapy and RNAi agents for treatment of drug-resistant cancers

Tumor cells exhibit drug resistant phenotypes that decrease the efficacy of chemotherapeutic treatments. The drug resistance has a genetic basis that is caused by an abnormal gene expression. There are several types of drug resistance: efflux pumps reducing the cellular concentration of the drug, alterations in membrane lipids that reduce cellular uptake, increased or altered drug targets, metabolic alteration of the drug, inhibition of apoptosis, repair of the damaged DNA, and alteration of the cell cycle checkpoints ( and ). siRNA is used to silence the drug resistant phenotype and prevent this drug resistance response. Of the listed types of drug resistance, pump-type resistance (e.g., high expression of ATP-binding cassette transporter proteins such as P-glycoproteins (Pgp; also known as multi-drug resistance protein 1 or MDR1, encoded by the ATP-Binding Cassette Sub-Family B Member 1 (ABCB1) gene)) and apoptosis inhibition (e.g., expression of anti-apoptotic proteins such as Bcl-2) are the most frequently targeted for gene silencing. The co-delivery of siRNA and chemotherapeutic drugs has a synergistic effect, but many of the current projects do not control the drug release from the nanocarrier. This means that the drug payload is released before the drug resistance proteins have degraded and the drug resistance phenotype has been silenced. Current research focuses on cross-linking the carrier's polymers to prevent premature drug release, but these carriers still rely on environmental cues to release the drug payload, and the drug may be released too early. In this review, we studied the release kinetics of siRNA and chemotherapeutic drugs from a broad range of carriers. We also give examples of carriers used to co-deliver siRNA and drugs to drug-resistant tumor cells, and we examine how modifications to the carrier affect the delivery. Lastly, we give our recommendations for the future directions of the co-delivery of siRNA and chemotherapeutic drug treatments.     

Cytokeratin 18-mediated disorganization of intermediate filaments is induced by degradation of plectin in human liver cells

Rho GDP dissociation inhibitor 2 (RhoGDI2) is a regulator of the Rho family GTPases. Recent work from our laboratory suggests that RhoGDI2 expression potentially enhances resistance to cisplatin as well as promotes tumor growth and malignant progression in gastric cancer. In this study, we demonstrate that phospholipase C-gamma (PLCγ) is required for RhoGDI2-mediated cisplatin resistance and cancer cell invasion in gastric cancer. The levels of phosphorylated PLCγ are markedly enhanced in RhoGDI2-overexpressing SNU-484 cells and, by contrast, repressed in RhoGDI2-depleted MKN-28 cells. Depletion of PLCγ expression or inhibition of its activity not only significantly increases cisplatin-induced apoptosis but also suppresses the invasive ability of RhoGDI2-overexpressing SNU-484 cells. Taken together, our results suggest that PLCγ plays a key role in RhoGDI2-mediated cisplatin resistance and cell invasion in gastric cancer cells.     

LAMP2A overexpression in breast tumors promotes cancer cell survival via chaperone-mediated autophagy

Lysosome-associated membrane protein type 2A (LAMP2A) is a key protein in the chaperone-mediated autophagy (CMA) pathway. LAMP2A helps in lysosomal uptake of modified and oxidatively damaged proteins directly into the lumen of lysosomes for degradation and protein turnover. Elevated expression of LAMP2A was observed in breast tumor tissues of all patients under investigation, suggesting a survival mechanism via CMA and LAMP2A. Reduced expression of the CMA substrates, GAPDH and PKM, was observed in most of the breast tumor tissues when compared with the normal adjacent tissues. Reactive oxygen species (ROS) mediated oxidative stress damages regulatory cellular components such as DNA, proteins and/or lipids. Protein carbonyl content (PCC) is widely used as a measure of total protein oxidation in cells. Ectopic expression of LAMP2A reduces PCC and thereby promotes cell survival during oxidative stress. Furthermore, inhibition of LAMP2A stimulates accumulation of GAPDH, AKT1 phosphorylation, generation of ROS, and induction of cellular apoptosis in breast cancer cells. Doxorubicin, which is a chemotherapeutic drug, often becomes ineffective against tumor cells with time due to chemotherapeutic resistance. Breast cancer cells deficient of LAMP2A demonstrate increased sensitivity to the drug. Thus, inhibiting CMA activity in breast tumor cells can be exploited as a potential therapeutic application in the treatment of breast cancer.     

LAMP2A overexpression in breast tumors promotes cancer cell survival via chaperone-mediated autophagy

Lysosome-associated membrane protein type 2A (LAMP2A) is a key protein in the chaperone-mediated autophagy (CMA) pathway. LAMP2A helps in lysosomal uptake of modified and oxidatively damaged proteins directly into the lumen of lysosomes for degradation and protein turnover. Elevated expression of LAMP2A was observed in breast tumor tissues of all patients under investigation, suggesting a survival mechanism via CMA and LAMP2A. Reduced expression of the CMA substrates, GAPDH and PKM, was observed in most of the breast tumor tissues when compared with the normal adjacent tissues. Reactive oxygen species (ROS) mediated oxidative stress damages regulatory cellular components such as DNA, proteins and/or lipids. Protein carbonyl content (PCC) is widely used as a measure of total protein oxidation in cells. Ectopic expression of LAMP2A reduces PCC and thereby promotes cell survival during oxidative stress. Furthermore, inhibition of LAMP2A stimulates accumulation of GAPDH, AKT1 phosphorylation, generation of ROS, and induction of cellular apoptosis in breast cancer cells. Doxorubicin, which is a chemotherapeutic drug, often becomes ineffective against tumor cells with time due to chemotherapeutic resistance. Breast cancer cells deficient of LAMP2A demonstrate increased sensitivity to the drug. Thus, inhibiting CMA activity in breast tumor cells can be exploited as a potential therapeutic application in the treatment of breast cancer.     

Autophagy Plays a Critical Role in ChLym-1-Induced Cytotoxicity of Non-Hodgkin’s Lymphoma Cells

Autophagy is a critical mechanism in both cancer therapy resistance and tumor suppression. Monoclonal antibodies have been documented to kill tumor cells via apoptosis, antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). In this study, we report for the first time that chLym-1, a chimeric anti-human HLA-DR monoclonal antibody, induces autophagy in Raji Non-Hodgkin’s Lymphoma (NHL) cells. Interestingly, inhibition of autophagy by pharmacological inhibitors (3-methyladenine and NH₄Cl) or genetic approaches (siRNA targeting Atg5) suppresses chLym-1-induced growth inhibition, apoptosis, ADCC and CDC in Raji cells, while induction of autophagy could accelerate cytotoxic effects of chLym-1 on Raji cells. Furthermore, chLym-1-induced autophagy can mediate apoptosis through Caspase 9 activation, demonstrating the tumor-suppressing role of autophagy in antilymphoma effects of chLym-1. Moreover, chLym-1 can activate several upstream signaling pathways of autophagy including Akt/mTOR and extracellular signal-regulated kinase 1/2 (Erk1/2). These results elucidate the critical role of autophagy in cytotoxicity of chLym-1 antibody and suggest a potential therapeutic strategy of NHL therapy by monoclonal antibody chLym-1 in combination with autophagy inducer.     

E2F1 confers anticancer drug resistance by targeting ABC transporter family members and Bcl-2 via the p73/DNp73-miR-205 circuitry

Resistance to anti-neoplastic agents is the major cause of therapy failure, leading to disease recurrence and metastasis. E2F1 is a strong inducer of apoptosis in response to DNA damage through its capacity to activate p53/p73 death pathways. Recent evidence, however, showed that E2F1, which is aberrantly expressed in advanced malignant melanomas together with antagonistic p73 family members, drives cancer progression. Investigating mechanisms responsible for dysregulated E2F1 losing its apoptotic function, we searched for genomic signatures in primary and late clinical tumor stages to allow the prediction of downstream effectors associated with apoptosis resistance and survival of aggressive melanoma cells. We identified miR-205 as specific target of p73 and found that upon genotoxic stress, its expression is sufficiently abrogated by endogenous DNp73. Significantly, metastatic cells can be rescued from drug resistance by selective knockdown of DNp73 or overexpression of miR-205 in p73-depleted cells, leading to increased apoptosis and the reduction of tumor growth in vivo. Our data delineate an autoregulatory circuit, involving high levels of E2F1 and DNp73 to downregulate miR-205, which, in turn, controls E2F1 accumulation. Finally, drug resistance associated to this genetic signature is mediated by removing the inhibitory effect of miR-205 on the expression of Bcl-2 and the ATP-binding cassette transporters A2 (ABCA2) and A5 (ABCA5) related to multi-drug resistance and malignant progression. These results define the E2F1-p73/DNp73-miR-205 axis as a crucial mechanism for chemoresistance and, thus, as a target for metastasis prevention.     

E2F1 confers anticancer drug resistance by targeting ABC transporter family members and Bcl-2 via the p73/DNp73-miR-205 circuitry

Resistance to anti-neoplastic agents is the major cause of therapy failure, leading to disease recurrence and metastasis. E2F1 is a strong inducer of apoptosis in response to DNA damage through its capacity to activate p53/p73 death pathways. Recent evidence, however, showed that E2F1, which is aberrantly expressed in advanced malignant melanomas together with antagonistic p73 family members, drives cancer progression. Investigating mechanisms responsible for dysregulated E2F1 losing its apoptotic function, we searched for genomic signatures in primary and late clinical tumor stages to allow the prediction of downstream effectors associated with apoptosis resistance and survival of aggressive melanoma cells. We identified miR-205 as specific target of p73 and found that upon genotoxic stress, its expression is sufficiently abrogated by endogenous DNp73. Significantly, metastatic cells can be rescued from drug resistance by selective knockdown of DNp73 or overexpression of miR-205 in p73-depleted cells, leading to increased apoptosis and the reduction of tumor growth in vivo. Our data delineate an autoregulatory circuit, involving high levels of E2F1 and DNp73 to downregulate miR-205, which, in turn, controls E2F1 accumulation. Finally, drug resistance associated to this genetic signature is mediated by removing the inhibitory effect of miR-205 on the expression of Bcl-2 and the ATP-binding cassette transporters A2 (ABCA2) and A5 (ABCA5) related to multi-drug resistance and malignant progression. These results define the E2F1-p73/DNp73-miR-205 axis as a crucial mechanism for chemoresistance and, thus, as a target for metastasis prevention.     

Knockdown of EpCAM Enhances the Chemosensitivity of Breast Cancer Cells to 5-fluorouracil by Downregulating the Antiapoptotic Factor Bcl-2

Resistance to fluoropyrimidine-based chemotherapy is the main reason for the failure of cancer treatment, and drug resistance is associated with an inability of tumor cells to undergo apoptosis in response to treatment. Alterations in the expression of epithelial cell adhesion molecule (EpCAM) affect the sensitivity or resistance of tumor cells to anticancer treatment and the activity of intracellular signaling pathways. However, the role of EpCAM in the induction of apoptosis in breast cancer cells remains unclear. Here, we investigated the effect of EpCAM gene knockdown on chemosensitivity to 5-fluorouracil (5-FU) in MCF-7 cells and explored the underlying mechanisms. Our results showed that knockdown of EpCAM promoted apoptosis, inhibited cell proliferation and caused cell-cycle arrest. EpCAM knockdown enhanced the cytotoxic effect of 5-FU, promoting apoptosis by downregulating the expression of the anti-apoptotic protein Bcl-2 and upregulating the expression of the pro-apoptotic proteins Bax, and caspase3 via the ERK1/2 and JNK MAPK signaling pathways in MCF-7 cells. These results indicate that knockdown of EpCAM may have a tumor suppressor effect and suggest EpCAM as a potential target for the treatment of breast cancer.