Regulation of autophagic proteolysis by the N-recognin SQSTM1/p62 of the N-end rule pathway

In macroautophagy/autophagy, cargoes are collected by specific receptors, such as SQSTM1/p62 (sequestosome 1), and delivered to phagophores for lysosomal degradation. To date, little is known about how cells modulate SQSTM1 activity and autophagosome biogenesis in response to accumulating cargoes. In this study, we show that SQSTM1 is an N-recognin whose ZZ domain binds N-terminal arginine (Nt-Arg) and other N-degrons (Nt-Lys, Nt-His, Nt-Trp, Nt-Phe, and Nt-Tyr) of the N-end rule pathway. The substrates of SQSTM1 include the endoplasmic reticulum (ER)-residing chaperone HSPA5/GRP78/BiP. Upon N-end rule interaction with the Nt-Arg of arginylated HSPA5 (R-HSPA5), SQSTM1 undergoes self-polymerization via disulfide bonds of Cys residues including Cys113, facilitating cargo collection. In parallel, Nt-Arg-bound SQSTM1 acts as an inducer of autophagosome biogenesis and autophagic flux. Through this dual regulatory mechanism, SQSTM1 plays a key role in the crosstalk between the ubiquitin (Ub)-proteasome system (UPS) and autophagy. Based on these results, we employed 3D-modeling of SQSTM1 and a virtual chemical library to develop small molecule ligands to the ZZ domain of SQSTM1. These autophagy inducers accelerated the autophagic removal of mutant HTT (huntingtin) aggregates. We suggest that SQSTM1 can be exploited as a novel drug target to modulate autophagic processes in pathophysiological conditions.     

The C-terminal proteolytic fragment of the breast cancer susceptibility type 1 protein (BRCA1) is degraded by the N-end rule pathway

The breast cancer susceptibility type 1 gene product (BRCA1) is cleaved by caspases upon the activation of apoptotic pathways. After proteolysis the C-terminal fragment has been reported to translocate to the cytoplasm and promote cell death. Here we report that the C-terminal fragment is unstable in cells as it is targeted for degradation by the N-end rule pathway. The data reveals that mutating the wild type N-terminal aspartate, of the C-terminal fragment, to valine stabilizes the fragment. If the N terminus is mutated to another N-terminal destabilizing residue, like arginine, the C-terminal fragment remains unstable in cells. Last, the C-terminal fragment of BRCA1 is stable in cells lacking ATE1, a component of the N-end rule pathway.     

The arginylation branch of the N-end rule pathway positively regulates cellular autophagic flux and clearance of proteotoxic proteins

The N-terminal amino acid of a protein is an essential determinant of ubiquitination and subsequent proteasomal degradation in the N-end rule pathway. Using para-chloroamphetamine (PCA), a specific inhibitor of the arginylation branch of the pathway (Arg/N-end rule pathway), we identified that blocking the Arg/N-end rule pathway significantly impaired the fusion of autophagosomes with lysosomes. Under ER stress, ATE1-encoded Arg-tRNA-protein transferases carry out the N-terminal arginylation of the ER heat shock protein HSPA5 that initially targets cargo proteins, along with SQSTM1, to the autophagosome. At the late stage of autophagy, however, proteasomal degradation of arginylated HSPA5 might function as a critical checkpoint for the proper progression of autophagic flux in the cells. Consistently, the inhibition of the Arg/N-end rule pathway with PCA significantly elevated levels of MAPT and huntingtin aggregates, accompanied by increased numbers of LC3 and SQSTM1 puncta. Cells treated with the Arg/N-end rule inhibitor became more sensitized to proteotoxic stress-induced cytotoxicity. SILAC-based quantitative proteomics also revealed that PCA significantly alters various biological pathways, including cellular responses to stress, nutrient, and DNA damage, which are also closely involved in modulation of autophagic responses. Thus, our results indicate that the Arg/N-end rule pathway may function to actively protect cells from detrimental effects of cellular stresses, including proteotoxic protein accumulation, by positively regulating autophagic flux.     

miR-4429 sensitized cervical cancer cells to irradiation by targeting RAD51

Cervical cancer (CC) is a prevalent malignancy in women, with the feature of metastasis and easy recurrence is responsible for a large proportion of global cancer deaths. Radiotherapy is one of the common treatment tools for CC patients with unresectable tumors. However, radio-resistance in patients could be a major reason for recurrence. Therefore, it is of significance to tunnel the molecular mechanism of radio-resistance in CC. MicroRNAs (miRNAs) are increasingly reported in the regulation of cancer progression and cellular response to radiotherapy and chemotherapy. miR-4429 is a newly discovered miRNA acting as a tumor-suppressor gene in multiple cancers, but its function in CC has never been explored yet. The current study tried to explore the role of miR-4429 in cell radio-sensitivity in CC. First, we validated the downregulation of miR-4429 in CC cells. Importantly, the association of miR-4429 with radio-resistance was validated by identifying the downregulation of miR-4429 in radio-resistant CC cells. Gain- and loss-of-function assays validated that miR-4429 sensitized CC cells to irradiation. Through bioinformatics tools, RAD51 recombinase (RAD51) was identified to be a target for miR-4429. RAD51 is known to be a crucial regulator for DNA damage repair and has been reported to influence cell radio-resistance in cancers, including in CC. Luciferase reporter assay confirmed the interaction between miR-4429 and RAD51. Finally, rescue assays indicated that miR-4429 promoted CC cell radio-sensitivity through RAD51. Consequently, our study showed that miR-4429 sensitized CC cells to irradiation by targeting RAD51, providing a potential therapeutic target for CC patients.     

Doxorubicin induces cell death in breast cancer cells regardless of Survivin and XIAP expression levels

Breast cancer is the leading cause of deaths in women around the world. Resistance to therapy is the main cause of treatment failure and still little is known about predictive biomarkers for response to systemic therapy. Increasing evidence show that Survivin and XIAP overexpression is closely associated with chemoresistance and poor prognosis in breast cancer. However, their impact on resistance to doxorubicin (dox), a chemotherapeutic agent widely used to treat breast cancer, is poorly understood. Here, we demonstrated that dox inhibited cell viability and induced DNA fragmentation and activation of caspases-3, -7 and -9 in the breast cancer-derived cell lines MCF7 and MDA-MB-231, regardless of different p53 status. Dox exposure resulted in reduction of Survivin and XIAP mRNA and protein levels. However, when we transfected cells with a Survivin-encoding plasmid, we did not observe a cell death-resistant phenotype. XIAP and Survivin silencing, either alone or in combination, had no effect on breast cancer cells sensitivity towards dox. Altogether, we demonstrated that breast cancer cells are sensitive to the chemotherapeutic agent dox irrespective of Survivin and XIAP expression levels. Also, our findings suggest that dox-mediated modulation of Survivin and XIAP might sensitize cells to taxanes when used in a sequential regimen.     

Downregulation of lncRNA HCP5 has inhibitory effects on gastric cancer cells by regulating DDX21 expression

LncRNA HCP5 has been confirmed to play crucial roles in many types of cancers. However, the role of lncRNA HCP5 in regulating the occurrence and development of gastric cancer (GC) remains unknown. In the current study, we aimed to investigate the precise effects of lncRNA HCP5 on cell proliferation, migration and invasion and molecular mechanisms in gastric cancer. Using RT-qPCR analysis, we found that lncRNA HCP5 was differentially expressed in GC cell lines. CCK-8, wound healing and transwell assay indicated that the proliferation, migration and invasion of gastric cancer cells were inhibited by downregulation of lncRNA HCP5 and lncRNA HCP5 overexpression exhibited the opposite effects in gastric cancer cells. Mechanistically, RNA binding protein immunoprecipitation and dual luciferase reporter assay confirmed the interaction between lncRNA HCP5 and DDX21. The effects of lncRNA HCP5 overexpression the proliferation, migration and invasion of GC cells were partly rescued by DDX21 silencing. Taken together, downregulation of lncRNA HCP5 exerted inhibitory effects on GC cell proliferation, migration and invasion through modulation of DDX21 expression, demonstrating the function of lncRNA HCP5 and DDX21 in GC progression.     

Akt and XIAP regulate the sensitivity of human uterine cancer cells to cisplatin,doxorubicin and taxol

We have investigated the interrelationship between two anti-apoptotic factors, XIAP and Akt, and their role in chemoresistance of uterine cancer cells. We used one cervical cancer cell line (HeLa) and two endometrial cancer cell lines (KLE and Ishikawa) as a model. The three drugs decreased Akt and XIAP content and induced apoptosis in P-Akt-negative HeLa cells. In P-Akt1/3-positive Ishikawa cells apoptosis induction correlated with XIAP decrease. P-Akt1/2/3-positive KLE cells showed maximum chemoresistance as XIAP and Akt levels/phosphorylation remained stable in response to the three drugs, and only cisplatin could significantly induce apoptosis. We found that XIAP and Akt were functionally linked in uterine cancer cells, as downregulation of XIAP with RNAi decreased P-Akt levels, and inhibition of PI3-K/Akt activity using LY294002 decreased XIAP content. Overexpression of constitutively active Akt isoforms in HeLa cells induced isoform-specific sensitivity to doxorubicin and taxol but not cisplatin. XIAP RNAi increased the cell-specific sensitivity to cisplatin and doxorubicin but not taxol. Finally, we found P-Akt immunoreactivity in epithelial cells from multiple human endometrial carcinoma tumors, suggesting that Akt may also regulate chemosensitivity in uterine cancers in vivo. Altogether these results highlight an intertwined role for specific Akt isoforms and XIAP in chemoresistance of uterine cancer cells.     

Role of XIAP in inhibiting cisplatin-induced caspase activation in non-small cell lung cancer cells: a small molecule Smac mimic sensitizes for chemotherapy-induced apoptosis by enhancing caspase-3 activation

X-linked IAP (XIAP) suppresses apoptosis by binding to initiator caspase-9 and effector caspases-3 and -7. Smac/DIABLO that is released from mitochondria during apoptosis can relieve its inhibitory activity. Here we investigated the role of XIAP in the previously found obstruction of chemotherapy-induced caspase-9 activation in non-small cell lung cancer (NSCLC) cells. Endogenously expressed XIAP bound active forms of both caspase-9 and caspase-3. However, downregulation of XIAP using shRNA or disruption of XIAP/caspase-9 interaction using a small molecule Smac mimic were unable to significantly induce caspase-9 activity, indicating that despite a strong binding potential of XIAP to caspase-9 it is not a major determinant in blocking caspase-9 in NSCLC cells. Although unable to revert caspase-9 blockage, the Smac mimic was able to enhance cisplatin-induced apoptosis, which was accompanied by increased caspase-3 activity. Additionally, a more detailed analysis of caspase activation in response to cisplatin indicated a reverse order of activation, whereby caspase-3 cleaved caspase-9 yielding an inactive form. Our findings indicate that the use of small molecule Smac mimic, when combined with an apoptotic trigger, may have therapeutic potential for the treatment of NSCLC.     

Depletion of JMJD5 sensitizes tumor cells to microtubule-destabilizing agents by altering microtubule stability

Microtubules play essential roles in mitosis, cell migration, and intracellular trafficking. Drugs that target microtubules have demonstrated great clinical success in cancer treatment due to their capacity to impair microtubule dynamics in both mitotic and interphase stages. In a previous report, we demonstrated that JMJD5 associated with mitotic spindle and was required for proper mitosis. However, it remains elusive whether JMJD5 could regulate the stability of cytoskeletal microtubules and whether it affects the efficacy of microtubule-targeting agents. In this study, we find that JMJD5 localizes not only to the nucleus, a fraction of it also localizes to the cytoplasm. JMJD5 depletion decreases the acetylation and detyrosination of α-tubulin, both of which are markers of microtubule stability. In addition, microtubules in JMJD5-depleted cells are more sensitive to nocodazole-induced depolymerization, whereas JMJD5 overexpression increases α-tubulin detyrosination and enhances the resistance of microtubules to nocodazole. Mechanistic studies revealed that JMJD5 regulates MAP1B protein levels and that MAP1B overexpression rescued the microtubule destabilization induced by JMJD5 depletion. Furthermore, JMJD5 depletion significantly promoted apoptosis in cancer cells treated with the microtubule-targeting anti-cancer drugs vinblastine or colchicine. Together, these findings suggest that JMJD5 is required to regulate the stability of cytoskeletal microtubules and that JMJD5 depletion increases the susceptibility of cancer cells to microtubule-destabilizing agents.     

Chloride ions control the G1/S cell-cycle checkpoint by regulating the expression of p21 through a p53-independent pathway in human gastric cancer cells

The aim of the present study is to investigate whether the chloride affects cell growth and cell-cycle progression of cancer cells. In human gastric cancer MKN28 cells, the culture in the Cl⁻-replaced medium (replacement of Cl⁻ by NO₃⁻) decreased the intracellular chloride concentration ([Cl⁻]_i) and inhibited cell growth. The inhibition of cell growth was due to cell-cycle arrest at the G₀/G₁ phase caused by diminution of CDK2 and phosphorylated Rb. The culture of cells in the Cl⁻-replaced medium significantly increased expressions of p21 mRNA and protein without any effects on p53. These observations indicate that chloride ions play important roles in cell-cycle progression by regulating the expression of p21 through a p53-independent pathway in human gastric cancer cells, leading to a novel, unique therapeutic strategy for gastric cancer treatment via control of [Cl⁻]_i.     

慢病毒载体介导RNAi稳定抑制XIAP表达及对胰腺癌细胞增殖、凋亡影响的实验研究

目的:探讨运用慢病毒载体介导的RNA干扰技术对X-连锁凋亡抑制蛋白(XIAP)的抑制效率及对胰腺癌细胞增殖、凋亡的影响,建立XIAP表达稳定抑制的胰腺癌细胞株.方法:应用pGJCSIL-PUR慢病毒载体构建针对XIAP的ShRNA载体,转染包装细胞293T,收集病毒上清转染胰腺癌细胞系SW1990,经嘌呤霉素(puromycin)筛选并扩大培养得到稳定克隆;实时荧光定量PCR和western-blot免疫印迹检测癌细胞内XIAP的表达:四甲基偶氮唑盐(MTT)比色法检测细胞增殖;caspase3/7活性测定和DAPI染色检测细胞凋亡.结果:成功构建3个XIAP-ShRNA慢病毒栽体(X1、X2、X3)及XIAP表达稳定抑制的胰腺癌细胞株,对XIAP的抑制效率均达70%以上;MTT检测显示X1、X3稳定抑制XIAP后胰腺癌细胞增殖明显减慢,但caspase3/7活性及细胞凋亡并没有明显增加.结论:慢病毒栽体介导的靶向XIAP的RNAi可有效抑制XIAP表达,降低胰腺癌细胞的增殖能力;成功建立的XIAP表达稳定抑制的胰腺癌细胞株为进一步研究打下基础.     

MiR‐3116 sensitizes glioma cells to temozolomide by targeting FGFR1 and regulating the FGFR1/PI3K/AKT pathway

Glioma is a brain tumour that is often diagnosed, and temozolomide (TMZ) is a common chemotherapeutic drug used in glioma. Yet, resistance to TMZ is a chief hurdle towards curing the malignancy. The current work explores the pathways and involvement of miR‐3116 in the TMZ resistance. miR‐3116 and FGFR1 mRNA were quantified by real‐time PCR in malignant samples and cell lines. Appropriate assays were designed for apoptosis, viability, the ability to form colonies and reporter assays to study the effects of the miR‐3116 or FGFR1. The involvement of PI3K/AKT signalling was assessed using Western blotting. Tumorigenesis was evaluated in an appropriate xenograft mouse model in vivo. This work revealed that the levels of miR‐3116 dipped in samples resistant to TMZ, while increased miR‐3116 caused an inhibition of the tumour features mentioned above to hence augment TMZ sensitivity. miR‐3116 was found to target FGFR1. When FGFR1 was overexpressed, resistance to TMZ was augmented and reversed the sensitivity caused by miR‐3116. Our findings further confirmed PI3K/AKT signalling pathway is involved in this action. In conclusion, miR‐3116 sensitizes glioma cells to TMZ through FGFR1 downregulation and the PI3K/AKT pathway inactivation. Our results provide a strategy to overcome TMZ resistance in glioma treatment.     

AS160 controls eukaryotic cell cycle and proliferation by regulating the CDK inhibitor p21

AS160 (TBC1D4) has been implicated in multiple biological processes. However, the role and the mechanism of action of AS160 in the regulation of cell proliferation remain unclear. In this study, we demonstrated that AS160 knockdown led to blunted cell proliferation in multiple cell types, including fibroblasts and cancer cells. The results of cell cycle analysis showed that these cells were arrested in the G1 phase. Intriguingly, this inhibition of cell proliferation and the cell cycle arrest caused by AS160 depletion were glucose independent. Moreover, AS160 silencing led to a marked upregulation of the expression of the cyclin-dependent kinase inhibitor p21. Furthermore, whereas AS160 overexpression resulted in p21 downregulation and rescued the arrested cell cycle in AS160-depeleted cells, p21 silencing rescued the inhibited cell cycle and proliferation in the cells. Thus, our results demonstrated that AS160 regulates glucose-independent eukaryotic cell proliferation through p21-dependent control of the cell cycle, and thereby revealed a molecular mechanism of AS160 modulation of cell cycle and proliferation that is of general physiological significance.     

Novel Polypyridyl chelators deplete cellular zinc and destabilize the X-linked inhibitor of apoptosis protein (XIAP) prior to induction of apoptosis in human prostate and breast cancer cells

X-linked inhibitor of apoptosis protein (XIAP), inhibits the initiation and execution phases of the apoptotic pathway. XIAP is the most potent member of the inhibitor of apoptosis protein (IAP) family of the endogenous caspase inhibitors. Therefore, targeting XIAP may be a promising strategy for the treatment of apoptosis-resistant malignancies. In this study, we systematically studied the relationships of chemical structures of several novel ligands to their zinc (Zn)-binding ability, molecular target XIAP, and tumor cell death-inducing activity. We show that treatment of PC-3 prostate cancer and MDA-MB-231 breast cancer cells with these membrane-permeable Zn-chelators with different Zn affinities results in varying degrees of XIAP depletion. Following decreased level of XIAP expression, we also show apoptosis-related caspase activation and cellular morphological changes upon treatment with strong Zn-chelators N4Py and BnTPEN. Addition of Zn has a full protective effect on the cells treated with these chelators, while iron (Fe) addition has only partial protection that, however, can be further increased to a comparable level of protection as Zn by inhibition of ROS generation, indicating that cell death effects mediated by Fe- but not Zn-complexes involve redox cycling. These findings suggest that strong Zn-chelating agents may be useful in the treatment of apoptosis-resistant human cancers.     

Deubiquitinase PSMD7 promotes the proliferation,invasion, and cisplatin resistance of gastric cancer cells by stabilizing RAD23B

Ubiquitination, a crucial post-translational modification, controls substrate degradation and can be reversed by deubiquitinases (DUBs). An increasing number of studies are showing that DUBs regulate the malignant behavior and chemotherapy resistance of gastric cancer (GC) by stabilizing various proteins. However, the expression level and biological function of the DUB, proteasome 26S subunit, non-ATPase 7 (PSMD7), in GC remains unknown. Herein, we report for the first time that PSMD7 is frequently overexpressed in GC tissues. Elevated levels of PSMD7 were also detected in GC cell lines. Notably, the upregulation of PSMD7 closely correlated with malignant clinical parameters and reduced the survival of GC patients. Functionally, we found that PSMD7 knockdown consistently suppressed the proliferation, migration, and invasion of AGS and SGC-7901 cells. Ectopic expression of PSMD7 facilitated GC cell proliferation and mobility. Based on protein-protein interaction prediction, RAD23 homolog B (RAD23B) protein was identified as a candidate substrate of PSMD7. PSMD7 positively regulated the abundance of RAD23B and xeroderma pigmentosum, complementation group C (XPC) protein in GC cells. The interaction between PSMD7 and RAD23B was confirmed using protein immunoprecipitation. PSMD7 knockdown enhanced the ubiquitination and degradation of RAD23B protein in GC cells. PSMD7 promoted cell viability, apoptosis resistance, and DNA damage repair in GC cells upon cisplatin (DDP) treatment. Moreover, PSMD7 silencing inhibited tumor growth and enhanced the sensitivity of GC cells to DDP treatment in mice. In summary, PSMD7 was highly expressed in GC and contributed to the malignant behavior and DDP resistance of tumor cells by stabilizing RAD23B.     

Metformin inhibits RANKL and sensitizes cancer stem cells to denosumab

The increased propensity of BRCA1 mutation carriers to develop aggressive breast tumors with stem-like properties begins to be understood in terms of osteoprotegerin (OPG)-unrestricted cross-talk between RANKL-overproducing progesterone-sensor cells and cancer-initiating RANK⁺ responder cells that reside within pre-malignant BRCA1^mut/+ breast epithelial tissue. We recently proposed that, in the absence of hormone influence, cancer-initiating cells might remain responsive to RANKL stimulation, and hence to the therapeutic effects of the anti-RANKL antibody denosumab because genomic instability induced by BRCA1 haploinsufficiency might suffice to cell-autonomously hyperactivate RANKL gene expression. Here we report that the biguanide metformin prevents BRCA1 haploinsufficiency-driven RANKL gene overexpression, thereby disrupting an auto-regulatory feedback control of RANKL-addicted cancer stem cell-like states within BRCA1^mut/? cell populations. Moreover, metformin treatment elicits a synergistic decline in the breast cancer-initiating cell population and its self-renewal capacity in BRCA1-mutated basal-like breast cancer cells with bone metastasis-initiation capacity that exhibit primary resistance to denosumab in mammosphere assays. The specific targeting of RANKL/RANK signaling with denosumab is expected to revolutionize prevention and treatment strategies currently available for BRCA1 mutation carriers. Our findings provide a rationale for new denosumab/metformin combinatorial strategies to clinically manage RANKL-related breast oncogenesis and metastatic progression.     

Cordycepin causes p21WAF1-mediated G2/M cell-cycle arrest by regulating c-Jun N-terminal kinase activation in human bladder cancer cells

Cordycepin (3′-deoxyadenosine), a bioactive compound of Cordycepsmilitaris, has many pharmacological activities. The present study reveals novel molecular mechanisms for the anti-tumor effects of cordycepin in two different bladder cancer cell lines, 5637 and T-24 cells. Cordycepin treatment, at a dose of 200 μM (IC₅₀) during cell-cycle progression resulted in significant and dose-dependent growth inhibition, which was largely due to G2/M-phase arrest, and resulted in an up-regulation of p21WAF1 expression, independent of the p53 pathway. Moreover, treatment with cordycepin-induced phosphorylation of JNK (c-Jun N-terminal kinases). Blockade of JNK function using SP6001259 (JNK-specific inhibitor) and small interfering RNA (si-JNK1) rescued cordycepin-dependent p21WAF1 expression, inhibited cell growth, and decreased cell cycle proteins. These results suggest that cordycepin could be an effective treatment for bladder cancer.