p62/SQSTM1 involved in cisplatin resistance in human ovarian cancer cells by clearing ubiquitinated proteins

Mechanisms of cisplatin resistance in cancer cells are not fully understood. Here, we show a critical role for the ubiquitin-binding protein p62/SQSTM1 in cisplatin resistance in human ovarian cancer cells (HOCCs). Specifically, we found that cisplatin-resistant SKOV3/DDP cells express much higher levels of p62 than do cisplatin-sensitive SKOV3 cells. The protein p62 binds ubiquitinated proteins for transport to autophagic degradation, reducing apoptosis induced by endoplasmic reticulum (ER) stress in SKOV3/DDP cells. Knockdown of p62 or inhibition of autophagy using 3-methyladenine resensitises SKOV3/DDP cells to cisplatin. Collectively, our data indicate that p62 acts as a receptor or adaptor for autophagic degradation of ubiquitinated proteins, and plays an important role in preventing ER stress-induced apoptosis, leading to cisplatin resistance in HOCCs.     

The NF‐κB‐modulated miR‐19a‐3p enhances malignancy of human ovarian cancer cells through inhibition of IGFBP‐3 expression

Ovarian cancer is the most lethal gynecologic malignancy due to the lack of symptoms until advanced stages, and new diagnosis and treatment strategy is in urgent need. In this study, we found higher expression of miR‐19a‐3p in ovarian cancer tissues compared with that in the adjacent normal tissues. By chromatin immunoprecipitation (ChIP) and electrophoretic mobility shift assay (EMSA) analysis, we showed that nuclear factor‐kappaB (NF‐κB) binds to the promoter of miR‐19a‐3p, leading to reduced expression in ovarian cancer cells. Further study indicated that miR‐19a‐3p inhibits the expression of insulin‐like growth factor binding protein‐3 (IGFBP‐3), resulting in enhanced growth and migration of ovarian cancer cells in vitro and tumor growth in vivo. These results showed that miR‐19a‐3p enhances the oncogenesis of ovarian cancer through inhibition of IGFBP‐3 expression, and which can be inhibited by NF‐κB, suggesting an NF‐κB/miR‐19a‐3p/IGFBP‐3 pathway in the oncogenesis of ovarian cancer, which expands our understanding of ovarian cancer and they may contribute to the development of new diagnosis and treatment of ovarian cancer.     

Inhibition of p62/SQSTM1 sensitizes small-cell lung cancer cells to cisplatin-induced cytotoxicity by targeting NEDD9 expression

Drug resistance is the leading cause for rapid progression and relapse in small-cell lung cancer (SCLC) patients. Thus overcoming drug resistance still remains to be urgently resolved during SCLC treatment. Here, we found p62/SQSTM1 was enriched in SCLC spheroids, a subpopulation possessing cancer stem-like properties, which is responsible for cancer relapse and metastasis. Subsequent functional assays in vitro showed that short hairpin RNA (shRNA)-mediated p62 knockdown increased sensitivity of SCLC cell lines to cisplatin (DDP), whereas lentivirus-mediated p62 ectopic overexpression diminished DDP-induced cytotoxicity in both NCI-H446 and NCI-H1688 cell lines. Moreover, ectopic p62 overexpression promoted DDP resistance of NCI-H446 cells-derived tumor xenografts in immunodeficient mice in vivo, as indicated by accelerated tumor growth rate and reduced fluorescent activity of cleaved caspase-3. Gene expression profiling analysis revealed that p62 was positively correlated with neuronal precursor cell-expressed, developmentally downregulated gene 9 (NEDD9) expression level. Consistently, NEDD9 messenger RNA (mRNA) level was decreased upon p62 suppression by small interfering RNA (siRNA) and increased with p62 transient overexpression in SCLC cell lines, suggesting that p62 positively regulated NEDD9 mRNA. Depletion of NEDD9 by siRNA, to a large extent, reversed p62-overexpressed SCLC cells to DDP-induced cytotoxicity, implying NEDD9 might act as a downstream target which was in charge of p62-mediated DDP resistance. Taken together, our findings uncovered a previously unknown role of p62 in the regulation of SCLC drug resistance, assigning p62 as an attractive target for SCLC treatment.     

Involvement of Akt/NF‐κB pathway in N6‐isopentenyladenosine‐induced apoptosis in human breast cancer cells

N⁶‐isopentenyladenosine (i6A) inhibits the tumor cell growth by inducing cell apoptosis in various cancer cell lines. However, little is known regarding the mechanisms by which the drug induces cell apoptosis. In this study, we further explored the molecular mechanisms of i6A as an anticancer agent on a human breast cancer cell line MDA MB 231. Treatment with i6A decreased the cell proliferation of MDA MB 231 cells in a dose‐dependent manner by arresting the cells at G₀/G₁ phase. This effect was strongly associated with concomitant decrease in the level of cyclin D1, cyclin E, cdk2, and increase of p21waf1 and p27kip. In addition i6A also induced apoptotic cell death by increasing the expression of Bax, and decreasing the levels of Bcl‐2 and Bcl‐xL, and subsequently triggered mitochondria apoptotic pathway (release of cytochrome c and activation of caspase‐3). We observed that i6A suppressed the nuclear factor kappaB (NF‐κB) pathway and inhibited the Akt activation. The results of this study indicate that i6A decreases cell proliferation and induces apoptotic cell death in human breast cancer cells, possibly by decreasing signal transduction through the Akt/NF‐κB cell survival pathway. © 2010 Wiley‐Liss, Inc.     

DCLK1 promotes epithelial‐mesenchymal transition via the PI3K/Akt/NF‐κB pathway in colorectal cancer

Double cortin‐like kinase 1 (DCLK1) plays important roles during the epithelial‐mesenchymal transition (EMT) process in human colorectal cancer (CRC). However, the role of DCLK1 in regulating the EMT of CRC is still poorly understood. In this study, we report evidence that DCLK1 acts as a potent oncogene to drive its extremely malignant character of EMT in an NF‐κB‐dependent manner in CRC cells. Mechanistic investigations showed that DCLK1 induced the NF‐κBp65 subunit expression through the PI3K/Akt/Sp1 axis and activated NF‐κBp65 through the PI3K/Akt/IκBα pathway during the EMT of CRC cells. Moreover, we found that silencing the expression of DCLK1 inhibited the invasion and metastasis of CRC cells in vivo. Collectively, our findings identify DCLK1 as a pivotal regulator of an EMT axis in CRC, thus implicating DCLK1 as a potential therapeutic target for CRC metastasis.     

PARP14 promotes the proliferation and gemcitabine chemoresistance of pancreatic cancer cells through activation of NF‐κB pathway

Pancreatic cancer (PC) is the most fatal gastrointestinal malignancy in the world, with a 5‐year relative survival of only 8%. Poly(ADP‐ribose) polymerase (PARP)14, a member of the macro‐PARP subfamily proteins, has been reported to participate in various biologic and pathologic processes in multiple cancers. The roles and underlying molecular mechanisms of PARP14 in PC carcinogenesis, however, remain to be elucidated. In this study, we for the first time discovered that PARP14 was highly expressed in human primary PC specimens and significantly correlated with poor patient prognosis. Using loss‐of‐function studies in vitro and in vivo, we showed that the knockdown of PARP14 led to enhanced apoptosis, repressed proliferation, and gemcitabine (GEM) resistance of PC cells. Further investigations revealed that PARP14 was significantly overexpressed in GEM‐resistant PC cells (SW1990/GZ). And silencing of PARP14 significantly reversed the GEM resistance of SW1990/GZ cells. To the mechanism, PARP14 could stimulate PC progression by the activation of nuclear factor‐κB (NF‐κB) signaling pathway. And inhibition of NF‐κB signal could significantly reverse PARP14–overexpression triggered PC carcinogenesis. In conclusion, PARP14 could promote PC cell proliferation, antiapoptosis, and GEM resistance via NF‐κB signaling pathway, highlighting its potential role as a therapeutic target for PC.     

Downregulation of miR‐29 contributes to cisplatin resistance of ovarian cancer cells

Drug resistance is an obstacle to the treatment of ovarian cancer. Using a unique cell model, we have proven previously that a subpopulation of ovarian cancer cells is more resistant to cisplatin than are the original cells. MicroRNAs (miRNAs), small noncoding RNAs, are involved in many biological events in cancer cells. In our study, we explored whether miRNAs are involved in cisplatin resistance of ovarian cancer cells. Cisplatin‐resistant cells expressed a lower level of miR‐29a/b/c. Manipulation of microRNA‐29 (miR‐29) expression modulated cisplatin sensitivity of CP70, HeyC2, SKOV3 and A2780 ovarian cancer cells. Knockdown of miR‐29a/b/c increased the ability of cells to escape cisplatin‐induced cell death partly through upregulation of collagen type I alpha 1 (COL1A1) and increased the activation of extracellular signal‐regulated kinase 1/2 and inactivation of glycogen synthase kinase 3 beta. When combined with cisplatin treatment, knockdown of miR‐29 decreased the amount of the active form of caspase‐9 and caspase‐3. Ectopic expression of miR‐29 alone or in combination with cisplatin treatment efficaciously reduced the tumorigenicity of CP70 cells in vivo. Our data show that downregulation of miR‐29 increases cisplatin resistance in ovarian cancer cells. Taken together, these data suggest that overexpression of miR‐29 is a potential sensitizer to cisplatin treatment that may have therapeutic implications.     

miR-483-5p在上皮性卵巢癌中的表达及其对顺铂敏感性的影响

背景与目的：顺铂是目前临床上治疗上皮性卵巢癌的一线化疗药物之一,但许多患者对铂类药物耐药。miR-483-5p在肺癌中过表达,然而目前尚未见miR-483-5p在上皮性卵巢癌中的研究。该研究检测miR-483-5p在上皮性卵巢癌组织和上皮性卵巢癌细胞系中的表达并探讨其对上皮性卵巢癌细胞对顺铂敏感性的影响。方法：采用实时荧光定量聚合酶链反应(real-time fluorescent quantitative polymerase chain reaction,RTFQ-PCR)检测43例上皮性卵巢癌患者的肿瘤组织、8例正常卵巢组织和5种上皮性卵巢癌细胞系中miR-483-5p的表达情况;通过慢病毒上调或敲低卵巢癌细胞miR-483-5p表达,应用CCK-8实验检测miR-483-5p对上皮性卵巢癌细胞系顺铂敏感性的影响。结果：上皮性卵巢癌组织中miR-483-5p表达明显高于正常卵巢组织(P<0.01)。此外,miR-483-5p在晚期上皮性卵巢癌组织中的表达水平显著高于早期肿瘤组织(P<0.05)。5种上皮性卵巢癌细胞系中SKOV3细胞表达miR-483-5p的量最低;miR-483-5p在上皮性卵巢癌顺铂耐药A2780/CP细胞中表达量最高。上调SKOV3细胞中miR-483-5p的表达能够降低上皮性卵巢癌细胞对顺铂的敏感性,并下调p21及Bcl-2的表达;下调A2780/CP细胞miR-483-5p的表达能够增加细胞对顺铂的敏感性,并上调p21及Bcl-2的表达。结论：miR-483-5p在上皮性卵巢癌组织中高表达并对顺铂耐药,可以作为临床预测上皮性卵巢癌对顺铂敏感性的生物标志物之一。     

Association between activation of atypical NF‐κB1 p105 signaling pathway and nuclear β‐catenin accumulation in colorectal carcinoma

Recent studies have demonstrated that increased expression of coding region determinant‐binding protein (CRD‐BP) in response to β‐catenin signaling leads to the stabilization of β‐TrCP1, a substrate‐specific component of SCF E3 ubiquitin ligase complex, resulting in an accelerated degradation of IκBα and activation of canonical nuclear factor‐κB (NF‐κB) pathway. Here, we show that the noncanonical NF‐κB1 p105 pathway is constitutively activated in colorectal carcinoma specimens, being particularly associated with β‐catenin‐mediated increased expression of CRD‐BP and β‐TrCP1. In the carcinoma tissues exhibiting high levels of nuclear β‐catenin the phospho‐p105 levels were increased and total p105 amounts were decreased in comparison to that of normal tissue indicating an activation of this NF‐κB pathway. Knockdown of CRD‐BP in colorectal cancer cell line SW620 resulted in significantly higher basal levels of both NF‐κB inhibitory proteins, p105 and IκBα. Furthermore decreased NF‐κB binding activity was observed in CRD‐BP siRNA‐transfected SW620 cells as compared with those transfected with control siRNA. Altogether, our findings suggest that activation of NF‐κB1 p105 signaling in colorectal carcinoma might be attributed to β‐catenin‐mediated induction of CRD‐BP and β‐TrCP1. © 2009 Wiley‐Liss, Inc.     

Significance of NF‐κB activation in immortalization of nasopharyngeal epithelial cells

NF‐κB is a key regulator of inflammatory response and is frequently activated in human cancer including the undifferentiated nasopharyngeal carcinoma (NPC), which is common in Southern China including Hong Kong. Activation of NF‐κB is common in NPC and may contribute to NPC development. The role of NF‐κB activation in immortalization of nasopharyngeal epithelial (NPE) cells, which may represent an early event in NPC pathogenesis, is unknown. Examination of NF‐κB activation in immortalization of NPE cells is of particular interest as the site of NPC is often heavily infiltrated with inflammatory cellular components. We found that constitutive activation of NF‐κB signaling is a common phenotype in telomerase‐immortalized NPE cell lines. Our results suggest that NF‐κB activation promotes the growth of telomerase‐immortalized NPE cells, and suppression of NF‐κB activity inhibits their proliferation. Furthermore, we observed upregulation of c‐Myc, IL‐6 and Bmi‐1 in our immortalized NPE cells. Inhibition of NF‐κB downregulated expression of c‐Myc, IL‐6 and Bmi‐1, suggesting that they are downstream events of NF‐κB activation in immortalized NPE cells. We further delineated that EGFR/MEK/ERK/IKK/mTORC1 is the key upstream pathway of NF‐κB activation in immortalized NPE cells. Elucidation of events underlying immortalization of NPE cells may provide insights into early events in pathogenesis of NPC. The identification of NF‐κB activation and elucidation of its activation mechanism in immortalized NPE cells may reveal novel therapeutic targets for treatment and prevention of NPC.     

p62 promotes bladder cancer cell growth by activating KEAP1/NRF2‐dependent antioxidative response

p62 is associated with 2 major cellular defense mechanisms against metabolic and oxidative stress, autophagy and the Kelch‐like ECH‐associated protein 1 (KEAP1)‐nuclear factor‐E2‐related factor 2 (NRF2) system. Recent studies indicate that the p62‐KEAP1‐NRF2 pathway promotes tumorigenesis and tumor growth mediated by NRF2‐dependent antioxidative response. However, whether p62 is involved in bladder cancer (BCa) development remains unknown. Here, we found that p62 is overexpressed in BCa tissue and several BCa cell lines. The knockdown of p62 inhibits BCa cell growth both in vitro and in vivo, with increased intracellular reactive oxygen species level. Mechanically, p62 activates NRF2 signaling by sequestrating KEAP1, which leads to the upregulation of antioxidant genes (Gclc, Gstm5, and Gpx2), thus protecting BCa cells from oxidative stress. Our findings indicate that p62 might be involved in the development of BCa and serve as a potential therapeutic target.     

Akt promotes cisplatin resistance in human ovarian cancer cells through inhibition of p53 phosphorylation and nuclear function

Resistance to cisplatin-based chemotherapy is a major cause of treatment failure in human ovarian cancer. Wild-type TP53 status is often, but not always, associated with cisplatin sensitivity, suggesting that additional factors may be involved. Overexpression/activation of the phosphatidylinositol-3-kinase/Akt pathway is commonly observed in ovarian cancer, and Akt activation is a determinant of chemoresistance in ovarian cancer cells, an effect that may be due, in part, to its inhibitory actions on p53-dependent apoptosis. To that end, we examined the role and regulation of p53 in chemosensitive ovarian cancer cells, as well as in their chemoresistant counterparts, and investigated if and how Akt influences this pathway. Cisplatin induced apoptosis in chemosensitive, but not chemoresistant cells, and this was inhibited by downregulation of p53. Cisplatin upregulated PUMA in a p53-dependent manner, and the presence of PUMA was necessary, but not sufficient for cisplatin-induced apoptosis. p53 was phosphorylated on numerous N-terminal residues, including Ser15, Ser20, in response to cisplatin in chemosensitive, but not chemoresistant cells. Furthermore, activation of Akt inhibited the cisplatin-induced upregulation of PUMA, and suppressed cisplatin-induced p53 phosphorylation, while inhibition of Akt increased total and phospho-p53 contents and sensitized p53 wild-type, chemoresistant cells to cisplatin-induced apoptosis. Finally, mutation of Ser15 and/or Ser20, but not of Ser37, to alanine significantly attenuated the ability of p53 to facilitate CDDP-induced apoptosis, and this was independent of PUMA expression. These results support the hypothesis that p53 is a determinant of CDDP sensitivity, and suggest that Akt contributes to chemoresistance, in part, by attenuating p53-mediated PUMA upregulation and phosphorylation of p53, which are essential, but independent determinants of sensitivity to CDDP-induced apoptosis.     

Endoplasmic reticulum stress pathway PERK‐eIF2α confers radioresistance in oropharyngeal carcinoma by activating NF‐κB

Endoplasmic reticulum stress (ERS) plays an important role in the pathogenesis and development of malignant tumors, as well as in the regulation of radiochemoresistance and chemoresistance in many malignancies. ERS signaling pathway protein kinase RNA‐like endoplasmic reticulum kinase (PERK)‐eukaryotic initiation factor‐2 (eIF2α) may induce aberrant activation of nuclear factor‐κB (NF‐κB). Our previous study showed that NF‐κB conferred radioresistance in lymphoma cells. However, whether PERK‐eIF2α regulates radioresistance in oropharyngeal carcinoma through NF‐κB activation is unknown. Herein, we showed that PERK overexpression correlated with a poor prognosis for patients with oropharyngeal carcinoma (P < 0.01). Meanwhile, the percentage of the high expression level of PERK in oropharyngeal carcinoma patients resistant to radiation was higher than in patients sensitive to radiation (77.7 and 33.3%, respectively; P < 0.05). Silencing PERK and eIF2α increased the radiosensitivity in oropharyngeal carcinoma cells and increased radiation‐induced apoptosis and G2/M phase arrest. PERK‐eIF2α silencing also inhibited radiation‐induced NF‐κB phosphorylation and increased the DNA double strand break‐related proteins ATM phosphorylation. NF‐κB activator TNF‐α and the ATM inhibitor Ku55933 offset the regulatory effect of eIF2α on the expression of radiation‐induced cell apoptosis‐related proteins and the G2/M phase arrest‐related proteins. These data indicate that PERK regulates radioresistance in oropharyngeal carcinoma through NF‐kB activation‐mediated phosphorylation of eIF2α, enhancing X‐ray‐induced activation of DNA DSB repair, cell apoptosis inhibition and G2/M cell cycle arrest.     

cIAP2 promotes gallbladder cancer invasion and lymphangiogenesis by activating the NF‐κB pathway

Several studies have produced contradictory findings about the prognostic implications for inhibitor of apoptosis proteins (IAP) in different types of cancer. Cellular inhibitor of apoptosis 2 (cIAP2/BIRC) is one of the most extensively characterized human IAP. To date, no studies have focused on the expression level of cIAP2 in human gallbladder cancer (GBC), and the mechanism of cIAP2 in GBC invasion and lymphangiogenesis remains unclear. Therefore, in the present study, cIAP2 expression in GBC was detected using quantitative real‐time polymerase chain reaction and immunohistochemistry, and the relationship between cIAP2 levels in cancer tissues and the clinicopathological characteristics of patients was analyzed. The biological effect of cIAP2 in GBC cells was tested using the Cell Counting Kit‐8 Assay, Transwell assays and the ability of human dermal lymphatic endothelial cells (HDLEC) to undergo tube formation. The role of cIAP2 in activating the NF‐κB pathway was determined using a dual‐luciferase reporter assay, immunofluorescence staining, western blotting and ELISA. Finally, an animal model was used to further confirm the role of cIAP2 in lymphangiogenesis. We showed that cIAP2 expression was elevated in human GBC tissues and correlated with a negative prognosis for patients. Moreover, cIAP2 was identified as a lymphangiogenic factor of GBC cells and, thus, promoted lymph node metastasis in GBC cells. Our study is the first to suggest that cIAP2 can promote GBC invasion and lymphangiogenesis by activating the NF‐κB pathway.     

Prognostic significance of p62/SQSTM1 subcellular localization and LC3B in oral squamous cell carcinoma

Background:

Autophagy is a programmed cell survival mechanism that has a key role in both physiologic and pathologic conditions. The relationship between autophagy and cancer is complex because autophagy can act as either a tumour suppressor or as a tumour promoter. The role of autophagy in oral squamous cell carcinoma (OSCC) is controversial. Several studies have claimed that either a high or low expression of autophagy-related proteins was associated with poor prognosis of OSCCs. The aims of the study were to compare autophagy in OSCCs, verrucous hyperplasias, and normal oral mucosas, and to inspect the prognostic role of autophagy in OSCCs.

Methods:

We used the autophagosome marker, LC3B, and autophagy flux marker, p62/SQSTM1 (p62), by using immunohistochemistry, and examined p62 mRNA by RNA in situ hybridization, to evaluate autophagy in 195 OSCCs, 47 verrucous hyperplasias, and 37 normal oral mucosas. The prognostic roles of LC3B and p62 protein expressions in OSCCs were investigated.

Results:

We discovered that the normal oral mucosa exhibited limited LC3B punctae and weak cytoplasmic p62 staining, whereas the OSCCs exhibited a marked increase in LC3B punctae and cytoplasmic p62 expression. The expression pattern of LC3B and cytoplasmic p62 of the verrucous hyperplasias were between normal oral mucosas and OSCCs. The normal oral mucosas, verrucous hyperplasias, and OSCCs presented no differences in nuclear p62 expression and the p62 mRNA level. p62 mRNA expression was elevated in a minority of cases. High p62 mRNA expression was associated with high p62 protein expression in the cytoplasm. Increased LC3B punctae, high cytoplasmic p62, and low nuclear p62 expressions in OSCCs were associated with aggressive clinicopathologic features and unfavourable prognosis. In addition, low nuclear p62 expression was an independent prognostic factor for overall and disease-specific survival rates. Furthermore, we disclosed that high cytoplasmic p62 expression accompanied with either a low or high LC3B expression, which indicated autophagy impairment under basal or activated autophagic activity, was associated with aggressive behaviour in advanced OSCCs.

Conclusions:

We suggested that autophagy was altered during cancer initiation and progression. Autophagy impairment contributed to cancer progression in advanced OSCCs.