Withaferin A induces p53-dependent apoptosis by repression of HPV oncogenes and upregulation of tumor suppressor proteins in human cervical cancer cells

Cervical cancer is caused by human papilloma virus (HPV) expressing E6 and E7 oncoproteins, which are known to inactivate tumor suppressor proteins p53 and pRb, respectively. Repression of HPV oncoproteins would therefore result in reactivation of tumor suppressor pathways and cause apoptosis in cancer cells. Withaferin A (WA), the active component of the medicinal plant Withania Somnifera, has exhibited inhibitory effects against several different cancers. We examined the activity of WA on human cervical cancer cells in vitro and in vivo. WA potently inhibited proliferation of the cervical cancer cells, CaSki (IC(50) 0.45 ± 0.05 μM). Mechanistically, WA was found to (i) downregulate expression of HPV E6 and E7 oncoproteins, (ii) induce accumulation of p53, (iii) increase levels of p21(cip1/waf1) and its interaction with proliferating cell nuclear antigen (PCNA), (iv) cause G(2)/M cell cycle arrest, associated with modulation of cyclin B1, p34(cdc2) and PCNA levels, (v) decrease the levels of STAT3 and its phosphorylation at Tyr(705) and Ser(727) and (vi) alter expression levels of p53-mediated apoptotic markers-Bcl2, Bax, caspase-3 and cleaved PARP. In vivo, WA resulted in reduction of nearly 70% of the tumor volume in athymic nude mice with essentially similar trend in the modulation of molecular markers as in vitro. This is the first demonstration indicating that WA significantly downregulates expression of HPV E6/E7 oncogenes and restores the p53 pathway, resulting in apoptosis of cervical cancer cells. Together, our data suggest that WA can be exploited as a potent therapeutic agent for the treatment and prevention of cervical cancer without deleterious effects.     

HPV E6,p53与宫颈癌的关系及其在基因治疗中的应用

目前宫颈癌的发病率仍居全世界妇女常见恶性肿瘤的第二位.高危型人乳头瘤病毒（high-risk Human Papillomavirus,HR-HPV）感染是宫颈癌及其癌前病变最主要的诱因之一.抑癌基因p53能够有效地抑制细胞增殖和促进细胞凋亡,人类近半数的肿瘤都与p53基因的突变和失活有关,HPV E6蛋白降解p53蛋白,是宫颈癌发生的前提.选择性杀伤p53功能缺失的肿瘤细胞,同时保护正常细胞,发展HPV疫苗及联合用药是临床肿瘤免疫学家进行宫颈癌基因治疗的目标.     

p53 Loss synergizes with estrogen and papillomaviral oncogenes to induce cervical and breast cancers

Whereas the tumor suppressor p53 gene is frequently mutated in most human cancers, this is not the case in human papillomavirus (HPV)-associated cancers, presumably because the viral E6 oncoprotein inactivates the p53 protein. The ability of E6 to transform cells in tissue culture and induce cancers in mice correlates in part with its ability to inactivate p53. In this study, we compared the expression of the HPV16 E6 oncogene to the conditional genetic disruption of p53 in the context of a mouse model for cervical cancer in which estrogen is a critical cofactor. Nearly all of the K14Crep53(f/f) mice treated with estrogen developed cervical cancer, a stark contrast to its complete absence in like-treated K14E6(WT)p53(f/f) mice, indicating that HPV16 E6 must only partially inactivate p53. p53-independent activities of E6 also contributed to carcinogenesis, but in the female reproductive tract, these activities were manifested only in the presence of the HPV16 E7 oncogene. Interestingly, treatment of K14Crep53(f/f) mice with estrogen also resulted in mammary tumors after only a short latency, many of which were positive for estrogen receptor alpha. The majority of these mammary tumors were of mixed cell types, suggestive of their originating from a multipotent progenitor. Furthermore, a subset of mammary tumors arising in the estrogen-treated, p53-deficient mammary glands exhibited evidence of an epithelial to mesenchymal transition. These data show the importance of the synergy between estrogen and p53 insufficiency in determining basic properties of carcinogenesis in hormone-responsive tissues, such as the breast and the reproductive tract.     

Enhanced specificity of the p53 family proteins-based adenoviral gene therapy in uterine cervical cancer cells with E2F1-responsive promoters

p63 and p73, the p53 family proteins, are similar to p53 in many aspects: structural homology, transactivation of p53-downstream genes, and induction of apoptosis. Interestingly, they also differ from p53; in particular, they are not inhibited by viral oncoproteins such as HPV E6. This feature would be an advantage over p53 in HPV-associated cancers and therefore, we evaluated the therapeutic potentials of various p53 family proteins (p73alpha, p73beta, p63alpha and p63gamma) against HPV-infected cervical cancers. In clonogenic assay, exogenous expression of p73alpha, p73beta and p63gamma inhibited the colony formation of HPV-positive cervical cancer cells under G418- selection while p53 could not. Recombinant adenoviruses Ad/CMVp73alpha, Ad/CMVp73beta and Ad/CMVp63gamma induced potent apoptosis in all infected cervical cancers (CasKi, SiHa, HeLa, C33A, SNU682, SNU17, SNU1005, SNU703), irrespective of their HPV-infection status. Unfortunately however, Ad/CMVp73alpha, Ad/CMVp73beta, and Ad/CMVp63gamma inhibited also normal cells such as endothelial cells, fibroblasts, and keratinocytes thus, tumorspecific promoter was indispensable to the p53 family proteins-based therapy. Here we report a stringent tumor killing by p73beta in combination with ESM6, a synthetic promoter targeting the DNA tumor virusassociated cancers. Recombinant adenoviruses encoding p73beta by ESM6 (Ad/ESM6p73beta and Ad/ESM6p73bENH) expressed p73beta and induced apoptosis only in the cancer cells but not in normal cells. Collectively, we suggest that the p53 family proteins are potent therapeutic agents for HPV-associated uterine cervical cancers and ESM6 mediated expression of the p53 family proteins would be a safe and strong tumor targeting strategy.     

The cytotoxicity of chemotherapy drugs varies in cervical cancer cells depending on the p53 status

Metastatic cervical cancer remains a clinical problem. The development of more efficient treatment modalities and the optimal use of chemo- and radiotherapy require better understanding of their impact on regulation of cell survival and apoptosis, but the issue is insufficiently explored. Human papillomavirus (HPV) E6 protein is present in nearly all cervical cancers, targeting the p53 tumor suppressor protein for degradation. We studied the role of p53 in mediating the cytotoxic effects of 31 chemotherapy compounds. SiHa cervical cancer cells, carrying wild type (wt) p53 and HPV16 genome, were stably transfected with dominant negative p53 (DDp53) or ectopic HPV16 E6 in order to inhibit p53 function. The effects of chemotherapy drugs in these cells were compared to the effects in cells retaining endogenous residual p53 activity. Twenty-ight out of 31 drugs reduced the amount of E6 mRNA, but only some induced marked p53 activation. In clonogenic cell survival experiments, the presence of DDp53 and ectopic E6 either increased or decreased cytotoxicity, depending on the drug. The decrease of E6 mRNA was necessary, but not sufficient event in the p53 activation by chemotherapy. The impact of p53 on clonogenic cell survival varied between 0-60%, indicating that p53 plays an important, but not crucial role in response to chemotherapy. The finding that chemosensitivity varies depending on the p53 status may have clinical implications, since early stage cervical cancer cells usually carry wt p53, whereas p53 mutations are frequently found in advanced disease.     

Therapeutic potential of an adenovirus expressing p73 beta, a p53 homologue, against human papilloma virus positive cervical cancer in vitro and in vivo

Human papilloma virus (HPV) infection is the most important risk factor for cervical cancer development. p53 based gene therapy is not suitable for cervical cancer because HPV oncoprotein E6 inactivates p53 protein by targeting it for ubiquitin mediated degradation. Here we evaluated the efficiency of Ad-p73, a replication deficient adenovirus expressing p73beta a p53 homologue, to inhibit the growth of HPV positive cervical cancer cells in vitro using tissue culture system and in vivo using human xenografts in nude mice. Ad-p73, but not Ad-p53 (p53 adenovirus), inhibited the growth in vitro of three different HPV positive cervical cancer cell lines, HeLa, ME180, and SiHa, efficiently, which correlated with stable expression of functional p73 protein. However, the growth of a HPV negative cervical cancer cell line, C33A, was inhibited equally by both Ad-p73 and Ad-p53. In addition, we show that Ad-p73 preinfected HeLa cells and HCT116 E6 cells, an E6 stable cell line, failed to form tumors in nude mice unlike Ad-p53 or Ad-LacZ preinfected cells. Moreover, Ad-p73, but not Ad-p53, inhibited completely the growth of already established tumors of HeLa or HCT116 E6 cells. Furthermore, the ability of p73 to inhibit the growth of these tumors correlated with the stable expression of p73 protein with the concomitant induction of its target gene p21(WAF1/CIP1) and induction of apoptosis in tumor cells. These results suggest that Ad-p73 inhibits efficiently the growth in vitro and tumorigenicity and tumor growth in vivo of HPV positive cervical cancer cells and that p73-based approach should be explored as a potential therapeutic model for the treatment of cervical cancer.     

Enhanced efficacy against cervical carcinomas through polymeric micelles physically incorporating the proteasome inhibitor MG132

Treatment of recurrent or advanced cervical cancer is still limited, and new therapeutic choices are needed for improving prognosis and quality of life of patients. Because human papilloma virus (HPV) infection is critical in cervical carcinogenesis, with the E6 and E7 oncogenes of HPV degrading tumor suppressor proteins through the ubiquitin proteasome system, the inhibition of the ubiquitin proteasome system appears to be an ideal target to suppress the growth of cervical tumors. Herein, we focused on the ubiquitin proteasome inhibitor MG132 (carbobenzoxy‐Leu‐Leu‐leucinal) as an anticancer agent against cervical cancer cells, and physically incorporated it into micellar nanomedicines for achieving selective delivery to solid tumors and improving its in vivo efficacy. These MG132‐loaded polymeric micelles (MG132/m) showed strong tumor inhibitory in vivo effect against HPV‐positive tumors from HeLa and CaSki cells, and even in HPV‐negative tumors from C33A cells. Repeated injection of MG132/m showed no significant toxicity to mice under analysis by weight change or histopathology. Moreover, the tumors treated with MG132/m showed higher levels of tumor suppressing proteins, hScrib and p53, as well as apoptotic degree, than tumors treated with free MG132. This enhanced efficacy of MG132/m was attributed to their prolonged circulation in the bloodstream, which allowed their gradual extravasation and penetration within the tumor tissue, as determined by intravital microscopy. These results support the use of MG132 incorporated into polymeric micelles as a safe and effective therapeutic strategy against cervical tumors.     

Efficient growth inhibition of HPV 16 E6-expressing cells by an adenovirus-expressing p53 homologue p73beta

Tumor suppressor p53 functions are downregulated in most cervical cancers, because the product of human papilloma virus (HPV) oncogene E6 binds to and inactivates p53 by promoting its degradation. p73, a p53 homologue, is similar to p53 in structure and function but yet not degraded by HPV E6 gene product. In this study, we have developed a replication-deficient recombinant adenovirus, which expresses p73beta (Ad-p73). Infection of human cancer cells with Ad-p73 results in several fold increase of p73beta levels as well as its known target genes like p21(WAF1/CIP1). Ad-p73-infected cells showed reduced cellular DNA synthesis, arrest in G1 phase of cell cycle and induction of apoptosis. Ad-p73 inhibited the growth of cancer cells of different types. More importantly, Ad-p73 inhibited the growth of cell lines carrying HPV E6 gene, which was introduced by stable integration, more efficiently in comparison to an Ad-p53. Furthermore, Ad-p73 also inhibited the growth of HeLa cells, a cell line derived from cervical cancer, very efficiently. The ability of Ad-p73 to inhibit the growth of HPV E6-expressing cells and HeLa cells correlated with the stable expression of functional p73 in the presence of E6. These results suggest that Ad-p73 could be used as a potential gene therapy agent against cervical cancer.     

MiR-125a suppresses tumor growth,invasion and metastasis in cervical cancer by targeting STAT3

MiR-125a has been characterized as a tumor suppressor in several cancers. However, the role of miR-125a in cervical cancer is unknown. In this study, we found the expression of miR-125a was downregulated in cervical cancer patients, and negatively correlated with the tumor size, FIGO stage, and preoperative metastasis. Kaplan-Meier analysis showed that miR-125a expression predicted favorable outcome for cervical cancer patients. Dual luciferase assays identified the STAT3 gene as a novel direct target of miR-125a. Functional studies showed that miR-125a overexpression significantly suppressed the growth, invasion and epithelial-mesenchymal transition (EMT) of cervical cancer cells both in vitro and in vivo via decreasing STAT3 expression. Moreover, miR-125a conferred to G2/M cell cycle arrest, accompanied by inhibition of several G2/M checkpoint proteins. Mechanistically, inactivation of miR-125a during cervical carcinogenesis was caused by HPV suppression of p53 expression. Clinically, STAT3, the expression of which, predicted poorer outcome, was inversely correlated with miR-125a in cervical cancer. These data highlight the importance of miR-125a in the cell proliferation and progression of cervical cancer, and indicate that miR-125a may be a useful therapeutic target for cervical cancer.     

Proteasome inhibitor MG132 sensitizes HPV-positive human cervical cancer cells to rhTRAIL-induced apoptosis

In cervical carcinogenesis, the p53 tumor suppressor pathway is disrupted by HPV (human papilloma virus) E6 oncogene expression. E6 targets p53 for rapid proteasome-mediated degradation. We therefore investigated whether proteasome inhibition by MG132 could restore wild-type p53 levels and sensitize HPV-positive cervical cancer cell lines to apoptotic stimuli such as rhTRAIL (recombinant human TNF-related apoptosis inducing ligand). In a panel of cervical cancer cell lines, CaSki was highly, HeLa intermediate and SiHa not sensitive to rhTRAIL-induced apoptosis. MG132 strongly sensitized HeLa and SiHa to rhTRAIL-induced apoptosis in a caspase-dependent and time-dependent manner. MG132 massively induced TRAIL receptor DR4 and DR5 membrane expression in HeLa, whereas in SiHa only DR5 membrane expression was upregulated from almost undetectable to high levels. Antagonistic DR4 antibody partially inhibited apoptosis induction by rhTRAIL and MG132 in HeLa but had no effect on apoptosis in SiHa. Inhibition of E6-mediated p53 proteasomal degradation by MG132 resulted in elevated levels of active p53 as demonstrated by p53 small interfering RNA (siRNA) sensitive p21 upregulation. Although p53 siRNA partially inhibited MG132-induced DR5 upregulation in HeLa and SiHa, no effect on rhTRAIL-induced apoptosis was observed. MG132 plus rhTRAIL enhanced caspase 8 and caspase 3 activation and concomitant cleavage of X-linked inhibitor of apoptosis (XIAP), particularly in HeLa. In addition, caspase 9 activation was only observed in HeLa. Downregulation of XIAP using siRNA in combination with rhTRAIL induced high levels of apoptosis in HeLa, whereas MG132 had to be added to the combination of XIAP siRNA plus rhTRAIL to induce apoptosis in SiHa. In conclusion, proteasome inhibition sensitized HPV-positive cervical cancer cell lines to rhTRAIL independent of p53. Our results indicate that not only DR4 and DR5 upregulation but also XIAP inactivation contribute to rhTRAIL sensitization by MG132 in cervical cancer cell lines. Combining proteasome inhibitors with rhTRAIL may be therapeutically useful in cervical cancer treatment.     

Chemoradiation of cervical cancer cells: targeting human papillomavirus E6 and p53 leads to either augmented or attenuated apoptosis depending on the platinum carrier ligand

Recent clinical trials comparing concurrent chemotherapy and radiation with radiation alone in cervical cancer have shown that chemoradiation reduces the risk of death by 30-50%. Despite the clinical success, treatment responses at the cellular level are still inadequately explored. A key event in cervical carcinogenesis is the disruption of p53 tumor suppressor pathway by human papillomavirus (HPV) E6 oncogene. We found that regardless of the HPV type in SiHa (HPV 16+) CaSki (HPV 16+), HeLa (HPV 18+), and UT-DEC-1 (HPV 33+) cell lines, cisplatin, carboplatin, and a novel platinum compound, oxaliplatin, activated a p53 reporter and reduced the HPV E6 mRNA. Carboplatin and oxaliplatin treatment led also to stabilization of p53, whereas none of the platinums changed p73 levels. After irradiation (IR) alone, a decrease in HPV E6 mRNA levels and an activation of the p53-reporter were detected in SiHa, CaSki, and HeLa cells, but not in UT-DEC-1 cells. Concomitant platinum treatment and IR led to poly(ADP-ribose) polymerase cleavage as a sign of caspase-3 activation and apoptosis. Clonogenic survival was enhanced by expressing a dominant negative p53 or ectopic HPV16 E6 in SiHa and HeLa cells treated with IR, carboplatin, or oxaliplatin or with a combination of IR + carboplatin or oxaliplatin. In contrast, dominant negative p53 or ectopic HPV 16 E6 sensitized the cells to cisplatin. Pt chemotherapeutics and radiation had a synergistic cytotoxic effect as determined by Bliss independence criterion. Taken together, p53 has a significant role in the cellular response to chemoradiation treatment in cervical cancer cell lines, but p53 activity may have a dramatically different effect on cell survival depending on the platinum carrier ligand.     

Homozygous arginine at codon 72 of p53 has no prognostic significance in cervical cancer.

Mutation of p53, a tumour suppressor gene, is uncommon in cervical cancer but the detection of human papillomavirus (HPV) DNA in cervical cancer is common. The findings of increased susceptibility to degradation of p53 by E6 protein of HPV16/18 in cervical cancer with homozygous arginine at codon 72 (HA72) of p53 led to this study on whether cervical cancers with HA72 were more aggressive with the increase in the rate of loss of p53 function. In 102 cervical cancers, 76.5% were HPV16/18 positive and 30% had HA72. No survival difference was detected between HA72 and non-HA72 tumours irrespective of HPV16/18 status. Furthermore, the detection of HPV16/18 in cervical cancer was found not to be of prognostic significance in this study.     

BAG3 down-modulation sensitizes HPV18 HeLa cells to PEITC-induced apoptosis and restores p53

BAG3 is a multi-functional component of tumor cell pro-survival machinery, and its biological functions have been largely associated to proteasome system. Here, we show that BAG3 down-modulation resulted in reduced cell viability and enhanced PEITC-induced apoptosis largely more extensively in HeLa (HPV18⁺) rather than in C33A (HPV⁻) cervical carcinoma cell lines. Moreover, we demonstrate that BAG3 suppression led to a decrease of viral E6 oncoprotein and a concomitant recovery of p53 tumor suppressor, the best recognized target of E6 for proteasome degradation. E6 and p53 expression were modulated at protein level, since their respective mRNAs were unaffected. Taken together our findings reveal a novel role for BAG3 as host protein contributing to HPV18 E6-activated pro-survival strategies, and suggest a possible relevance of its expression levels in drug/radiotherapy-resistance of HPV18-bearing cervical carcinomas.     

p53-independent abrogation of a postmitotic checkpoint contributes to human papillomavirus E6-induced polyploidy

Polyploidy is often an early event during cervical carcinogenesis, and it predisposes cells to aneuploidy, which is thought to play a causal role in tumorigenesis. Cervical and anogenital cancers are induced by the high-risk types of human papillomavirus (HPV). The HPV E6 oncoprotein induces polyploidy in human keratinocytes, yet the mechanism is not known. It was believed that E6 induces polyploidy by abrogating the spindle checkpoint after mitotic stress. We have tested this hypothesis using human keratinocytes in which E6 expression induces a significant amount of polyploidy. We found that E6 expression does not affect the spindle checkpoint. Instead, we provide direct evidence that E6 is capable of abrogating the subsequent G(1) arrest after adaptation of the mitotic stress. E6 targets p53 for degradation, and previous studies have shown an important role for p53 in modulation of the G(1) arrest after mitotic stress. Importantly, we have discovered that E6 mutants defective in p53 degradation also induce polyploidy, although with lower efficiency. These results suggest that E6 is able to induce polyploidy via both p53-dependent and p53-independent mechanisms. Therefore, our studies highlight a novel function of HPV E6 that may contribute to HPV-induced carcinogenesis and improve our understanding of the onset of the disease.     

P53: an ubiquitous target of anticancer drugs

The p53 tumor suppressor can induce growth arrest, apoptosis and cell senescence. Not surprisingly, p53 is an appealing target for therapeutic intervention. Although current anticancer agents do not directly interact with p53, these agents (including DNA damaging drugs, antimetabolites, microtubule-active drugs and inhibitors of the proteasome) cause accumulation of wt p53. Depending on the p53 status of cancer cells, diverse therapeutic strategies are under development. These include pharmacological rescue of mutant p53 function and reactivation of wt p53 in E6-expressing cells. For protection of normal cells, strategies range from abrogation of wt p53 induction, thereby decreasing the toxicity of DNA damaging agents, to activation of wt p53-dependent checkpoints, thereby protecting cells against cell cycle-dependent therapeutics.     

Selective targeting of HPV-16 E6/E7 in cervical cancer cells with a potent oncolytic adenovirus and its enhanced effect with radiotherapy in vitro and vivo

Recent studies have shown that oncolytic adenovirus specifically targeted tumor cells while sparing normal cells. Here, we report a novel E1A-mutant adenovirus (M6) with antisense HPV16 E6 E7 DNA inserted into the deleted 6.7 K/gp19 K region of E3. The target effects of M6 on HPV16-positive cervical cancer cells were evaluated in vivo and in vitro. By using cytopathic effect (CPE) and viral replication assays, we verified M6 was competent to selectively replicate in cervical cancer cells in vitro. Moreover, we found infection of M6 was able to inhibit the expression of HPV16 E6 and E7 oncogenes and induce apoptosis of HPV16-positive cervical cancer cells. Further analysis in vitro revealed that the invasive ability of SiHa cells was significantly inhibited by M6. To determine if M6 synergized with radiotherapy-induced anti-tumor activity against HPV16-related cancer cells, we transfected SiHa cells with M6 followed by a single exposure to radiation. A significantly suppression of cell growth and induced apoptosis was observed in SiHa cells received M6 transfection combined with radiotherapy. Animal experiments showed that M6 transfection notably improved the survival of tumor-bearing mice in combination with radiotherapy, much superior to that of those treated by Adv5/dE1A plus radiation or M6 alone. These findings indicated the anti-tumoral efficacy of M6 on HPV16-positive cervical cancer cells and its synergic therapeutic application in radiation for cervical cancer.