Qinglei Gao

Genome-wide profiling of HPV integration in cervical cancer identifies clustered genomic hot spots and a potential microhomology-mediated integration mechanism

Human papillomavirus (HPV) integration is a key genetic event in cervical carcinogenesis. By conducting whole-genome sequencing and high-throughput viral integration detection, we identified 3,667 HPV integration breakpoints in 26 cervical intraepithelial neoplasias, 104 cervical carcinomas and five cell lines. Beyond recalculating frequencies for the previously reported frequent integration sites POU5F1B (9.7%), FHIT (8.7%), KLF12 (7.8%), KLF5 (6.8%), LRP1B (5.8%) and LEPREL1 (4.9%), we discovered new hot spots HMGA2 (7.8%), DLG2 (4.9%) and SEMA3D (4.9%). Protein expression from FHIT and LRP1B was downregulated when HPV integrated in their introns. Protein expression from MYC and HMGA2 was elevated when HPV integrated into flanking regions. Moreover, microhomologous sequence between the human and HPV genomes was significantly enriched near integration breakpoints, indicating that fusion between viral and human DNA may have occurred by microhomology-mediated DNA repair pathways. Our data provide insights into HPV integration-driven cervical carcinogenesis.

Resistance to chemotherapy-induced apoptosis via decreased caspase-3 activity and overexpression of antiapoptotic proteins in ovarian cancer

GoalsResistance to cisplatin is the main reason for treatment failure in ovarian cancer. Apoptosis is the main mechanism of action of most cancer chemotherapeutic agents. The apoptosis-associated proteins expressed in cisplatin-sensitive (A2780, COC1) and -resistant (A2780/DDP, COC1/DDP) ovarian cancer cell lines, as well as their effects on caspase-3 activity in these cells, were studied by reverse transcriptase polymerase chain reaction and Western blot analysis.MethodsThe apoptotic ratios of A2780, COC1, A2780/DDP, and COC1/DDP cells after treatment with cisplatin were measured by flow cytometry.ResultsExpression of Bcl-2 and Bcl-XL in A2780/DDP and COC1/DDP cells was significantly higher than that in A2780 and COC1 cells, respectively. Expression of Bax and Bcl-Xs did not differ in cisplatin-resistant and -sensitive cells. Caspase-3 activity was reduced markedly and apoptotic ratios were significantly lower in A2780/DDP and COC1/DDP cells than in A2780 and COC1 cells after treatment with cisplatin.ConclusionWe conclude that overexpression of antiapoptotic proteins Bcl-2 and Bcl-XL and down-regulation of caspase-3 activity may be associated with cisplatin resistance in human ovarian cancer.

Adjuvant Adenovirus-Mediated Delivery of Herpes Simplex Virus Thymidine Kinase Administration Improves Outcome of Liver Transplantation in Patients with Advanced Hepatocellular Carcinoma

Purpose: Previous poor results of liver transplantation (LT) have been confirmed in patients with advanced hepatocellular carcinoma (HCC). Adenovirus-mediated delivery of herpes simplex virus thymidine kinase (ADV-TK) therapy is an established adjuvant treatment in cancer, and we evaluated its potential as an adjuvant treatment for HCC patients who underwent LT. Experimental Design: Forty-five HCC patients with tumors >5 cm in diameter participated in the study over a follow-up period of 50 months. Among these patients, 22 received LT only, and 23 received LT combined with ADV-TK therapy. All HCC patients enrolled in this study had tumors >5 cm in diameter and no metastasis in lungs or bones was detected by computed tomography or magnetic resonance imaging scans. Results: The recurrence-free survival and the overall survival in the LT plus ADV-TK therapy group were 43.5% and 69.6%, respectively, at 3 years; both values were significantly higher than those in the LT-only group (9.1% and 19.9%, respectively). In the nonvascular invasion subgroup, overall survival was 100% and recurrence-free survival was 83.3% in the patients receiving LT plus ADV-TK, significantly higher than the patients receiving LT only. Conclusions: HCC patients with no vascular invasion could be selected for LT followed by adjuvant ADV-TK therapy, regardless of intrahepatic huge or diffuse tumor. We propose that the current criteria for LT based on tumor size may be expanded if accompanied by ADV-TK therapy due to improved prognosis.

Silencing of the STAT3 signaling pathway reverses the inherent and induced chemoresistance of human ovarian cancer cells.

Ovarian cancer is the leading cause of gynecologic cancer deaths among women. Although platinum-based chemotherapy is the first-line treatment for human ovarian cancer, chemoresistance remains a major obstacle to successful treatment, and there are currently no approved molecularly targeted therapies. Recent evidence indicates that signal transducer and activator of transcription-3 (STAT3) is a determinant of chemoresistance and is related to tumor recurrence in a large number of solid malignancies. In this study, we demonstrated that high levels of pSTAT3 were associated with chemoresistance in human ovarian cancer cells. Targeting STAT3 by siRNA technology markedly enhanced cisplatin-induced apoptosis in cisplatin-resistant ovarian cancer cells that expressed a high level of pSTAT3. Interleukin-6 (IL-6) could induce STAT3 activation in cisplatin-sensitive ovarian cancer cells and led to protection against cisplatin. The STAT3 siRNA treatment also blocked IL-6-induced STAT3 phosphorylation, resulting in the attenuation of the anti-apoptotic activity of IL-6. We found that the combination of cisplatin and STAT3 siRNA resulted in the collapse of the mitochondrial membrane potential, attenuated the expression of Bcl-xL and Bcl-2, and increased the release of cytochrome C and expression of Bax. Taken together, these results suggest that the pharmacological inhibition of STAT3 may be a promising therapeutic strategy for the management of chemoresistance in ovarian cancer.

TALEN-mediated targeting of HPV oncogenes ameliorates HPV-related cervical malignancy

Persistent HPV infection is recognized as the main etiologic factor for cervical cancer. HPV expresses the oncoproteins E6 and E7, both of which play key roles in maintaining viral infection and promoting carcinogenesis. While siRNA-mediated targeting of E6 and E7 transcripts temporarily induces apoptosis in HPV-positive cells, it does not eliminate viral DNA within the host genome, which can harbor escape mutants. Here, we demonstrated that specifically targeting E6 and E7 within host DNA with transcription activator-like effector nucleases (TALENs) induces apoptosis, inhibits growth, and reduces tumorigenicity in HPV-positive cell lines. TALEN treatment efficiently disrupted E6 and E7 oncogenes, leading to the restoration of host tumor suppressors p53 and retinoblastoma 1 (RB1), which are targeted by E6 and E7, respectively. In the K14-HPV16 transgenic mouse model of HPV-driven neoplasms, direct cervical application of HPV16-E7-targeted TALENs effectively mutated the E7 oncogene, reduced viral DNA load, and restored RB1 function and downstream targets transcription factor E2F1 and cycling-dependent kinase 2 (CDK2), thereby reversing the malignant phenotype. Together, the results from our study suggest that TALENs have potential as a therapeutic strategy for HPV infection and related cervical malignancy.

Abrogation of constitutive Stat3 activity circumvents cisplatin resistant ovarian cancer

The aim of the present study was to investigate the role of Stat3 in cisplatin resistant ovarian cancer. It was first demonstrated that higher activated Stat3 was detected in cisplatin-resistant ovarian cancer cell lines. To provide evidence that supported the hypothesis that phosphorylated-Stat3 expression may promote cisplatin resistance, ectopic Stat3 was expressed by IL-6 stimulation that partially abrogates Stat3, as opposed to the knock-down of Stat3 by specific siRNA that restores cisplatin sensitivity against ovarian cancer cells. This hypothesis was further confirmed by clinical tumor specimens of ovarian cancer obtained from patients with cisplatin-resistance. Based on these premises, Stattic, an effective small molecular inhibitor of Stat3, was used to inhibit Stat3 activation. The data presented here show that Stattic restored the sensitivity to cisplatin in chemoresistant ovarian cancer by significant reductions in the expression of the anti-apoptosis protein Bcl-2, Bcl-XL, Survivin protein and phosphorylated-Akt levels. Consistent with these observations, this experiment demonstrated the first evidence of Stattic circumvented cisplatin resistance of orthotopic xenograft ovarian cancer in vivo. Altogether, these findings emphasize the importance of Stat3 in cisplatin resistance in ovarian cancer and provide a further impetus to clinically evaluate biological modifiers that may circumvent cisplatin resistance in patients with chemoresistant ovarian cancer.

Inhibition of ovarian cancer metastasis by adeno-associated virus-mediated gene transfer of nm23H1 in an orthotopic implantation model

Ovarian cancer is one of the most threatening malignant tumors in females due to the frequent occurrence of metastasis that precedes diagnosis. The present study explored the possibility of preventing ovarian cancer metastasis by promoting nm23H1 expression through adeno-associated virus (AAV)-mediated gene transfer. A cell line of high metastatic potential, SW626-M4, was derived by in vivo selection and used to establish an ovarian cancer metastasis model in the mouse. Liver metastasis and animal survival time were measured after transfer of a recombinant adeno-associated viral vector expressing nm23H1 (AAV-nm23H1) into the aforementioned model. Intraperitoneal injection of AAV-nm23H1 into this orthotopic implantation model of ovarian cancer resulted in (1) expression of the exogenous gene in more than 95% of tumor cells in situ in nude mice; (2) a 60% reduction in the number of animals developing liver metastases; and (3) a 35-day prolongation of median survival time compared with the untreated host group. In conclusion, the results support the feasibility of induction of nm23H1 expression through gene transfer as a therapeutic strategy for preventing metastases and prolonging host survival time, and indicate that AAV vectors deserve attention in the design of future gene therapy approaches to achieving long-term expression of curative genes in vivo.

Novel Oncolytic Adenovirus Selectively Targets Tumor-Associated Polo-Like Kinase 1 and Tumor Cell Viability

Purpose: Polo-like kinase 1 (plk1) is a serine/threonine protein kinase essential for multiple mitotic processes. Previous observations have validated plk1 as a promising therapeutic target. Despite being conceptually attractive, the potency and specificity of current plk1-based therapies remain limited. We sought to develop a novel plk1-targeting strategy by constructing an oncolytic adenovirus to selectively silence plk1 in tumor cells. Experimental Design: Two artificial features were engineered into one wild-type adenovirus type 5 (wt-Adv5) genome to generate a new oncolytic adenovirus (M1). First, M1 contains a 27-bp deletion in E1A region, which confers potent, oncolytic efficacy. Second, M1 is armed with a fragment of antisense plk1 cDNA that substitutes the E3 region encoding 6.7K and gp19K. In this design, tumor-selective replication of M1 would activate the native adenovirus E3 promoters to express the antisense plk1 cDNA preferentially in tumor cells and silence tumor-associated plk1 protein. Results: By virtue of combining oncolysis with plk1 targeting, M1 exhibited potent antitumoral efficacy in vitro and in vivo. Systemic administration of M1 plus cisplatin induced complete tumor regression in 80% of orthotopic hepatic carcinoma model mice that were otherwise resistant to cisplatin and disseminated metastases. Conclusions: Coupling plk1 targeting with oncolysis had shown superior antitumor efficacy. Present findings would benefit the development of novel oncolytic adenoviruses generally applicable to a wide range of molecule-based therapeutics.

SIX1 Promotes Tumor Lymphangiogenesis by Coordinating TGFβ Signals That Increase Expression of VEGF-C

Lymphatic vessels are one of the major routes for the dissemination of cancer cells. Malignant tumors release growth factors such as VEGF-C to induce lymphangiogenesis, thereby promoting lymph node metastasis. Here, we report that sine oculis homeobox homolog 1 (SIX1), expressed in tumor cells, can promote tumor lymphangiogenesis and lymph node metastasis by coordinating with TGFβ to increase the expression of VEGF-C. Lymphangiogenesis and lymph node metastasis in cervical cancer were closely correlated with higher expression of SIX1 in tumor cells. By enhancing VEGF-C expression in tumor cells, SIX1 could augment the promoting effect of tumor cells on the migration and tube formation of lymphatic endothelial cells (LEC) in vitro and lymphangiogenesis in vivo. SIX1 enhanced TGFβ-induced activation of SMAD2/3 and coordinated with the SMAD pathway to modulate VEGF-C expression. Together, SIX1 and TGFβ induced much higher expression of VEGF-C in tumor cells than each of them alone. Despite its effect in promoting VEGF-C expression, TGFβ could inhibit lymphangiogenesis by directly inhibiting tube formation by LECs. However, the increased production of VEGF-C not only directly promoted migration and tube formation of LECs but also thwarted the inhibitory effect of TGFβ on LECs. That is, tumor cells that expressed high levels of SIX1 could promote lymphangiogenesis and counteract the negative effects of TGFβ on lymphangiogenesis by increasing the expression of VEGF-C. These findings provide new insights into tumor lymphangiogenesis and the various roles of TGFβ signaling in tumor regulation. Our results also suggest that SIX1/TGFβ might be a potential therapeutic target for preventing lymph node metastasis of tumor.

Elaiophylin, a novel autophagy inhibitor, exerts antitumor activity as a single agent in ovarian cancer cells.

Currently, targeting the autophagic pathway is regarded as a promising new strategy for cancer drug discovery. Here, we screened the North China Pharmaceutical Group Corporations pure compound library of microbial origin using GFP-LC3B-SKOV3 cells and identified elaiophylin as a novel autophagy inhibitor. Elaiophylin promotes autophagosome accumulation but blocks autophagic flux by attenuating lysosomal cathepsin activity, resulting in the accumulation of SQSTM1/p62 in various cell lines. Moreover, elaiophylin destabilizes lysosomes as indicated by LysoTracker Red staining and CTSB/cathepsin B and CTSD/ cathepsin D release from lysosomes into the cytoplasm. Elaiophylin eventually decreases cell viability, especially in combination with cisplatin or under hypoxic conditions. Furthermore, administration of a lower dose (2 mg/kg) of elaiophylin as a single agent achieves a significant antitumor effect without toxicity in an orthotopic ovarian cancer model with metastasis; however, high doses (8 mg/kg) of elaiophylin lead to dysfunction of Paneth cells, which resembles the intestinal phenotype of ATG16L1-deficient mice. Together, these results provide a safe therapeutic window for potential clinical applications of this compound. Our results demonstrate, for the first time, that elaiophylin is a novel autophagy inhibitor, with significant antitumor efficacy as a single agent or in combination in human ovarian cancer cells, establishing the potential treatment of ovarian cancer by this compound.