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Fig. S2 Flowcytometric analysis of CD49f and CD71 expression on SiHa (HPV16+ve), HeLa (HPV18+ve) and C33a (HPV-ve) parent cultures.

Purpose: Perturbation of keratinocyte differentiation by E6/E7 oncoproteins of high-risk human papillomaviruses that drive oncogenic transformation of cells in squamocolumnar junction of the uterine cervix may confer “stem-cell like” characteristics. However, the crosstalk between E6/E7 and stem cell signaling during cervical carcinogenesis is not well understood. We therefore examined the role of viral oncoproteins in stem cell signaling and maintenance of stemness in cervical cancer.Experimental Design: Isolation and enrichment of cervical cancer stem–like cells (CaCxSLCs) was done from cervical primary tumors and cancer cell lines by novel sequential gating using a set of functional and phenotypic markers (ABCG2, CD49f, CD71, CD133) in defined conditioned media for assessing sphere formation and expression of self-renewal and stemness markers by FACS, confocal microscopy, and qRT-PCR. Differential expression level and DNA-binding activity of Notch1 and its downstream targets in CaCxSLCs as well as silencing of HPVE6/Hes1 by siRNA was evaluated by gel retardation assay, FACS, immunoblotting, and qRT-PCR followed by in silico and in vivo xenograft analysis.Results: CaCxSLCs showed spheroid-forming ability, expressed self-renewal and stemness markers Oct4, Sox2, Nanog, Lrig1, and CD133, and selectively overexpressed E6 and HES1 transcripts in both cervical primary tumors and cancer cell lines. The enriched CaCxSLCs were highly tumorigenic and did recapitulate primary tumor histology in nude mice. siRNA silencing of HPVE6 or Hes1 abolished sphere formation, downregulated AP-1-STAT3 signaling, and induced redifferentiation.Conclusions: Our findings suggest the possible mechanism by which HPVE6 potentially regulate and maintain stem-like cancer cells through Hes1. Clin Cancer Res; 22(16); 4170–84. ©2016 AACR.

Table 1: List of real-time RT-PCR primers used in the study Table 2: Result of cervical cancer cell line or primary culture derived xenografting in nude mice Table 3: Physical state of HPV16 DNA in CINI/II, CINIII/IV cervical cancer lesions and primary xenograft

Aberrant activation of Wnt/β-catenin axis occurs in several gastrointestinal malignancies due to inactivating mutations of adenomatous polyposis coli (in colorectal cancer) or activating mutations of β-catenin itself [in hepatocellular carcinoma (HCC)]. These lead to β-catenin stabilization, increase in β-catenin/T-cell factor (TCF)-mediated transcriptional activation, and target gene expression, many of which are involved in tumor progression. While studying pharmaceutical agents that can target β-catenin in cancer cells, we observed that the plant compound berberine (BBR), a potent activator of AMP-activated protein kinase (AMPK), can reduce β-catenin expression and downstream signaling in HCC cells in a dose-dependent manner. More in-depth analyses to understand the mechanism revealed that BBR-induced reduction of β-catenin occurs independently of AMPK activation and does not involve transcriptional or post-translational mechanisms. Pretreatment with protein synthesis inhibitor cycloheximide antagonized BBR-induced β-catenin reduction, suggesting that BBR affects β-catenin translation. BBR treatment also antagonized mammalian target of rapamycin (mTOR) activity and was associated with increased recruitment of eukaryotic translation initiation factor 4E-binding protein (4E-BP) 1 in the translational complex, which was revealed by 7-methyl-cap-binding assays, suggesting inhibition of cap-dependent translation. Interestingly, knocking down 4E-BP1 and 4E-BP2 significantly attenuated BBR-induced reduction of β-catenin levels and expression of its downstream target genes. Moreover, cells with 4E-BP knockdown were resistant to BBR-induced cell death and were resensitized to BBR after pharmacological inhibition of β-catenin. Our findings indicate that BBR antagonizes β-catenin pathway by inhibiting β-catenin translation and mTOR activity and thereby reduces HCC cell survival. These also suggest that BBR could be used for targeting HCCs that express mutated/activated β-catenin variants that are currently undruggable. SIGNIFICANCE STATEMENT: β-catenin signaling is aberrantly activated in different gastrointestinal cancers, including hepatocellular carcinoma, which is currently undruggable. In this study we describe a novel mechanism of targeting β-catenin translation via utilizing a plant compound, berberine. Our findings provide a new avenue of targeting β-catenin axis in cancer, which can be utilized toward the designing of effective therapeutic strategies to combat β-catenin-dependent cancers.

Incidence of human papillomavirus (HPV)-associated oral cancers is on the rise. However, epidemiological data of this subset of cancers are limited. Dental hospital poses a unique advantage in detection of HPV-positive oral malignancies. We assessed the utility of formalin-fixed paraffin-embedded (FFPE) tissues, which are readily available, for evaluation of high-risk HPV infection in oral cancer. For protocol standardization, we used 20 prospectively collected paired FFPE and fresh tissues of histopathologically confirmed oral cancer cases reported in Oral Medicine department of a dental hospital for comparative study. Only short PCRs (~ 200 bp) of DNA isolated using a modified xylene-free method displayed a concordant HPV result. For HPV analysis, we used additional 30 retrospectively collected FFPE tissues. DNA isolated from these specimens showed an overall 23.4% (11/47) HPV positivity with detection of HPV18. Comparison of HPV positivity from dental hospital FFPE specimens with overall HPV positivity of freshly collected oral cancer specimens (n = 55) from three cancer care hospitals of the same region showed notable difference (12.7%; 7/55). Further, cancer hospital specimens showed HPV16 positivity and displayed a characteristic difference in reported sub-sites and patient spectrum. Overall, using a xylene-free FFPE DNA isolation method clubbed with short amplicon PCR, we showed detection of HPV-positive oral cancer in dental hospitals.

Incidence of human papillomavirus (HPV)-associated oral cancers is on the rise. However, epidemiological data of this subset of cancers are limited. Dental hospital poses a unique advantage in detection of HPV-positive oral malignancies. We assessed the utility of formalin-fixed paraffin-embedded (FFPE) tissues, which are readily available, for evaluation of high-risk HPV infection in oral cancer. For protocol standardization, we used 20 prospectively collected paired FFPE and fresh tissues of histopathologically confirmed oral cancer cases reported in Oral Medicine department of a dental hospital for comparative study. Only short PCRs (~ 200 bp) of DNA isolated using a modified xylene-free method displayed a concordant HPV result. For HPV analysis, we used additional 30 retrospectively collected FFPE tissues. DNA isolated from these specimens showed an overall 23.4% (11/47) HPV positivity with detection of HPV18. Comparison of HPV positivity from dental hospital FFPE specimens with overall HPV positivity of freshly collected oral cancer specimens (n = 55) from three cancer care hospitals of the same region showed notable difference (12.7%; 7/55). Further, cancer hospital specimens showed HPV16 positivity and displayed a characteristic difference in reported sub-sites and patient spectrum. Overall, using a xylene-free FFPE DNA isolation method clubbed with short amplicon PCR, we showed detection of HPV-positive oral cancer in dental hospitals.

Incidence of human papillomavirus (HPV)–associated oral cancers is on the rise. However, epidemiological data of this subset of cancers is limited. Dental hospital pose a unique advantage in detection of HPV-positive oral malignancies. We assessed the utility of formalin-fixed, paraffin-embedded (FFPE) tissues, which are readily available, for evaluation of high-risk HPV infection in oral cancer. For protocol standardization, we used 20 prospectively-collected, paired FFPE and fresh tissues of histopathologically-confirmed oral cancer cases reported in Oral Medicine department of a dental hospital for comparative study. Only short-PCRs (~200bp) of DNA isolated using a modified xylene-free method displayed a concordant HPV result. For HPV analysis, we used additional 30 retrospectively-collected FFPE tissues. DNA isolated from these specimens showed an overall 23.4% (11/47) HPV positivity with detection of HPV18. Comparison of HPV positivity from dental hospital FFPE-specimens with overall HPV positivity of freshly-collected oral cancer specimens (n-55) from three cancer care hospitals of the same region showed notable difference (12.7%; 7/55). Further, cancer hospital specimens showed HPV16 positivity and displayed a characteristic difference in reported sub-sites and patient spectrum. Overall, using a xylene-free FFPE-DNA isolation method clubbed with short amplicon PCR, we showed detection of HPV-positive oral cancer in dental hospitals.

Hepatocellular carcinoma (HCC) is an inflammation-induced malignant disease of the liver. Abundant expression of immune checkpoint proteins has been reported in HCCs, which contribute to immune cell dysfunction and HCC progression. Immune checkpoint inhibitors as monotherapy or combination therapy have been approved by Food and Drug Administration for advanced HCCs. However, the median survival has not significantly improved, suggesting the need for exploring additional mechanisms to increase efficacy. Metabolic reprogramming is one of the mechanisms by which checkpoint proteins promote tumor growth and immune cell dysfunction. This review provides an insight into the role of immune checkpoint proteins on metabolic reprogramming in tumor and immune cells. An in-depth understating of these could help in the development of more efficacious and long-term therapies for HCC.

Cancer is a multi-step process and requires constitutive expression/activation of transcription factors (TFs) for growth and survival. Many of the TFs reported so far are critical for carcinogenesis. These include pro-inflammatory TFs, hypoxia-inducible factors (HIFs), cell proliferation and epithelial–mesenchymal transition (EMT)-controlling TFs, pluripotency TFs upregulated in cancer stem-like cells, and the nuclear receptors (NRs). Some of those, including HIFs, Myc, ETS-1, and β-catenin, are multifunctional and may regulate multiple other TFs involved in various pro-oncogenic events, including proliferation, survival, metabolism, invasion, and metastasis. High expression of some TFs is also correlated with poor prognosis and chemoresistance, constituting a significant challenge in cancer treatment. Considering the pivotal role of TFs in cancer, there is an urgent need to develop strategies targeting them. Targeting TFs, in combination with other chemotherapeutics, could emerge as a better strategy to target cancer. So far, targeting NRs have shown promising results in improving survival. In this review, we provide a comprehensive overview of the TFs that play a central role in cancer progression, which could be potential therapeutic candidates for developing specific inhibitors. Here, we also discuss the efforts made to target some of those TFs, including NRs.

Aberrant activation of Wnt/β-catenin axis occurs in several gastrointestinal malignancies due to inactivating mutations of adenomatous polyposis coli (in colorectal cancer) or activating mutations of β-catenin itself [in hepatocellular carcinoma (HCC)]. These lead to β-catenin stabilization, increase in β-catenin/T-cell factor (TCF)–mediated transcriptional activation, and target gene expression, many of which are involved in tumor progression. While studying pharmaceutical agents that can target β-catenin in cancer cells, we observed that the plant compound berberine (BBR), a potent activator of AMP-activated protein kinase (AMPK), can reduce β-catenin expression and downstream signaling in HCC cells in a dose-dependent manner. More in-depth analyses to understand the mechanism revealed that BBR-induced reduction of β-catenin occurs independently of AMPK activation and does not involve transcriptional or post-translational mechanisms. Pretreatment with protein synthesis inhibitor cycloheximide antagonized BBR-induced β-catenin reduction, suggesting that BBR affects β-catenin translation. BBR treatment also antagonized mammalian target of rapamycin (mTOR) activity and was associated with increased recruitment of eukaryotic translation initiation factor 4E–binding protein (4E-BP) 1 in the translational complex, which was revealed by 7-methyl-cap–binding assays, suggesting inhibition of cap-dependent translation. Interestingly, knocking down 4E-BP1 and 4E-BP2 significantly attenuated BBR-induced reduction of β-catenin levels and expression of its downstream target genes. Moreover, cells with 4E-BP knockdown were resistant to BBR-induced cell death and were resensitized to BBR after pharmacological inhibition of β-catenin. Our findings indicate that BBR antagonizes β-catenin pathway by inhibiting β-catenin translation and mTOR activity and thereby reduces HCC cell survival. These also suggest that BBR could be used for targeting HCCs that express mutated/activated β-catenin variants that are currently undruggable. SIGNIFICANCE STATEMENT: β-catenin signaling is aberrantly activated in different gastrointestinal cancers, including hepatocellular carcinoma, which is currently undruggable. In this study we describe a novel mechanism of targeting β-catenin translation via utilizing a plant compound, berberine. Our findings provide a new avenue of targeting β-catenin axis in cancer, which can be utilized toward the designing of effective therapeutic strategies to combat β-catenin–dependent cancers.

Lymphatic filariasis (LF) affects 120 million people around the world and another 856 million people are at risk of acquiring the infection. Mass Drug Administration (MDA) spearheaded by the World Health Organization is the only current strategy to control this infection. Recent reports suggest that despite several rounds of MDA, elimination has not been achieved and there is a need for more stringent control strategies for control of LF. An effective prophylactic vaccine combined with MDA has significant potential. Initial trials using a prophylactic trivalent recombinant Brugia malayi heat shock protein 12.6, abundant larval transcript -2 and tetraspanin large extra-cellular loop (rBmHAT) vaccine developed in our laboratory conferred only 35% protection in macaques. Therefore, the focus of the present study was to improve the current vaccine formulation to obtain better protection in non-human primates. We made two modifications to the current formulation: (i) the addition of another antigen, thioredoxin peroxidase-2 (TPX-2) to make it a tetravalent vaccine (rBmHAXT) and (ii) the inclusion of an adjuvant; AL019 (alum plus glucopyranosyl lipid adjuvant-stable emulsion) that is known to promote a balanced Th1/Th2 response. A double-blinded vaccination trial was performed with 40 macaques that were divided into three treatment groups and one control group (n = 10/group). Vaccinated animals received 4 immunisations at 1 month intervals with 150 µg/ml of rBmHAT plus alum, rBmHAT plus AL019 or rBmHAXT plus AL019. Control animals received AL019 only. All vaccinated macaques developed significant (P ≤ 0.003) titers of antigen-specific IgG antibodies (1:20,000) compared with the controls. One month after the last dose, all macaques were challenged s.c. with 130–180 B. malayi L3s. Our results showed that seven out of 10 (70%) of macaques given the improved rBmHAXT vaccine did not develop the infection compared with AL019 controls, of which seven out of 10 macaques developed the infection. Titers of antigen-specific IgG1 and IgG2 antibodies were significantly (P ≤ 0.01) higher in vaccinated animals and there was an increase in the percentage of IL-4 and IFN-γ secreting antigen-responding memory T cells. These studies demonstrated that the improved formulation (rBmHAXT plus AL019) is a promising vaccine candidate against human lymphatic filariasis.

Prior studies established constitutively active AP‐1, NF‐κB, and STAT3 signaling in oral cancer. Differential expression/activation of specific members of these transcription factors has been documented in HPV‐positive oral lesions that respond better to therapy. We performed a comprehensive analysis of differentially expressed, transcriptionally active members of these pivotal signaling mediators to develop specific signatures of HPV‐positive and HPV‐negative oral lesions by immunohistochemical method that is applicable in low‐resource settings. We examined a total of 31 prospective and 30 formalin‐fixed, paraffin‐embedded tissues from treatment‐naïve, histopathologically and clinically confirmed cases diagnosed as oral or oropharyngeal squamous cell carcinoma (OSCC/OPSCC). Following determination of their HPV status by GP5 + /GP6 + PCR, the sequential sections of the tissues were evaluated for expression of JunB, JunD, c‐Fos, p50, p65, STAT3, and pSTAT3(Y705), along with two key regulatory proteins pEGFR and p16 by IHC. Independent analysis of JunB and p65 showed direct correlation with HPV positivity, whereas STAT3 and pSTAT3 were inversely correlated. A combined analysis of transcription factors revealed a more restrictive combination, characterized by the presence of AP‐1 and NF‐κB lacking involvement of STAT3 that strongly correlated with HPV‐positive tumors. Presence of STAT3/pSTAT3 with NF‐κB irrespective of the presence or absence of AP‐1 members was present in HPV‐negative lesions. Expression of pSTAT3 strongly correlated with all the AP‐1/NF‐κB members (except JunD), its upstream activator pEGFRY 1092, and HPV infection‐related negative regulator p16. Overall, we show a simple combination of AP‐1, NF‐κB, and STAT3 members’ expression that may serve as molecular signature of HPV‐positive lesions or more broadly the tumors that show better prognosis.

Etiological role of viral proteins E6 and E7 of high-risk HPV in cervical carcinogenesis is well established. However, their contribution in chemoresistance and epithelial-mesenchymal transition (EMT) that leads to advanced metastatic lesions and chemoresistance is poorly defined. In the present study, contribution of viral oncoproteins in acquisition of EMT character during onset of chemoresistance was assessed. A chemoresistant cell line (SiHaCR) was developed from an established HPV16-positive cervical cancer cell line, SiHa, by escalating selection pressure of 5-fluorouracil (5-FU). Expression of Survivin, ABCG2, Snail, Slug, Twist, and Vimentin was examined in SiHa and SiHaCR cells by reverse transcriptase-PCR (RT-PCR) and immunoblotting assays. Mesenchymal phenotype in SiHaCR cells was confirmed by assessment of migration and invasion potentials. SiHaCR cells displayed elevated level of functional and molecular markers associated with chemoresistance (Survivin, ABCG2) and EMT (Snail, Slug, Twist, Vimentin) and reduced E-cadherin. SiHaCR also showed increased levels of HPV16 E6 and E7 transcripts. Specific silencing of HPV16 E6, but not E7 using corresponding siRNA, demonstrated a differential involvement of HPV oncogenes in manifestation of EMT. HPV16 E6 silencing resulted in reduction of Slug and Twist expression. However, the expression of Snail and Vimentin was only marginally affected. In contrast, there was an increase in the expression of E-cadherin. A reduced migration and invasion capabilities were observed only in E6-silenced SiHaCR cells, which further confirmed functional contribution of HPV16 E6 in manifestation of EMT. Taken together, our study demonstrated an active involvement of HPV16 E6 in regulation of EMT, which promotes chemoresistance in cervical cancer.

Sorafenib is the only first line and FDA-approved drug available for the treatment of advanced hepatocellular carcinoma (HCC). Despite an increased overall patient survival of about 3-5 months with sorafenib, majority of the HCC patients acquire sorafenib-resistance, thus making sorafenib treatment ineffective in long-term. Therefore, there is an urgent need to explore the mechanisms underlying sorafenib-resistance to develop better treatment strategies to combat this deadly disease. To achieve this goal, we first established sorafenib-resistant cells by repeated treatment of different HCC cell lines (Huh7, Hep3B and HepG2) with increasing concentrations of sorafenib up to 6µM. MTT assays confirmed that all three cell types treated with sorafenib achieved resistance and were termed soraR to distinguish them from their sorafenib-sensitive (soraS) counterparts. Further characterization of the soraR cells showed increased expression of mesenchymal markers including Slug, Snail, Zeb2, vimentin, and reduced expression of epithelial marker (E-cadherin), indicative of Epithelial-Mesenchymal Transition (EMT). Moreover soraR cells were found to have high migratory potential in wound healing assay and higher sphere-forming capability compared to the soraS cells. To find out the signaling pathways which could be responsible for promoting sorafenib-resistance, RT2-profiler PCR Array analysis for cytokine and chemokines (PAHS-150Z from Qiagen) were performed, which revealed an increase in the expression of BMP6 and BMP7 mRNA levels in the soraR cells. The results were validated by qPCR. In addition, western blot analyses showed an increase in pSmad1, 5, 9 levels in the soraR cells, suggesting a potential activation of BMP6, 7 axis in sorafenib-resistance. Effect of inhibition of BMP6 and BMP7 signaling in re-sensitizing the SoraR cells towards sorafenib was determined next. Flow cytometric analysis of Hep3B-SoraR cells treated with DMH1, a small molecule inhibitor of BMP receptors in combination with sorafenib showed increased cell death which was accompanied with increased expression of cleaved PARP and cleaved caspase 3 in western blot. BMP6 and BMP7 inhibition also reduced migration capacity of soraR cell types as examined by wound healing assay. The results were further confirmed by treating soraR cells with recombinant BMP6 and BMP7 and examining their effect on migration and sorafenib-mediated apoptosis. Interestingly, exogenous BMP6 and BMP7 treatment was found to enhance migration capacity and rescue the soraS cells from sorafenib-mediated cell death. These findings indicated an important role of BMP6 and BMP7 pathways in mediating sorafenib-resistance in HCC and suggested that pharmacological targeting of BMP6 and BMP7 signaling could be an effective strategy to ameliorate sorafenib-resistance in advanced HCC. Citation Format: Kanchan Vishnoi, Rong Ke, Randhir Kumar, Navin Viswakarma, Ajay Rana, Basabi Rana. Elucidation of the molecular mechanisms mediating sorafenib-resistance in Hepatocellular carcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2614.

Hepatocellular carcinoma (HCC) is one of the most common gastrointestinal (GI) malignancies and a major cause of death worldwide, with limited therapeutic options. Evidence suggest that Mixed Lineage Kinase 3 (MLK3), a member of MAPK pathway, is a key regulator of tumorigenesis and metastasis in different cancer-types. Previous studies from our laboratory indicated that, knocking out MLK3 in mice alleviates hepatocarcinogenesis when treated with Diethylnitrosamine (DEN) in combination with phenobarbital (PB). While these suggested an involvement of MLK3 in HCC progression, the detailed mechanism by which this is accomplished is still unclear. To address the mechanism of MLK3-induced HCC, liver cancer cells stably overexpressing MLK3 wildtype (WT) or kinase dead (KA) in a Doxycycline (Dox)-inducible manner, were established. Our RT2 Profiler PCR array analysis with a cytokine and chemokine array (PAHS-150Z from QIAGEN) showed that MLK3-WT overexpression can significantly upregulate mRNA levels of inflammatory cytokines, such as IL11, BMP6, TNFα and IL6, which were absent with MLK3-KA overexpression. Q-PCR and ELISA analysis confirmed the increased IL11 mRNA levels and IL11 release following MLK3 overexpression. Interestingly, STAT3, the downstream signal transducer of Janus kinases (JAKs), which are activated by cytokine signals was also activated by MLK3 overexpression, suggesting that MLK3 may have a novel crosstalk with JAK-STAT3 signaling pathway. To explore this possibility, cells were treated with inhibitors of JAKs (AZD1480 and Ruxolitinib) in the presence of Dox (to induce MLK3), which significantly decreased STAT3 activation as well as IL11 release, suggesting the involvement of JAKs in MLK3-induced IL11 signaling. Furthermore, we also demonstrated a significant reduction of IL11 release following treatment with the JNK inhibitor, SP600125. To elucidate the detailed mecahnism and identify potential transcription factors that mediate the effect of MLK3 overexpression on IL11 promoter activity, deletion mutants of pGL4.16 IL11-luciferase reporter (-877/+169), (-249/+169), (-153/+169) and (-78/+169) were generated. Luciferase assays confirmed that MLK3-WT overexpression indeed increased IL11 promoter reporter activity, which required the fragment from -153 to -78. More studies are currently undergoing with site-directed mutagenesis to explore the detailed mechanism. In addition, our studies demonstrated an induction of epithelial to mesenchymal transition (EMT) during later stages of Dox treatment, suggesting that MLK3-WT overexpression may regulate EMT, cell migration and invasion. These results indicated that both JAKs, and MLK3/JNK signaling converge to mediate IL11 release in HCC, but the mechanism of crosstalk between MLK3 and JAK-STAT3 signaling pathway is still unclear. Taken together, our current studies indicate that overexpression of MLK3 may not only activate JNK/c-Jun pathway, but also regulate JAK-STAT3 signaling pathway to induce IL11 release and downstream signaling in hepatocellular carcinoma. Citation Format: Rong Ke, Navin Viswakarma, Kanchan Vishnoi, Sandeep Kumar, Ajay Rana, Basabi Rana. Elucidation of MLK3-induced signaling pathways in hepatocellular carcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 108.

Our previous studies indicated that combination of Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and PPARγ ligand Troglitazone (TZD), can induce significant apoptosis in various TRAIL-resistant prostate and hepatocellular carcinoma (HCC) cells. These also suggested serine/threonine kinase AMP-activated protein kinase (AMPK) to be a mediator of TRAIL-TZD-induced apoptosis. To further validate AMPK’s role in TRAIL sensitization, we determined the apoptotic potential of TRAIL in combination with the natural compound Berberine (BBR), the latter being a potent activator of AMPK. These demonstrated a significant reduction of cell viability and induction of apoptosis (increased cleavage of caspase 3, 8, 9) when treated with TRAIL-BBR combination. This apoptosis is attenuated in cells overexpressing AMPKα-dominant negative (DN) or following AMPKα knockdown, confirming involvement of AMPK. To identify potential downstream mediators involved, an apoptosis RT2 PCR array analysis was performed. These showed induction of several genes including TNFRSF10B (expresses DR5) and Harakiri following BBR treatment, which were further validated by qPCR analysis. Furthermore, knocking down DR5 expression significantly attenuated TRAIL-BBR-induced apoptosis, suggesting DR5 to be a mediator of this apoptosis. Our studies indicate that combination of TRAIL and AMPK activator BBR might be an effective means of ameliorating TRAIL-resistance involving DR5 in advanced cancer.

Aberrantly expressed survivin and STAT3 signaling have emerged as major determinants of chemoresistance in gastric cancer. We evaluated effects of potent herbal derivatives curcumin, berberine, and quercetin on STAT3 signaling, survivin expression, and response to 5-fluorouracil (5-FU) treatment in gastric cancer cells (AGS). Cytotoxic and inhibitory effects of berberine, curcumin, and quercetin alone or in combination with 5-FU were examined by MTT assay, and their effect on survivin, STAT3, and the phosphorylated active STAT3 (pSTAT3) expression was examined by western blotting. Effect of these herbal derivatives on STAT3 DNA binding activity was measured by electrophoretic mobility shift assay. Curcumin, berberine, and quercetin effectively downregulated pSTAT3 levels, survivin expression, and gastric cancer cells viability in a dose-dependent manner (with corresponding IC50 values of 40.3μM, 29.2μM and 37.5μM, respectively). Berberine was more effective in inhibiting survivin expression as compared to other herbal agents. 5-FU in combination with berberine or curcumin showed a synergistic inhibition of survivin and STAT3 level resulting in enhanced cell death in gastric cancer cells. Overall, our data suggest use of berberine and curcumin as adjunct therapeutics to overcome chemoresistance during treatment of gastric malignancies.

Current strategy for cancer treatment involves surgery, radiotherapy, and chemotherapy. Among them, either chemotherapy is the only option or it is administered in conjunction with other modalities for treatment for almost all cancers. Anticancer drugs used for the treatment belong to diverse groups such as cell-cycle inhibitors, alkylating agents, antimetabolites, specific monoclonal antibodies, and small molecule inhibitors that can selectively target either rapidly proliferating cells or specific signature molecules on these cells. Naive tumors respond to these chemotherapeutic agents initially, but later, they develop resistance to most of these agents. This phenomenon makes tumors refractory, and management of the disease becomes difficult irrespective of the tumor type. Resistance may be intrinsic to the tumor clones or acquired during the course of treatment. Principal mechanisms of chemoresistance may include increased activity of the drug efflux transporters, decreased drug activation, altered drug targets, increased drug degradation, enhanced DNA-damage repair, and failure of cells to undergo apoptosis as a result of alterations in p53. Several attempts have been made by researchers to develop strategies to target chemoresistance. Among these, the use of nutraceuticals being most notable as they are safe and economic and have the capability of targeting multiple pathways of chemoresistance, and many of them also show independent anticancer activities. Taking this into consideration, several natural products are in different phases of clinical evaluation and have shown positive results in both preclinical and clinical studies. The results demonstrate that nutraceuticals may be developed as clinically useful anticancer chemosensitizers for adjuvant therapy in combination with existing chemotherapy to increase the treatment efficacy.

Viral oncoproteins E6/E7 play key oncogenic role in human papillomavirus (HPV)-mediated cervical carcinogenesis in conjunction with aberrant activation of cellular signaling events. GLI-signaling has been implicated in metastasis and tumor recurrence of cervical cancer. However, the interaction of GLI-signaling with HPV oncogenes is unknown. We examined this relationship in established HPV-positive and HPV-negative cervical cancer cell lines using specific GLI inhibitor, cyclopamine and HPVE6/E7 siRNAs. Cervical cancer cell lines showed variable expression of GLI-signaling components. HPV16-positive SiHa cells, overexpressed GLI1, Smo and Patch. Inhibition by cyclopamine resulted in dose-dependent reduction of Smo and GLI1 and loss of cell viability with a higher magnitude in HPV-positive cells. Cyclopamine selectively downregulated HPVE6 expression and resulted in p53 accumulation, whereas HPVE7 and pRb level remained unaffected. siRNA-mediated silencing of HPV16E6 demonstrated reduced GLI1 transcripts in SiHa cells. Cervical cancer stem-like cells isolated by side population analysis, displayed retention of E6 and GLI1 expression. Fraction of SP cells was reduced in cyclopamine-treated cultures. When combined with E6-silencing cyclopamine resulted in loss of SP cell’s sphere-forming ability. Co-inhibition of GLI1 and E6 in cervical cancer cells showed additive anti-cancer effects. Overall, our data show existence of a cooperative interaction between GLI signaling and HPVE6.

Lymphatic filariasis (LF) affects 120 million people around the world and another 856 million people are at risk of acquiring the infection. Mass Drug Administration (MDA) spearheaded by the World Health Organization is the only current strategy to control this infection. Recent reports suggest that despite several rounds of MDA, elimination has not been achieved and there is a need for more stringent control strategies for control of LF. An effective prophylactic vaccine combined with MDA has significant potential. Initial trials using a prophylactic trivalent recombinant Brugia malayi heat shock protein 12.6, abundant larval transcript -2 and tetraspanin large extra-cellular loop (rBmHAT) vaccine developed in our laboratory conferred only 35% protection in macaques. Therefore, the focus of the present study was to improve the current vaccine formulation to obtain better protection in non-human primates. We made two modifications to the current formulation: (i) the addition of another antigen, thioredoxin peroxidase-2 (TPX-2) to make it a tetravalent vaccine (rBmHAXT) and (ii) the inclusion of an adjuvant; AL019 (alum plus glucopyranosyl lipid adjuvant-stable emulsion) that is known to promote a balanced Th1/Th2 response. A double-blinded vaccination trial was performed with 40 macaques that were divided into three treatment groups and one control group (n = 10/group). Vaccinated animals received 4 immunisations at 1 month intervals with 150 µg/ml of rBmHAT plus alum, rBmHAT plus AL019 or rBmHAXT plus AL019. Control animals received AL019 only. All vaccinated macaques developed significant (P ≤ 0.003) titers of antigen-specific IgG antibodies (1:20,000) compared with the controls. One month after the last dose, all macaques were challenged s.c. with 130-180 B. malayi L3s. Our results showed that seven out of 10 (70%) of macaques given the improved rBmHAXT vaccine did not develop the infection compared with AL019 controls, of which seven out of 10 macaques developed the infection. Titers of antigen-specific IgG1 and IgG2 antibodies were significantly (P ≤ 0.01) higher in vaccinated animals and there was an increase in the percentage of IL-4 and IFN-γ secreting antigen-responding memory T cells. These studies demonstrated that the improved formulation (rBmHAXT plus AL019) is a promising vaccine candidate against human lymphatic filariasis.

Sorafenib is the only FDA-approved drug currently available for the treatment of advanced hepatocellular carcinoma (HCC). However, development of sorafenib-resistance in patients poses a major challenge to make this treatment successful in long-term. There is an urgent need to explore the mechanisms underlying sorafenib-resistance and to develop strategies towards overcoming sorafenib-resistance. Aberrant activation of Wnt/β-catenin signaling in HCC is suggested to maintain tumor initiating cells, drug-resistance, tumor progression, and metastasis. Combining β-catenin inhibitors with sorafenib might thus be an effective therapeutic strategy to target drug-resistant HCC. To explore this, we used wnt/β-catenin pathway inhibitor XAV-939 in combination with sorafenib to study their effect on the apoptotic potential of HCC cell lines Huh7, Hep3B and HepG2. XAV-939 was demonstrated to effectively reduce the expression of β-catenin in wild-type expressing Huh7 and Hep3B cells. Flow cytometric analysis of these cells pre-treated with XAV-939 followed by sorafenib treatment showed a significant increase in apoptosis in a time dependent manner. Furthermore, these cells revealed increased mitochondrial depolarization which was accompanied by a parallel increase in cytosolic cytochrome c release and downregulation of pro-survival protein Mcl-1. These effects of XAV-939 were further validated following knockdown of endogenous β-catenin, which showed a potentiation of sorafenib effects with β-catenin knockdown. Further, to understand the mechanism associated with sorafenib-resistance, we developed sorafenib-resistant HCC cells by repeated exposure to increasing concentration of sorafenib. To our surprise the combination of sorafenib and XAV-939 was ineffective in increasing the apoptotic potential of these resistant cells, suggesting a differential role of wnt/β-catenin axis in sorafenib-resistance. Taken together, these results suggested that combination of wnt/β-catenin pathway inhibitor and sorafenib could be effective in sensitizing HCC cells to apoptosis initially, but might be ineffective once these cells acquire sorafenib-resistance. Citation Format: Kanchan Vishnoi, Rong Ke, Navin Vishwakarma, Ajay Rana, Basabi Rana. XAV-939, a Wnt/beta-catenin pathway inhibitor, sensitizes liver cancer cells to sorafenib: Implications in sorafenib resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2338.

BACKGROUND: Localization and differential expression of STAT3 and survivin in cancer cells are often related to distinct cellular functions. The involvement of survivin and STAT3 in gastric cancer has been reported in separate studies but without clear understanding of their kinetics in cancer progression. METHODS: We examined intracellular distribution of STAT3 and survivin in gastric adenocarcinoma and compared it with normal and precancer tissues using immunoblotting and immunohistochemistry. RESULTS: Analysis of a total of 156 gastric samples comprising 61 histologically normal, 30 precancerous tissues (comprising intestinal metaplasia and dysplasia), and 65 adenocarcinomas, collected as endoscopic biopsies from treatment naïve study participants, revealed a significant (P

Purpose: Perturbation of keratinocyte differentiation by E6/E7 oncoproteins of high-risk human papillomaviruses that drive oncogenic transformation of cells in squamocolumnar junction of the uterine cervix may confer “stem-cell like” characteristics. However, the crosstalk between E6/E7 and stem cell signaling during cervical carcinogenesis is not well understood. We therefore examined the role of viral oncoproteins in stem cell signaling and maintenance of stemness in cervical cancer. Experimental Design: Isolation and enrichment of cervical cancer stem–like cells (CaCxSLCs) was done from cervical primary tumors and cancer cell lines by novel sequential gating using a set of functional and phenotypic markers (ABCG2, CD49f, CD71, CD133) in defined conditioned media for assessing sphere formation and expression of self-renewal and stemness markers by FACS, confocal microscopy, and qRT-PCR. Differential expression level and DNA-binding activity of Notch1 and its downstream targets in CaCxSLCs as well as silencing of HPVE6/Hes1 by siRNA was evaluated by gel retardation assay, FACS, immunoblotting, and qRT-PCR followed by in silico and in vivo xenograft analysis. Results: CaCxSLCs showed spheroid-forming ability, expressed self-renewal and stemness markers Oct4, Sox2, Nanog, Lrig1, and CD133, and selectively overexpressed E6 and HES1 transcripts in both cervical primary tumors and cancer cell lines. The enriched CaCxSLCs were highly tumorigenic and did recapitulate primary tumor histology in nude mice. siRNA silencing of HPVE6 or Hes1 abolished sphere formation, downregulated AP-1-STAT3 signaling, and induced redifferentiation. Conclusions: Our findings suggest the possible mechanism by which HPVE6 potentially regulate and maintain stem-like cancer cells through Hes1. Clin Cancer Res; 22(16); 4170–84. ©2016 AACR.

Resistance towards standard therapeutic regimens and evasion of apoptosis are some of the hallmarks of advanced forms of cancer, which include Sorafenib-resistance in hepatocellular carcinoma (HCC), castration-resistance in prostate cancer. Development of effective therapeutic strategies that can target these resistant forms is critically needed. In an effort to understand the molecular mechanism mediating resistance in cancer, in previous studies utilizing Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-resistant cancer cells we showed that combination of TRAIL along with the PPARγ ligand Troglitazone (TZD) can sensitize them towards apoptosis. Using more molecular approaches these studies identified the serine/threonine protein kinase, AMP-activated protein kinase (AMPK) as a mediator of TRAIL-TZD-induced apoptosis. Since AMPK seemed to be a major mediator of this apoptosis, in the current studies we utilized the natural compound Berberine (BBR), a known activator of AMPK in combination with TRAIL. These demonstrated a significant reduction of cell viability (MTT assay) and induction of apoptosis (caspase activation) when treated with a combination of TRAIL and BBR. This apoptosis is attenuated in cells overexpressing AMPKα-dominant negative (DN), suggesting an involvement of AMPK in mediating this. To understand the downstream targets and the mechanism involved, an apoptosis PCR array analysis was performed, which suggested induction of TNFRSF10B (DR5) gene expression by BBR. In addition, knockdown of DR5 expression attenuated TRAIL-BBR-induced apoptosis, suggesting DR5 to be a potential target of BBR in this apoptotic cascade. Future studies will include determining any crosstalk of AMPK in BBR signaling to modulate DR5 pathway. Our earlier studies also demonstrated an involvement of β-catenin in mediating cancer cell resistance. Stabilizing mutations and overexpression of β-catenin has been reported in many cancers, most profoundly in HCC leading to an activation of Wnt/β-catenin signaling. To understand any role of β-catenin in TRAIL-BBR-induced apoptosis, we determined the effect of BBR on β-catenin pathway. These revealed a significant attenuation of β-catenin protein expression by BBR in various HCC cells in a time and dose-dependent manner. BBR treatment also attenuated β-catenin/TCF-induced transcriptional activity, indicating antagonism of β-catenin pathway. Our studies indicate that combination of TRAIL and AMPK activator BBR might be an effective means of antagonizing β-catenin pathway and ameliorating TRAIL resistance via DR5 in advanced forms of cancer. Citation Format: Basabi Rana, Rong Ke, Kanchan Vishnoi, Navin Viswakarma, Subhasis Das, Ajay Rana. Elucidation of the signaling pathways that mediate berberine-induced effects in cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3322. doi:10.1158/1538-7445.AM2017-3322

Despite well-established human papillomavirus (HPV)-mediated etiology of cervical cancer, it has remained a major cause of cancer-related mortality of women from developing countries. Factors that contribute to high mortality rate primarily include disease diagnosis in advanced stages and failure of conventional treatment offered to these patients resulting in tumor relapse. Cancer stem cells (CSCs), in spite of controversies on their existence, are emerging as important players that initiate and maintain the tumor phenotype, govern tumor relapse, and chemo-/radioresistance, and thus considered as main factor that decides the treatment outcome. Recent studies provide evidence not only for presence of CSCs but also involvement of stemness-related signaling pathways and transcription factors in cervical cancer tissues and cell lines. The investigations made till date, however, lack evidence demonstrating a direct involvement of HPV oncoprotein(s) in modulation of CSC properties, relative proportions, or stem cell signaling in tumor tissues. Available literature, however, does support the involvement of HPV in these events. In the present article, we attempt to collate available data that demonstrates presence of CSC in cervical cancer, specific markers used to derive cervical CSC, and explored possibility of a potential cross talk of HPV oncoproteins with CSC signaling particularly with the known self-renewal signaling pathways like Notch, Hedgehog (Hh), and Wnt as well as stemness-related transcription factor like Oct4, Nanog, Sox2, and STAT3 that maintain CSC phenotype and functionality. Overall, present article provides rationale for detailed investigation of HPV role in regulation of CSC in cervical tumor tissues for an effective targeting of refractory lesions and to prevent tumor relapse.

Signal transducer and activator of transcription 3 (STAT3) is an oncogenic transcription factor constitutively active and aberrantly expressed in cervical cancer. However, the functional role of STAT3 in regulation of HPV's viral oncogene expression and downstream events associated with cervical carcinogenesis is not known. Our present study performed on HPV16-positive cervical cancer cell lines (SiHa and CaSki) and primary tumor tissues revealed a strong positive correlation of constitutively active STAT3 with expression of HPV16 E6 and E7 oncoproteins and a negative association with levels of p53 and pRB. Pharmacologic targeting of STAT3 expression in cervical cancer cell lines either by STAT3-specific siRNA or blocking its tyrosine phosphorylation by AG490 or curcumin led to dose-dependent accumulation of p53 and pRb in cervical cancer cells. Interestingly, the suppression of STAT3 expression or activation was associated with the gradual loss of HPV16 E6 and E7 expression and was accompanied by loss of cell viability. The viability loss was specifically high in HPV16-positive cells as compared to HPV negative C33a cells. These findings substantiate the regulatory role of STAT3 in HPV16-mediated cervical carcinogenesis. Leads obtained from the present study provide a strong rationale for developing novel STAT3-based approaches for therapeutic interventions against HPV infection to control cervical cancer.

Background & Objectives: Persistent infection of high-risk human papillomaviruses (HR-HPV) is a key etiological factor responsible for the development of cervical cancer. Deregulated expression of two viral oncoproteins E6 and E7 drive oncogenic transformation and tumour progression which may occur upon targeted infection of specific basal cervical cells with stem cell properties. However, how oncoproteins impact on putative CSCs, the stem cell signaling and their downstream functions, is not known. In this study, we have identified and characterized cervical cancer stem-like cells and unraveled the crosstalk between HPV oncoproteins and stem cell signaling that allowed maintenance of stemness in cancer cells. Experimental Procedure: Cervical cancer stem-like cells (CaCxSLCs) were isolated and enriched by sequential gating from HPV+ve and HPV-ve human cervical cancer cell lines (SiHa, HeLa and C33a) using a set of functional and phenotypic markers (ABCG2, CD49f, CD71, CD133) in defined conditioned media (DCM) with intermittent culturing. CaCxSLCs were also assessed for their cervicosphere forming ability, exclusion of DCV dye for generating SP cells, self renewability and quiescenceness. Moreover, differential expression level and DNA-binding activity of CSL-specific Notch1 and its downstream targets in CaCxSLCs cells along with HPVE6 specific siRNA mediated gene silencing was evaluated by gel retardation assay, immunobloting qRT-PCR followed by in vivo tumor-initiating capacity in athymic nude mice. Results: CaCxSLCs cells isolated from HPV+ve cells showed high expression of CD49f, Lrig1, Nanog, Sox2, Oct4, ABCG2 and low Notch1 DNA binding activity as compared to non- CaCxSLCs cells. Enriched CaCxSLCs were also found to be more tumorigenic and showed high expression of HPVE6, HES1, CD133 transcripts with high percentage of SP cells. CaCxSLCs differed from their parental/non-CaCxSLCs as they overexpressed E6 oncogene. Knocking down of HPVE6 by RNA interference resulted in abolition of spheroid formation through downregulation of Hes1 and induction of re-differentiation while interference of Hes-1 expression resulted in reduced cervicosphere formation through down regulation of E6 and Nanog suggesting loss of self-renewing ability. Conclusions: Our findings suggest a potential regulatory role of HPVE6 in induction and maintenance of cervical cancer stem-like cells through Hes1 expression which could be utilized as a putative target for designing drugs that can promote differentiation of cancer stem cells making the therapy most effective. Note: This abstract was not presented at the meeting. Citation Format: Bhudev C. Das, Abhishek Tyagi, Kanchan Vishnoi, Sutapa Mahata, Gaurav Verma, Alok C. Bharti. Role of HPVE6 oncoprotein in the maintenance of cervical cancer stem cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4079. doi:10.1158/1538-7445.AM2015-4079

To investigate the low gastric cancer incidence rate relative to the highly prevalent Helicobacter pylori (H. pylori) infection; data relevant to H. pylori infection during gastric carcinogenesis in Indian patients is currently lacking.The present study examines the prevalence of H. pylori infection in DNA derived from 156 endoscopic gastric biopsies of different disease groups that represent gastric pre-cancer [intestinal metaplasia (n = 15), dysplasia (n = 15)], cancer [diffuse adenocarcinoma (n = 44), intestinal adenocarcinoma (n = 21)], and symptomatic but histopathologically-normal controls (n = 61). This was done by generic ureC polymerase chain reaction (PCR) and cagA-specific PCR that could specifically identify the carcinogenic H. pylori strain.Our analysis showed the presence of H. pylori infection in 61% of symptomatic histopathologically-normal individuals, however only 34% of control tissues were harboring the cagA(+) H. pylori strain. A similar proportion of H. pylori infection (52%) and cagA (26%) positivity was observed in the tumor tissue of the gastric cancer group. In comparison, H. pylori infection (90%) and cagA positivity (73%) were the highest in gastric pre-cancer lesions. In relation to tobacco and alcohol abuse, H. pylori infection showed an association with tobacco chewing, whereas we did not observe any association between tobacco smoking or alcohol abuse with prevalence of H. pylori infection in the tissue of any of the patient groups studied.High incidence of H. pylori infection and carcinogenic cagA positive strain in pre-cancer lesions during gastric carcinogenesis may be associated with the habit of chewing tobacco.