MLK3 promotes prooncogenic signaling in hepatocellular carcinoma via TGFß pathway.

Advanced hepatocellular carcinoma (HCC) is a lethal disease, with limited therapeutic options. Mixed Lineage Kinase 3 (MLK3) is a key regulator of liver diseases, although its role in HCC remains unclear. Analysis of TCGA databases suggested elevated MAP3K11 (MLK3 gene) expression, and TMA studies showed higher MLK3 activation in human HCCs. To understand MLK3's role in HCC, we utlized carcinogen-induced HCC model and compared between wild-type and MLK3 knockout (MLK3-/-) mice. Our studies showed that MLK3 kinase activity is upregulated in HCC, and MLK3 deficiency alleviates HCC progression. MLK3 deficiency reduced proliferation in vivo and MLK3 inhibition reduced proliferation and colony formation in vitro. To obtain further insight into the mechanism and identify newer targets mediating MLK3-induced HCCs, RNA-sequencing analysis was performed. These showed that MLK3 deficiency modulates various gene signatures, including EMT, and reduces TGFB1&2 expressions. HCC cells overexpressing MLK3 promoted EMT via autocrine TGFß signaling. Moreover, MLK3 deficiency attenuated activated hepatic stellate cell (HSC) signature, which is increased in wild-type. Interestingly, MLK3 promotes HSC activation via paracrine TGFß signaling. These findings reveal TGFß playing a key role at different steps of HCC, downstream of MLK3, implying MLK3-TGFß axis to be an ideal drug target for advanced HCC management.

Ets1 mediates sorafenib resistance by regulating mitochondrial ROS pathway in hepatocellular carcinoma.

The incidence and mortality of hepatocellular carcinoma (HCC) are on a rise in the Western countries including US, attributed mostly to late detection. Sorafenib has been the first-line FDA-approved drug for advanced unresectable HCC for almost a decade, but with limited efficacy due to the development of resistance. More recently, several other multi-kinase inhibitors (lenvatinib, cabozantinib, regorafenib), human monoclonal antibody (ramucirumab), and immune checkpoint inhibitors (nivolumab, pembrolizumab) have been approved as systemic therapies. Despite this, the median survival of patients is not significantly increased. Understanding of the molecular mechanism(s) that govern HCC resistance is critically needed to increase efficacy of current drugs and to develop more efficacious ones in the future. Our studies with sorafenib-resistant (soraR) HCC cells using transcription factor RT2 Profiler PCR Arrays revealed an increase in E26 transformation-specific-1 (Ets-1) transcription factor in all soraR cells. HCC TMA studies showed an increase in Ets-1 expression in advanced HCC compared to the normal livers. Overexpression or knocking down Ets-1 modulated sorafenib resistance-related epithelial-mesenchymal transition (EMT), migration, and cell survival. In addition, the soraR cells showed a significant reduction of mitochondrial damage and mitochondrial reactive oxygen species (mROS) generation, which were antagonized by knocking down Ets-1 expression. More in-depth analysis identified GPX-2 as a downstream mediator of Ets-1-induced sorafenib resistance, which was down-regulated by Ets-1 knockdown while other antioxidant pathway genes were not affected. Interestingly, knocking down GPX2 expression significantly increased sorafenib sensitivity in the soraR cells. Our studies indicate the activation of a novel Ets-1-GPX2 signaling axis in soraR cells, targeting which might successfully antagonize resistance and increase efficacy.

Dysregulation of immune checkpoint proteins in hepatocellular carcinoma: Impact on metabolic reprogramming.

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.

Detection of human papillomavirus infection in oral cancers reported at dental facility: assessing the utility of FFPE tissues.

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.

Berberine Represses ß-Catenin Translation Involving 4E-BPs in Hepatocellular Carcinoma Cells.

Aberrant activation of Wnt/ß-catenin axis occurs in several gastrointestinal malignancies due to inactivating mutations of adenomatous polyposis coli (in colorectal cancer) or activating mutations of ß-catenin itself [in hepatocellular carcinoma (HCC)]. These lead to ß-catenin stabilization, increase in ß-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 ß-catenin in cancer cells, we observed that the plant compound berberine (BBR), a potent activator of AMP-activated protein kinase (AMPK), can reduce ß-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 ß-catenin occurs independently of AMPK activation and does not involve transcriptional or post-translational mechanisms. Pretreatment with protein synthesis inhibitor cycloheximide antagonized BBR-induced ß-catenin reduction, suggesting that BBR affects ß-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 ß-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 ß-catenin. Our findings indicate that BBR antagonizes ß-catenin pathway by inhibiting ß-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 ß-catenin variants that are currently undruggable. SIGNIFICANCE STATEMENT: ß-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 ß-catenin translation via utilizing a plant compound, berberine. Our findings provide a new avenue of targeting ß-catenin axis in cancer, which can be utilized toward the designing of effective therapeutic strategies to combat ß-catenin-dependent cancers.

Transcription Factors in Cancer Development and Therapy.

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 ß-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.

Prospects of developing a prophylactic vaccine against human lymphatic filariasis - evaluation of protection in non-human primates.

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.

Involvement of AMP-activated protein kinase and Death Receptor 5 in TRAIL-Berberine-induced apoptosis of cancer cells.

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.

Differentially localized survivin and STAT3 as markers of gastric cancer progression: Association with Helicobacter pylori.

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 < .001) increase in overall protein levels. Survivin expression was detectable in both cytoplasmic (90.8%) and nuclear (87.7%) compartments in gastric adenocarcinomas lesions. Precancerous dysplastic gastric lesions exhibited a moderate survivin expression (56.7%) localized in cytoplasmic compartment. Similarly, STAT3 and pSTAT3 expression was detected at high level in gastric cancer lesions. The levels of compartmentalized expression of survivin and STAT3/pSTAT3 correlated in precancerous and adenocarcinoma lesions. Although overexpression of these proteins was found associated with the tobacco use and alcohol consumption, their expression invariably and strongly correlated with concurrent Helicobacter pylori infection. Receiver operating characteristic analysis of nuclear survivin, STAT3, and pSTAT3 in different study groups showed acceptable positive and negative predictive values with area under the curve above 0.8 (P < .001). CONCLUSION: Overall, our results suggest that overall increase in survivin and STAT3 and their subcellular localization are key determinants of gastric cancer progression, which can be collectively used as potential disease biomarkers and therapeutic targets for gastric cancer.

Cervical cancer stem cells manifest radioresistance: Association with upregulated AP-1 activity.

Transcription factor AP-1 plays a central role in HPV-mediated cervical carcinogenesis. AP-1 has also been implicated in chemo-radio-resistance but the mechanism(s) remained unexplored. In the present study, cervical cancer stem-like cells (CaCxSLCs) isolated and enriched from cervical cancer cell lines SiHa and C33a demonstrated an elevated AP-1 DNA-binding activity in comparison to non-stem cervical cancer cells. Upon UV-irradiation, CaCxSLCs showed a UV exposure duration-dependent higher proliferation and highly increased AP-1 activity whereas it was completely abolished in non-stem cancer cells. CaCxSLCs also showed differential overexpression of c-Fos and c-Jun at transcript as well as in protein level. The loss of AP-1 activity and expression was accompanied by decrease in cell viability and proliferation in UV-irradiated non-stem cancer cells. Interestingly, CaCxSLCs treated with curcumin prior to UV-irradiation abolished AP-1 activity and a concomitant reduction in SP cells leading to abrogation of sphere forming ability, loss of proliferation, induction of apoptosis and the cells were poorly tumorigenic. The curcumin pre-treatment abolished the expression of c-Fos and c-Jun but upregulated Fra-1 expression in UV-irradiated CaCxSLCs. Thus, the study suggests a critical role of AP-1 protein in the manifestation of radioresistance but targeting with curcumin helps in radiosensitizing CaCxSLCs through upregulation of Fra-1.

Characterization of key transcription factors as molecular signatures of HPV-positive and HPV-negative oral cancers.

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 pEGFRY1092 , 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.

Cross-talk between Human Papillomavirus Oncoproteins and Hedgehog Signaling Synergistically Promotes Stemness in Cervical Cancer Cells.

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.

Human papillomavirus oncoproteins differentially modulate epithelial-mesenchymal transition in 5-FU-resistant cervical cancer cells.

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.

Cervical Cancer Stem Cells Selectively Overexpress HPV Oncoprotein E6 that Controls Stemness and Self-Renewal through Upregulation of HES1.

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.

Berberine and Curcumin Target Survivin and STAT3 in Gastric Cancer Cells and Synergize Actions of Standard Chemotherapeutic 5-Fluorouracil.

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.

Carcinogenic Helicobacter pylori in gastric pre-cancer and cancer lesions: association with tobacco-chewing.

AIM: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Functional regulatory role of STAT3 in HPV16-mediated cervical carcinogenesis.

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.