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.

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.

Background Angiogenic switch is a hallmark feature of transition from low-grade to high-grade cervical intraepithelial neoplasia (CIN) in cervical cancer progression. Therefore, early events leading to locally-advanced cervical metastatic lesions demand a greater understanding of the underlying mechanisms. Recent leads indicate the role of tumor-derived exosomes in altering the functions of endothelial cells in cervical cancer, which needs further investigation. Methods Exosomes isolated from cervical cancer cell lines were assessed for their angiogenic effect on the human umbilical vein endothelial cells (HUVEC) using tube formation and wound healing assay. The exosomal uptake by HUVEC cells was monitored using PKH-67 labelling followed by fluorescence microscopy. Alterations in Hh-GLI signaling components, PTCH1 and GLI1, in HUVEC were measured by immunoblotting. Changes in angiogenesis-related transcripts of vascular endothelial growth factor VEGF-A, VEGF-B, VEGFR2 and angiopoietin-1, angiopoietin-2, osteopontin were measured in exosome-treated HUVEC and in the exosomal RNA by RT-PCR. Results Enhanced tube formation, with an increased number of nodes and branching was observed in HUVEC’s treated with exosomes derived from different cervical cancer cell lines. HPV-positive (SiHa and HeLa) cells’ exosomes were more angiogenic. Exosome-treated HUVEC showed increased migration rate. PKH-67 labelled exosomes were found internalized in HUVEC. A high level of PTCH1 protein was detected in the exosome—treated endothelial cells. Subsequent RT-PCR analysis showed increased transcripts of Hh-GLI downstream target genes VEGF-A, VEGFR2, angiopoietin-2, and decreased expression of VEGF-B, and angiopoietin-1, suggestive of active Hh-GLI signaling. These effects were more pronounced in HUVEC’s treated with exosomes of HPV-positive cells. However, these effects were independent of tumor-derived VEGF-A as exosomal cargo lacked VEGF-A transcripts or proteins. Conclusion Overall, the data showed cervical cancer exosomes promote pro-angiogenic response in endothelial cells via upregulation of Hh-GLI signaling and modulate downstream angiogenesis-related target genes. The study provides a novel exosome-mediated mechanism potentially favoring cervical angiogenesis and thus identifies the exosomes as potential pharmacological targets against locally-advanced metastatic cervical lesions. Graphic abstract

Human papillomavirus (HPV) infection is linked to development of cancer of cervix, vagina, vulva, penis, ano-genital and non-genital oro-pharyngeal sites. HPV being a sexually transmitted virus infects both genders equally but with higher chances of pathological outcome in women. In the absence of organized screening programs, women report HPV-infected lesions at relatively advanced stages where they are subjected to standard treatments that are not HPV-specific. HPV infection-driven lesions usually take 10-20 years for malignant progression and are preceded by well-characterized pre-cancer stages. Despite availability of window for pharmacological intervention, therapeutic that could eradicate HPV from infected lesions is currently lacking. A variety of experimental approaches have been made to address this lacuna and there has been significant progress in a number of lead molecules which are in different stages of clinical and pre-clinical development. Present review provides a brief overview of the magnitude of the problem and current status of research on promising lead molecules, formulations and therapeutic strategies that showed potential to translate to clinically-viable HPV therapeutics to counteract this reproductive health challenge.

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.

Head and neck squamous cell carcinoma (HNSCC) is a heterogeneous group of cancers. Collectively, HNSCC ranks sixth in incidence rate worldwide. Apart from classical risk factors like tobacco and alcohol, infection of human papillomavirus (HPV) is emerging as a discrete risk factor for HNSCC. HPV-positive HNSCC represent a distinct group of diseases that differ in their clinical presentation. These lesions are well-differentiated, occur at an early age, and have better prognosis. Epidemiological studies have demonstrated a specific increase in the proportions of the HPV-positive HNSCC. HPV-positive and HPV-negative HNSCC lesions display different disease progression and clinical response. For tumorigenic-transformation, HPV essentially requires a permissive cellular environment and host cell factors for induction of viral transcription. As the spectrum of host factors is independent of HPV infection at the time of viral entry, presumably entry of HPV only selects host cells that are permissive to establishment of HPV infection. Growing evidence suggest that HPV plays a more active role in a subset of HNSCC, where they are transcriptionally-active. A variety of factors provide a favorable environment for HPV to become transcriptionally-active. The most notable are the set of transcription factors that have direct binding sites on the viral genome. As HPV does not have its own transcription machinery, it is fully dependent on host transcription factors to complete the life cycle. Here, we review and evaluate the current evidence on level of a subset of host transcription factors that influence viral genome, directly or indirectly, in HNSCC. Since many of these transcription factors can independently promote carcinogenesis, the composition of HPV permissive transcription factors in a tumor can serve as a surrogate marker of a separate molecularly-distinct class of HNSCC lesions including those cases, where HPV could not get a chance to infect but may manifest better prognosis.

Plasmodium falciparum heat shock proteins and proteases are known for their indispensable roles in parasite virulence and survival in the host cell. They neutralize various host-derived stress responses that are deleterious for parasite growth and invasion. We report identification and functional characterization of the first DegP from an apicomplexan (P. falciparum).To determine the molecular identity and functions of the parasite-encoded DegP, we complemented the Escherichia coli degP null mutant with a putative PfdegP gene, and the results showed that PfDegP complements the growth defect of the temperature sensitive DegP-deficient mutant and imparts resistance to non-permissive temperatures and oxidative stress. Molecular interaction studies showed that PfDegP exists as a complex with parasite-encoded heat shock protein 70, iron superoxide dismutase and enolase. DegP expression is significantly induced in parasite culture upon heat shock/oxidative stress. Our data suggest that the PfDegP protein may play a role in the growth and development of P. falciparum through its ability to confer protection against thermal/oxidative stress. Antibody against DegP showed anti-plasmodial activity against blood-stage parasites in vitro, suggesting that PfDegP and its associated complex may be a potential focus for new anti-malarial therapies. Structured digital abstract• PfDegP physically interacts with PfHsp70 and PfEno by anti-bait co-immunoprecipitation (View interaction) • PfDegP physically interacts with PfEno, PfSod, PfOat, PfHsp70, PfLDH and PfGpi by antibait co-immunoprecipitation (View interaction) • PfHsp-70 and PfDegP co-localize by fluorescence microscopy (View interaction) • PfDegP physically interacts with PfOat, PfHsp70, PfEno, PfSod, PfGpi and PfLDH by surface plasmon resonance (View interaction) • PfEno and PfDegP co-localize by fluorescence microscopy (View interaction) • PfDegP and PfHsp70 co-localize by co-sedimentation through density gradient (View interaction)

Our earlier studies indicated an important role of inducible transcription factor STAT3 in the establishment of persistent infection of human papillomavirus (HPV) type 16 and promotion of cervical carcinogenesis. Since HPV load and its physical state are two potential determinants of this virally-induced carcinogensis, though with some exceptions, we extended our study to examine the role of active STAT3 level in cervical precancer and cancer lesions and it’s association with HPV viral load and physical state. An elevated level of active STAT3 was measured by assessing phospho-STAT3-Y705 (pSTAT3), in tumor tissues harboring higher viral load irrespective of the disease grade. Physical state analysis of HPV16 by assessing the degree of amplification of full length E2 and comparing it with E6 (E2:E6 ratio), which predominantly represent episomal form of HPV16, revealed low or undetectable pSTAT3. A strong pSTAT3 immunoreactivity was found in tissues those harbored either mixed or predominantly integrated form of viral genome. Cumulative analysis of pSTAT3 expression, viral load and physical state demonstrated a direct correlation between pSTAT3 expression, viral load and physical state of HPV. The study suggests that there exists a strong clinical correlation between level of active STAT3 expression and HPV genome copy number, and integrated state of the virus that may play a pivotal role in promotion/maintanence of tumorigenic phenotype.