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

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.

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.

Background Exosomes play a key role in cell-to-cell communication and are integral component of the tumor microenvironment. Recent observations suggest transfer of RNA through tumor-derived exosomes that can potentially translate into regulatory proteins in the recipient cells. Role of cervical cancer-derived exosomes and their transcript cargo is poorly understood. Materials and methods The total RNA of exosomes from HPV-positive (SiHa and HeLa) and HPV-negative (C33a) cervical cancer cell lines were extracted and the transcripts were estimated using Illumina HiSeq X. Further, validation of HPV transcripts were performed using RT-PCR. Results 3099 transcripts were found to be differentially-exported in HPV-positive vs. HPV-negative exosomes (p value <0.05). Analysis of top 10 GO terms and KEGG pathways showed enrichment of transcripts belonging to axon guidance and tumor innervation in HPV-positive exosomes. Among top 20 overexpressed transcripts, EVC2, LUZP1 and ANKS1B were the most notable due to their involvement in Hh signaling, cellular migration and invasion, respectively. Further, low levels of HPV-specific reads were detected. RT-PCR validation revealed presence of E6*I splice variant of HPV18 in exosomal RNA of HeLa cells. The E6*I transcripts were consistently retained in exosomes obtained from HeLa cells undergoing 5-FU and cisplatin-induced oxidative stress. Conclusion Our data suggests the enrichment of poly-A RNA transcripts in the exosomal cargo of cervical cancer cells, which includes pro-tumorigenic cellular RNA and viral transcripts such as HPV E6, which may have clinical utility as potential exosomal biomarkers of cervical cancer.

Reversion of tumor to a normal differentiated cell once considered a dream is now at the brink of becoming a reality. Different layers of molecules/events such as microRNAs, transcription factors, alternative RNA splicing, post-transcriptional, post-translational modifications, availability of proteomics, genomics editing tools, and chemical biology approaches gave hope to manipulation of cancer cells reversion to a normal cell phenotype as evidences are subtle but definitive. Regardless of the advancement, there is a long way to go, as customized techniques are required to be fine-tuned with precision to attain more insights into tumor reversion. Tumor regression models using available genome-editing methods, followed by in vitro and in vivo proteomics profiling techniques show early evidence. This review summarizes tumor reversion developments, present issues, and unaddressed challenges that remained in the uncharted territory to modulate cellular machinery for tumor reversion towards therapeutic purposes successfully. Ongoing research reaffirms the potential promises of understanding the mechanism of tumor reversion and required refinement that is warranted in vitro and in vivo models of tumor reversion, and the potential translation of these into cancer therapy. Furthermore, therapeutic compounds were reported to induce phenotypic changes in cancer cells into normal cells, which will contribute in understanding the mechanism of tumor reversion. Altogether, the efforts collectively suggest that tumor reversion will likely reveal a new wave of therapeutic discoveries that will significantly impact clinical practice in cancer therapy.

Head and neck cancer (HNC) usually arises from squamous cells of the upper aerodigestive tract that line the mucosal surface in the head and neck region. In India, HNC is common in males, and it is the sixth most common cancer globally. Conventionally, HNC attributes to the use of alcohol or chewing tobacco. Over the past four decades, portions of human papillomavirus (HPV)-positive HNC are increasing at an alarming rate. Identification based on the etiological factors and molecular signatures demonstrates that these neoplastic lesions belong to a distinct category that differs in pathological characteristics and therapeutic response. Slow development in HNC therapeutics has resulted in a low 5-year survival rate in the last two decades. Interestingly, HPV-positive HNC has shown better outcomes following conservative treatments and immunotherapies. This raises demand to have a pre-therapy assessment of HPV status to decide the treatment strategy. Moreover, there is no HPV-specific treatment for HPV-positive HNC patients. Accumulating evidence suggests that phytochemicals are promising leads against HNC and show potential as adjuvants to chemoradiotherapy in HNC. However, only a few of these phytochemicals target HPV. The aim of the present article was to collate data on various leading phytochemicals that have shown promising results in the prevention and treatment of HNC in general and HPV-driven HNC. The review explores the possibility of using these leads against HPV-positive tumors as some of the signaling pathways are common. The review also addresses various challenges in the field that prevent their use in clinical settings.

Background: Present study examines phytochemical preparation that uses berberine’s plant source B. aquifolium root for availability of similar anti-cervical cancer (CaCx) and anti-HPV activities to facilitate repurposing of the B. aquifolium based drug in the treatment of CaCx.Purpose: To evaluate therapeutic potential of different concentrations of ethanolic extract of B. aquifolium root mother tincture (BAMT) against HPV-positive (HPV16: SiHa, HPV18: HeLa) and HPV-negative (C33a) CaCx cell lines at molecular oncogenic level.Materials and Methods: BAMT was screened for anti-proliferative activity by MTT assay. Cell cycle progression was analyzed by flowcytometry. Then, the expression level of STAT3, AP-1, HPV E6 and E7 was detected by immunoblotting, whereas nuclear localization was observed by fluorescence microscopy. Phytochemicals reportedly available in BAMT were examined for their inhibitory action on HPV16 E6 by in silico molecular docking.Results: BAMT induced a dose-dependent decline in CaCx cell viability in all cell types tested. Flowcytometric evaluation of BAMT-treated cells showed a small but specific cell growth arrest in G1-phase. BAMT-treatment resulted in reduced protein expression of key transcription factors, STAT3 with a decline of its active form pSTAT3 (Y705); and components of AP-1 complex, JunB and c-Jun. Immunocytochemistry revealed that BAMT did not prevent the entry of remnant active transcription factor to the nucleus, but loss of overall transcription factor activity resulted in reduced availability of transcription factors in the cancer cells. These changes were accompanied by gradual loss of HPV E6 and E7 protein in BAMT-treated HPV-positive cells. Molecular docking of reported active phytochemicals in B. aquifolium root was performed, which indicated a potential interference of HPV16 E6’s interaction with pivotal cellular targets p53, E6AP or both by constituent phytochemicals. Among these, berberine, palmatine and magnoflorine showed highest E6 inhibitory potential.Conclusion: Overall, BAMT showed multi-pronged therapeutic potential against HPV infection and cervical cancer and the study described the underlying molecular mechanism of its action.

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