Identification of microRNAs that stabilize p53 in HPV-positive cancer cells

Etiologically, 5% of all cancers worldwide are caused by the high-risk human papillomaviruses (hrHPVs). These viruses encode two oncoproteins (E6 and E7) whose expression is required for cancer initiation and maintenance. Among their cellular targets are the p53 and the retinoblastoma tumor suppressor proteins. Inhibition of the hrHPV E6-mediated ubiquitylation of p53 through the E6AP ubiquitin ligase results in the stabilization of p53, leading to cellular apoptosis. We utilized a live cell high throughput screen to determine whether exogenous microRNA (miRNA) transfection had the ability to stabilize p53 in hrHPV-positive cervical cancer cells expressing a p53-fluorescent protein as an in vivo reporter of p53 stability. Among the miRNAs whose transfection resulted in the greatest p53 stabilization was 375-3p that has previously been reported to stabilize p53 in HeLa cells, providing validation of the screen. The top 32 miRNAs in addition to 375-3p were further assessed using a second cell-based p53 stability reporter system as well as in non-reporter HeLa cells to examine their effects on endogenous p53 protein levels, resulting in the identification of 23 miRNAs whose transfection increased p53 levels in HeLa cells. While a few miRNAs that stabilized p53 led to decreases in E6AP protein levels, all targeted HPV oncoprotein expression. We further examined subsets of these miRNAs for their abilities to induce apoptosis and determined whether it was p53-mediated. The introduction of specific miRNAs revealed surprisingly heterogeneous responses in different cell lines. Nonetheless, some of the miRNAs described here have potential as therapeutics for treating HPV-positive cancers. Importance Human papillomaviruses cause approximately 5% of all cancers worldwide and encode genes that contribute to both the initiation and maintenance of these cancers. The viral oncoprotein E6 is expressed in all HPV-positive cancers and functions by targeting the degradation of p53 through the engagement of the cellular ubiquitin ligase E6AP. Inhibiting the degradation of p53 leads to apoptosis in HPV-positive cancer cells. Using a high throughput live cell assay we identified several miRNAs whose transfection stabilize p53 in HPV-positive cells. These miRNAs have the potential to be used in the treatment of HPV-positive cancers.

Paradoxical effects of DNA tumor virus oncogenes on epithelium-derived tumor cell fate during tumor progression and chemotherapy response

Epstein-Barr virus (EBV) and human papillomavirus (HPV) infection is the risk factors for nasopharyngeal carcinoma and cervical carcinoma, respectively. However, clinical analyses demonstrate that EBV or HPV is associated with improved response of patients, although underlying mechanism remains unclear. Here, we reported that the oncoproteins of DNA viruses, such as LMP1 of EBV and E7 of HPV, inhibit PERK activity in cancer cells via the interaction of the viral oncoproteins with PERK through a conserved motif. Inhibition of PERK led to increased level of reactive oxygen species (ROS) that promoted tumor and enhanced the efficacy of chemotherapy in vivo. Consistently, disruption of viral oncoprotein-PERK interactions attenuated tumor growth and chemotherapy in both cancer cells and tumor-bearing mouse models. Our findings uncovered a paradoxical effect of DNA tumor virus oncoproteins on tumors and highlighted that targeting PERK might be an attractive strategy for the treatment of NPC and cervical carcinoma.

Androgen receptor transactivates KSHV noncoding RNA PAN to promote lytic replication–mediated oncogenesis: A mechanism of sex disparity in KS

Kaposi’s sarcoma-associated herpesvirus (KSHV) preferentially infects and causes Kaposi’s sarcoma (KS) in male patients. However, the biological mechanisms are largely unknown. This study was novel in confirming the extensive nuclear distribution of the androgen receptor (AR) and its co-localization with viral oncoprotein of latency-associated nuclear antigen in KS lesions, indicating a transcription way of AR in KS pathogenesis. The endogenous AR was also remarkably higher in KSHV-positive B cells than in KSHV-negative cells and responded to the ligand treatment of 5α-dihydrotestosterone (DHT), the agonist of AR. Then, the anti-AR antibody-based chromatin immunoprecipitation (ChIP)-associated sequencing was used to identify the target viral genes of AR, revealing that the AR bound to multiple regions of lytic genes in the KSHV genome. The highest peak was enriched in the core promoter sequence of polyadenylated nuclear RNA (PAN), and the physical interaction was verified by ChIP–polymerase chain reaction (PCR) and the electrophoretic mobility shift assay (EMSA). Consistently, male steroid treatment significantly transactivated the promoter activity of PAN in luciferase reporter assay, consequently leading to extensive lytic gene expression and KSHV production as determined by real-time quantitative PCR, and the deletion of nuclear localization signals of AR resulted in the loss of nuclear transport and transcriptional activity in the presence of androgen and thus impaired the expression of PAN RNA. Oncogenically, this study identified that the AR was a functional prerequisite for cell invasion, especially under the context of KSHV reactivation, through hijacking the PAN as a critical effector. Taken together, a novel mechanism from male sex steroids to viral noncoding RNA was identified, which might provide a clue to understanding the male propensity in KS.

Expression of the E5 Oncoprotein of HPV16 Impacts on the Molecular Profiles of EMT-Related and Differentiation Genes in Ectocervical Low-Grade Lesions

Infection with human papillomavirus type 16 (HPV16) is one of the major risk factors for the development of cervical cancer. Our previous studies have demonstrated the involvement of the early oncoprotein E5 of HPV16 (16E5) in the altered isoform switch of fibroblast growth factor receptor 2 (FGFR2) and the consequent expression in human keratinocytes of the mesenchymal FGFR2c isoform, whose aberrant signaling leads to EMT, invasiveness, and dysregulated differentiation. Here, we aimed to establish the possible direct link between these pathological features or the appearance of FGFR2c and the expression of 16E5 in low-grade squamous intraepithelial lesions (LSILs). Molecular analysis showed that the FGFR2c expression displayed a statistically significant positive correlation with that of the viral oncoprotein, whereas the expression values of the epithelial FGR2b variant, as well as those of the differentiation markers keratin 10 (K10), loricrin (LOR) and involucrin (INV), were inversely linked to the 16E5 expression. In contrast, the expression of EMT-related transcription factors Snail1 and ZEB1 overlapped with that of 16E5, becoming a statistically significant positive correlation in the case of Snail2. Parallel analysis performed in human cervical LSIL-derived W12 cells, containing episomal HPV16, revealed that the depletion of 16E5 by siRNA was able to counteract these molecular events, proving to represent an effective strategy to identify the specific role of this viral oncoprotein in determining LSIL oncogenic and more aggressive profiles. Overall, coupling in vitro approaches to the molecular transcript analysis in ectocervical early lesions could significantly contribute to the characterization of specific gene expression profiles prognostic for those LSILs with a greater probability of direct neoplastic progression.

Snapshot of Clinical Implications of p16 Overexpression in Carcinogenesis

Molecular markers are needed to decide the treatment plans of certain cancer types when the histological and other clinical diagnoses are not sufficient to decide the tumor nodular metastasis (TNM) stage. The ubiquitous p16 gene is one of them gained popularity by fulfilling criteria to be a useful biomarker. Over expression of p16, to compensate the inactivity of another two tumor suppressor genes (TSG)s, pRb and p53 due to the integration of E7 and E6 high risk Human Papilloma viral oncoprotein respectively into the host keratinocytes is useful to consider the clinical impact of p16 biomarker in carcinogenesis. The p16 immunohistochemistry helps the diagnosis as well as prognosis of cervical and oropharyngeal squamous cell carcinoma, though there are ambiguities in the cutoff values of p16 positivity. There is also a re-emerging interest on clinical impact of p16 positivity in lung, breast, and colorectal cancer types. High risk HPV genotypes have been already established as the aetiological agents of cervical, other rare ano-genital and oropharyngeal (especially tonsils and base of the tongue) cancers. The HPV associated subset of head and neck cancers demonstrate a unique tumor biology, when compared with HPV non associated ones thus, most effective treatment planning including counselling is much needed to maximize the overall survival of HPV associated cancer patients, in the era of personalized precision medicine. In the shed of light, this communication glimpses on clinical implications of p16 overexpression in carcinogenesis not limiting to cervical and a subset of head and neck carcinomas (HNSCC).

Nuclear receptor corepressor (NCoR) is a positive prognosticator for cervical cancer

Purpose Enzymes with epigenetic functions play an essential part in development of cancer. However, the significance of epigenetic changes in cervical carcinoma as a prognostic factor has not been fully investigated. Nuclear receptor corepressor (NCoR) presents itself as a potentially important element for epigenetic modification and as a potential prognostic aspect in cervical cancer. Methods By immunohistochemical staining of 250 tumor samples, the expression strength of NCoR was measured and evaluated by immunoreactive score (IRS) in the nucleus and cytoplasm. Results A low expression of NCoR in our patients was a disadvantage in overall survival. Expression of NCoR was negatively correlated with viral oncoprotein E6, acetylated histone H3 acetyl K9 and FIGO status, and positively correlated to p53. Conclusions Our study has identified epigenetic modification of tumor cells thus seems to be of relevance in cervical cancer as well for diagnosis, as a marker or as a potential therapeutic target in patients with advanced cervical carcinoma.

Crk and CrkL as Therapeutic Targets for Cancer Treatment

Crk and CrkL are cellular counterparts of the viral oncoprotein v-Crk. Crk and CrkL are overexpressed in many types of human cancer, correlating with poor prognosis. Furthermore, gene knockdown and knockout of Crk and CrkL in tumor cell lines suppress tumor cell functions, including cell proliferation, transformation, migration, invasion, epithelial-mesenchymal transition, resistance to chemotherapy drugs, and in vivo tumor growth and metastasis. Conversely, overexpression of tumor cells with Crk or CrkL enhances tumor cell functions. Therefore, Crk and CrkL have been proposed as therapeutic targets for cancer treatment. However, it is unclear whether Crk and CrkL make distinct or overlapping contributions to tumor cell functions in various cancer types because Crk or CrkL have been examined independently in most studies. Two recent studies using colorectal cancer and glioblastoma cells clearly demonstrated that Crk and CrkL need to be ablated individually and combined to understand distinct and overlapping roles of the two proteins in cancer. A comprehensive understanding of individual and overlapping roles of Crk and CrkL in tumor cell functions is necessary to develop effective therapeutic strategies. This review systematically discusses crucial functions of Crk and CrkL in tumor cell functions and provides new perspectives on targeting Crk and CrkL in cancer therapy.

Loss of interleukin-10 activates innate immunity to eradicate adult T cell leukemia initiating cells

Adult T cell leukemia/lymphoma (ATL) is associated to chronic human T cell leukemia virus type 1 (HTLV-1) infection and carries a poor prognosis. Arsenic trioxide (AS) and interferon-alpha (IFNα) together selectively trigger Tax viral oncoprotein degradation and cure Tax-driven murine ATL. AS/IFNα/zidovudine treatment achieves a high response rate in patients with chronic ATL. Interleukin 10 (IL-10) is an immuno-suppressive cytokine whose expression is activated by Tax. Here we show that, in ATL, AS/IFNα-induced abrogation of leukemia initiating cell activity requires IL-10 expression shutoff. Loss of IL-10 secretion drives production of inflammatory cytokines by the microenvironment, followed by innate immunity-mediated clearance of Taxdriven leukemic cells. Accordingly, anti-IL-10 monoclonal antibodies significantly increased the efficiency of AS/IFNα therapy. These results emphasize the sequential targeting of malignant ATL cells and their immune microenvironment in leukemia initiating cell (LIC) eradication and provide a strong rational to test AS/IFNα/anti-IL10 combination in ATL.

Equine Sarcoids—Causes, Molecular Changes, and Clinicopathologic Features: A Review

Equine sarcoid is the most common skin tumor of horses. Clinically, it occurs as a locally invasive, fibroblastic, wart-like lesion of equine skin, which has 6 clinical classes: occult, verrucose, nodular, fibroblastic, mixed, and malignant. Sarcoids may be single but multiple lesions are more frequent. The typical histological feature is increased density of dermal fibroblasts which form interlacing bundles and whorls within the dermis. Lesions are mostly persistent, resist therapy, and tend to recur following treatment. In general, sarcoids are not fatal but their location, size, and progression to the more aggressive form may lead to the withdrawal of a horse from use and serious infringement of their welfare leading to the loss of valuable animals. Bovine papillomavirus (BPV) type 1 and less commonly type 2 contribute to the development of equine sarcoid. The viral genome and proteins are detected in a high percentage of cases. Furthermore, viral oncoprotein activity leads to changes in the fibroblastic tissue similar to changes seen in other types of tumors. Equine sarcoids are characterized by a loss of tumor suppressor activity and changes allowing abnormal formation of the affected tissue, as well as y immune defense abnormalities that weaken the host’s immune response. This impaired immune response to BPV infection appears to be crucial for the development of lesions that do not spontaneously regress, as occurs in BPV-infected cows.