Protocadherin 7 is overexpressed in castration resistant prostate cancer and promotes aberrant MEK and AKT signaling.

BACKGROUND: Castrate resistant prostate cancer (CRPC) accounts for almost all prostate cancer (PCa) deaths. Aberrant activation of ERK/MEK and PI3K/AKT signaling pathways plays an important role in subsets of patients with CRPC. The role of protocadherin 7 (PCDH7) in modulating these signaling pathways is investigated for the first time in PCa in the present investigation. METHODS: PCDH7 expression was analyzed in CRPC/neuroendocrine prostate cancer (NEPC) dataset. Protein expression was assessed by Western blotting and immunohistochemistry, and messenger RNA (mRNA) by quantitative real-time polymerase chain reaction. Small hairpin ribonucleic acid was used to knockdown PCDH7. Colony formation, cell migration, and invasion studies were done using standard protocols. RESULTS: PCDH7 amplification/mRNA upregulation was observed in 41% of patients in CRPC/NEPC dataset. PCDH7 was also overexpressed in CRPC cells. Increased PCDH protein expression was observed during tumor progression in PCa tissues and in TRAMP mice. Epidermal growth factor treatment resulted in aberrant activation of ERK/AKT. Knockdown of PCDH7 decreased ERK, AKT, and RB phosphorylation and reduced colony formation, decreased cell invasion, and cell migration. CONCLUSIONS: These data show for the first time that PCDH7 is overexpressed in a large number of patients with CRPC and suggest that PCDH7 may be an attractive target in subsets of patients with CRPC for whom there is no cure to-date.

Alterations in microRNAs miR-21 and let-7a correlate with aberrant STAT3 signaling and downstream effects during cervical carcinogenesis.

BACKGROUND: Present study provides clinical evidence of existence of a functional loop involving miR-21 and let-7a as potential regulators of aberrant STAT3 signaling recently reported by our group in an experimental setup (Shishodia et al. BMC Cancer 2014, 14:996). The study is now extended to a set of cervical tissues that represent natural history of human papillomavirus (HPV)-induced tumorigenic transformation. MATERIALS AND METHODS: Cervical tissues from histopathologically-confirmed pre-cancer (23) and cancer lesions (56) along with the normal control tissues (23) were examined for their HPV infection status, expression level of miR-21 & let-7a and STAT3 & pSTAT3 (Y705) by PCR-based genotyping, quantitative real-time PCR and immunoblotting. RESULTS: Analysis of cancer tissues revealed an elevated miR-21 and reduced let-7a expression that correspond to the level of STAT3 signaling. While miR-21 showed direct association, let-7a expression was inversely related to STAT3 expression and its activation. In contrast, a similar reciprocal expression kinetics was absent in LSIL and HSIL tissues which overexpressed let-7a. miR-21 was found differentially overexpressed in HPV16-positive lesions with a higher oncoprotein E6 level. Overexpression of miR-21 was accompanied by elevated level of other STAT3-regulated gene products MMP-2 and MMP-9. Enhanced miR-21 was found associated with decreased level of STAT3 negative regulator PTEN and negative regulator of MMPs, TIMP-3. CONCLUSION: Overall, our study suggests that the microRNAs, miR-21 and let-7a function as clinically relevant integral components of STAT3 signaling and are responsible for maintaining activated state of STAT3 in HPV-infected cells during cervical carcinogenesis.

Deregulation of microRNAs Let-7a and miR-21 mediate aberrant STAT3 signaling during human papillomavirus-induced cervical carcinogenesis: role of E6 oncoprotein.

BACKGROUND: Aberrantly expressed and constitutively active STAT3 signaling plays a pivotal role in initiation and progression of human papillomavirus-induced cervical carcinogenesis. However, the underlying mechanism(s) responsible for pleiotropic effects of STAT3 signaling is poorly understood. In view of emerging regulatory role of microRNAs, Let-7a and miR-21 that may interact with STAT3 signaling and/or its downstream effectors, present study was designed in HPV16-positive cervical cancer cells to assess the functional contribution of these miRs in STAT3 signaling in cervical cancer. METHODS: Functional silencing of STAT3 signaling and HPV16 oncoprotein expression in SiHa cells was done by STAT3-specific and 16 E6 siRNAs. Pharmacological intervention of STAT3 was done using specific inhibitors like curcumin and stattic. Loss-of-function study of miR-21 using miR-21 inhibitor and gain-of-function study of let-7a was done using let-7a mimic in SiHa cells. RESULTS: Functional silencing of STAT3 signaling in SiHa cells by STAT3-specific siRNA resulted in a dose-dependent decrease in cellular miR-21 level. Pharmacological intervention of STAT3 using specific inhibitors like curcumin and Stattic that abrogated STAT3 activation resulted in loss of cellular miR-21 pool. Contrary to this, specific targeting of miR-21 using miR-21 inhibitor resulted in an increased level of PTEN, a negative regulator of STAT3, and reduced active pSTAT3 level. Besides miR-21, restoration of cellular Let-7a using chemically synthesized Let-7a mimic reduced overall STAT3 level. Abrogation of HPV oncoprotein E6 by specific siRNA resulted in increased Let-7a but loss of miR-21 and a correspondingly reduced pSTAT3/STAT3 and elevated the level of cellular PTEN. CONCLUSIONS: Our results demonstrate existence of a functional loop involving Let-7a, STAT3 and miR-21 which were found potentially regulated by viral oncoprotein E6. IMPLICATIONS: miR-21 and Let-7a along with STAT3 may prove useful targets for pharmacological intervention for management of cervical cancer.

Physical state & copy number of high risk human papillomavirus type 16 DNA in progression of cervical cancer.

BACKGROUND & OBJECTIVES: High-risk human papilloma virus (HR-HPV) infection and its integration in host genome is a key event in malignant transformation of cervical cells. HPV16 being a dominant HR-HPV type, we undertook this study to analyze if viral load and physical state of the virus correlated with each other in the absence of other confounding variables and examined their potential as predictors of progressive cervical lesions. METHODS: Both, viral load and integration status of HPV16 were determined by real time URR PCR and estimation of E2:E6 ratio in a total of 130 PGMY-RLB -confirmed, monotypic HPV16-infected cervical DNA samples from biopsies of cytology-confirmed low grade (LSIL, 30) and high grade (HSIL, 30), and invasive carcinoma, (squamous cell carcinoma SCC, 70) cases. RESULTS: Investigation of DNA samples revealed a gradual increase in HPV16 viral load over several magnitudes and increased frequency of integration from LSIL to HSIL and HSIL to invasive cancer in relation to the severity of lesions in monotypic HPV16-infected cervical tissues. In a substantial number of precancer (11/60) and cancer cases (29/70), HPV16 was detected in concomitant mixed form. The concomitant form of HPV16 genome carried significantly higher viral load. INTERPRETATION & CONCLUSIONS: Overall, viral load and integration increased with disease severity and could be useful biomarkers in disease progression, at least, in HPV16-infected cervical pre-cancer and cancer lesions.

Preparation and in vitro evaluation of folate-receptor-targeted SPION-polymer micelle hybrids for MRI contrast enhancement in cancer imaging.

Polymer-SPION hybrids were investigated for receptor-mediated localization in tumour tissue. Superparamagnetic iron oxide nanoparticles (SPIONs) prepared by high-temperature decomposition of iron acetylacetonate were monodisperse (9.27 ± 3.37 nm), with high saturation magnetization of 76.8 emu g(-1). Amphiphilic copolymers prepared from methyl methacrylate and PEG methacrylate by atom transfer radical polymerization were conjugated with folic acid (for folate-receptor specificity). The folate-conjugated polymer had a low critical micellar concentration (0.4 mg l(-1)), indicating stability of the micellar formulation. SPION-polymeric micelle clusters were prepared by desolvation of the SPION dispersion/polymer solution in water. Magnetic resonance imaging of the formulation revealed very good contrast enhancement, with transverse (T(2)) relaxivity of 260.4 mM(-1) s(-1). The biological evaluation of the SPION micelles included cellular viability assay (MTT) and uptake in HeLa cells. These studies demonstrated the potential use of these nanoplatforms for imaging and targeting.

Investigation of molecular mechanisms underlying JAK/STAT signaling pathway in HPV-induced cervical carcinogenesis using 'omics' approach.

The precise mechanism of action of Janus Kinases (JAK)/Signal Transducer and activator of Transcription (STAT) signaling in human papillomavirus (HPV)-associated cervical cancer (CaCx) is poorly defined. The present study dissected the underlying components of JAK/STAT signaling in HPV-positive cervical neoplasms. Whole transcriptome profile of CaCx cohort from TCGA database revealed elevated STAT3 and its impact on CaCx patients' survival. Using the RT2 Profiler PCR Array, we analyzed 84 genes of interest associated with JAK/STAT signaling in mRNA derived from HPV-negative and HPV-positive cervical lesions which revealed 21 differentially expressed genes (DEGs). Analyses of DEGs using the Database for Annotation, Visualization and Integrated Discovery tool indicated maximum genes enriched in immune response and negative regulation of apoptotic process. Protein-protein network analysis indicated IL4, STAT5A, STAT4, and JAK3 to be the key genes in the interaction network. Further, 7 key DEGs (IL4R, IRF1, EGFR, OAS1, PIAS1, STAT4, and STAT5A) were validated in TCGA cohort using R2 platform. These genes were differentially expressed among HPV-positive cervical tissues and their correlation with STAT3 was established. EGFR and IL4R showed a comparatively strong correlation with STAT3 that supports their involvement in pathogenesis of CaCx. Finally, the Kaplan-Meier analysis established the prognostic association of the key DEGs, in CaCx cohort. The STAT3 and associated key genes discovered from our study establish a strong pathogenic role of JAK/STAT3 pathway in HPV-mediated cervical carcinogenesis.

Pancreatic cancer: genetics, disease progression, therapeutic resistance and treatment strategies.

Pancreatic cancer is a deadly disease and the third-highest cause of cancer-related deaths in the United States. It has a very low five-year survival rate (< 5%) in the United States as well as in the world (about 9%). The current gemcitabine-based therapy soon becomes ineffective because treatment resistance and surgical resection also provides only selective benefit. Signature mutations in pancreatic cancer confer chemoresistance by deregulating the cell cycle and promoting anti-apoptotic mechanisms. The stroma-rich tumor microenvironment impairs drug delivery and promotes tumor-specific immune escape. All these factors render the current treatment incompetent and prompt an urgent need for new, improved therapy. In this review, we have discussed the genetics of pancreatic cancer and its role in tumor evolution and treatment resistance. We have also evaluated new treatment strategies for pancreatic cancer, like targeted therapy and immunotherapy.

4NQO enhances differential activation of DNA repair proteins in HPV positive and HPV negative HNSCC cells.

Tobacco exposure and human papillomavirus (HPV) infection are among the main risk factors for the development of head and neck squamous cell carcinoma (HNSCC). Interestingly, recent studies show that tumors from HPV positive (HPV+) smokers and non-smokers have similar mutational profiles, which suggests that HPV could prevent mutation induction or accumulation in the intermediate risk group composed of HPV+ smokers. Hence, we tested this observation by analyzing the effects of 4-Nitroquinoline N-oxide (4NQO), a mutagen and smoking mimetic, in NOK (normal oral keratinocytes), NOKE6.E7 (NOK cells transfected with E6.E7 oncogenes of HPV), HPV+ and HPV negative (HPV-) HNSCC cells. Oxidative DNA damage, γH2AX foci formation, DNA repair protein activation, cell cycle phase analysis, apoptotic cell death, cell viability and clonogenic cell survival were analyzed after 4NQO treatment in NOK, NOKE6.E7, HPV+ and HPV- HNSCC cells. 4NQO increased oxidative base damage and γH2AX foci formation in NOKE6.E7, HPV+ and HPV- HNSCC cells. Phosphorylation of homologous recombination (HR) repair proteins was higher in NOKE6.E7 and HPV+ HNSCC cells compared to NOK and HPV- HNSCC cells respectively. HPV+ and HPV- HNSCC cells showed differential activation of cell cycle regulatory proteins, increased apoptosis, and decreased cell viability upon 4NQO-induced DNA damage. Taken together, 4NQO (a smoking mimetic), induced higher activation of HR repair in HPV+ HNSCC cells compared to HPV- HNSCC cells. This may allow for increased mutational resistance and help explain why HPV+ smokers have a worse prognosis than HPV+ non-smokers.

Aberrant expression and constitutive activation of STAT3 in cervical carcinogenesis: implications in high-risk human papillomavirus infection.

BACKGROUND: Recent observations indicate potential role of transcription factor STAT3 in cervical cancer development but its role specifically with respect to HPV infection is not known. Present study has been designed to investigate expression and activation of STAT3 in cervical precancer and cancer in relation to HPV infection during cervical carcinogenesis. Established cervical cancer cell lines and prospectively-collected cervical precancer and cancer tissues were analyzed for the HPV positivity and evaluated for STAT3 expression and its phosphorylation by immunoblotting and immunohistochemistry whereas STAT3-specific DNA binding activity was examined by gel-shift assays. RESULTS: Analysis of 120 tissues from cervical precancer and cancer lesions or from normal cervix revealed differentially high levels of constitutively active STAT3 in cervical precancer and cancer lesions, whereas it was absent in normal controls. Similarly, a high level of constitutively active STAT3 expression was observed in HPV-positive cervical cancer cell lines when compared to that of HPV-negative cells. Expression and activity of STAT3 were found to change as a function of severity of cervical lesions from precancer to cancer. Expression of active pSTAT3 was specifically high in cervical precancer and cancer lesions found positive for HPV16. Interestingly, site-specific accumulation of STAT3 was observed in basal and suprabasal layers of HPV16-positive early precancer lesions which is indicative of possible involvement of STAT3 in establishment of HPV infection. In HPV16-positive cases, STAT3 expression and activity were distinctively higher in poorly-differentiated lesions with advanced histopathological grades. CONCLUSION: We demonstrate that in the presence of HPV16, STAT3 is aberrantly-expressed and constitutively-activated in cervical cancer which increases as the lesion progresses thus indicating its potential role in progression of HPV16-mediated cervical carcinogenesis.

Reactive oxygen species and cancer: A complex interaction.

Elevated levels of Reactive Oxygen Species (ROS), increased antioxidant ability and the maintenance of redox homeostasis can cumulatively contribute to tumor progression and metastasis. The sources and the role of ROS in a heterogeneous tumor microenvironment can vary at different stages of tumor: initiation, development, and progression, thus making it a complex subject. In this review, we have summarized the sources of ROS generation in cancer cells, its role in the tumor microenvironment, the possible functions of ROS and its important scavenger systems in tumor progression with special emphasis on solid tumors.

Level of phospho-STAT3 (Tyr705) correlates with copy number and physical state of human papillomavirus 16 genome in cervical precancer and cancer lesions.

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.

Tetrandrine (TET) Induces Death Receptors Apo Trail R1 (DR4) and Apo Trail R2 (DR5) and Sensitizes Prostate Cancer Cells to TRAIL-Induced Apoptosis.

TNF-related apoptosis-inducing ligand (TRAIL) selectively induces apoptosis in cancer cells, but not in normal cells; as such, it is a promising therapeutic agent. However, therapeutic resistance limits its clinical use in many malignancies, including prostate cancer. Strategies to sensitize cancer cells to TRAIL are urgently needed. We demonstrate here that small-molecule tetrandrine (TET) potentially sensitizes previously resistant (LNCaP and C4-2B cells) and mildly sensitive (PC3 cells) prostate cancer cells to TRAIL-induced apoptosis, and they do so by upregulating mRNA expression and protein levels of death receptors Apo Trail R1 (DR4) and Apo Trail R2 (DR5). Using shRNA knockdown, we show critical requirement of DR4 and DR5 in sensitization of prostate cancer cells to TRAIL. We show that double knockdown of DR4 and DR5 abrogated the apoptotic effects of TET and TRAIL. We also demonstrate that TET-induced DR4 and DR5 expression is independent of p53 status. Given that loss of p53 is associated with progression of prostate cancer to CRPC and NEPC, our results show that TET, by acting as a TRAIL-sensitizing agent in prostate cancer, could serve as a potential therapeutic agent in CRPC and NEPC, for which there is no cure to date. Mol Cancer Ther; 17(6); 1217-28. ©2018 AACR.

miRNA as viral transcription tuners in HPV-mediated cervical carcinogenesis.

High-risk human papillomaviruses (HPVs) are oncogenic DNA viruses that promote carcinogenic signaling by their oncoproteins mainly E6 and E7. A well-defined promoter regulates expression and enhancer region on HPV genome containing number of cis elements that essentially require a set of cognate host transcription factors to regulate viral promoter gene activity. Expression of these host factors is tightly regulated at multiple levels such as transcriptional, post-transcriptional and post-translational level. Discovery of microRNAs (miRs) in recent years and differential expression of a set of specific miRs in HPV infection and cervical lesions indicate that among various regulatory mechanisms, role of these differentially expressed miRs in the post-transcriptional control is pivotal. Present review analyses and attempts to compile currently available miR data related to HPV infection and cervical carcinogenesis with a special focus on miRs that may regulate expression of the host and viral factors particularly responsible for viral transcription leading to carcinogenic progression of the lesion. Further, the review attempts to assess the therapeutic potential of miR-based strategies in therapeutic targeting of HPV infection during cervical carcinogenesis.

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.