Interferon-beta induces S phase slowing via up-regulated expression of PML in squamous carcinoma cells.

Interferon-β induces S phase slowing via up-regulated expression of PML in squamous carcinoma cell

Role of the microenvironment in tumorigenesis. Focus on virus-induced tumors

Tumor microenvironment can differ considerably in various types of tumors in terms of cellular and cytokine networks and molecular drivers. The well known link between inflammation and cancer has recently found a number of genetic and molecular confirmations. In this respect, numerous reports have revealed that infection and chronic inflammation can contribute to cancer development, progression and control. Adhesion molecules, chemokines and proinflammatory cytokines, that enroll leukocytes, are persistently present in cancer microenvironment, thus increasing the risk for developing tumors. In this respect, cancer-derived microvescicles, in particular exosomes, exert an important role in the recruitment and reprogramming of components of tumor microenvironment. The relationship between cancer and virus infection has generated, in recent years, a great interest for studies aiming to better understand the role of the immune system in the control of these infections and of the immune co-factors in the promotion of the virus-induced neoplastic transformation. This suggests that virus-induced immune alterations may play a role to create an immunotolerogenic microenvironment during the carcinogenesis process

Pro-inflammatory cytokines analysis in HPV-positive cancer cells

Recent data expanded the concept that inflammation is a critical component of tumor progression. The tumor microenvironment is an indispensable participant in the neoplastic process, fostering proliferation, survival and migration. Tumor cells co-opted signalling molecules of the innate immune system, such as selectins, chemokines and their receptors for invasion, migration and metastasis. In this respect, Virus-induced tumors, like Papillomavirus-induced Squamous Cell Carcinomas (SCC), could represent a paradigmatic example of interplay between inflammatory responses and malignant transformation. To establish a tumorigenic role of inflammatory mediators in HPV+ SCC, we analyzed by real time RT-PCR the expression of inflammatory cytokines, chemokines and related molecules in HPV+ carcinoma cell lines, HPV- SCC (C33A) and in human foreskin keratinocytes transduced by E6 and E7 derived from mucosal HPV-16 or cutaneous HPV-38 genotypes. We also tested the effect of the IFN-β on the levels of these pro-inflammatory mediators. In HPV+ SCC the level of both IL-1β and IL6 mRNAs were augmented if compared to HPV- SCC. Interestingly, levels of cytokines in supernantants, tested by CBA assay, revealed that despite huge increase in mRNA levels, these cells didn’t secrete IL-1β, whereas IL-6 appeared to be released by HPV+ SCC

Interferon-β induces cellular senescence in cutaneous human papilloma virus-transformed human keratinocytes by affecting p53 transactivating activity

Interferon (IFN)-beta inhibits cell proliferation and affects cell cycle in keratinocytes transformed by both mucosal high risk Human Papilloma Virus (HPV) and cutaneous HPV E6 and E7 proteins. In particular, upon longer IFN-beta treatments, cutaneous HPV38 expressing cells undergo senescence. IFN-beta appears to induce senescence by upregulating the expression of the tumor suppressor PML, a well known IFN-induced gene. Indeed, experiments in gene silencing via specific siRNAs have shown that PML is essential in the execution of the senescence programme and that both p53 and p21 pathways are involved. IFN-beta treatment leads to a modulation of p53 phosphorylation and acetylation status and a reduction in the expression of the p53 dominant negative Delta Np73. These effects allow the recovery of p53 transactivating activity of target genes involved in the control of cell proliferation. Taken together, these studies suggest that signaling through the IFN pathway might play an important role in cellular senescence. This additional understanding of IFN antitumor action and mechanisms influencing tumor responsiveness or resistance appears useful in aiding further promising development of biomolecular strategies in the IFN therapy of cancer

IFN-β induces senescence in K38 cells.

<p>(A) Control keratinocytes (LXSN), K16, K38 cells and high risk HPV-positive squamous carcinoma cell lines ME-180, Caski, HeLa and SiHa were treated with IFN-β for 4 days and senescent cells were quantified by counting cells displaying senescent-associated β-galactosidase (SA-βgal) activity at pH 6.0. (B) Percentage of senescent cells in K16 and K38 cells treated with IFN-β for different time points. (C) SA-βgal-positive K38 blue cells observed under a light microscope after 4 days of IFN-β treatment. * = p<0.05; *** = p<0.001.</p

IFN-β effect on p53 post-translational modification and expression of its target genes.

<p>(A) WB analysis of p53 in control keratinocytes (LXSN), K16 and K38 cells and in high risk HPV-positive squamous carcinoma cell lines SiHa and ME-180 treated with IFN-β for 48 h. (B) WB analysis of p53 phosphorylated at different phosphorylation sites. (C) WB analysis of acetylated p53. (D) WB analysis of phosphorylated and acetylated p53 and ΔNp73 in K38 cells silenced by PML siRNA and treated with IFN-β for 48 h. (E) WB analysis of ΔNp73 in K38 cells treated with IFN-β for different time points. Whole cell extracts were resolved on SDS-PAGE and transferred onto PVDF membrane. Immunoblotting was performed as reported in M&M. (F) Real time PCR analysis of Bax and Pig3 was carried out on K38 cells treated with IFN-β for 48 h, also in the presence of PML siRNA. NT  =  not transfected. * = p<0.05; ** = p<0.01.</p

p53 and ΔNp73 co-localyze with IFN-β-induced PML into PML Nuclear Bodies.

<p>(A,B) For confocal microscopy analysis, K38 cells were cultured on glass bottom dishes (MatTek Corporation) and treated with IFN-β for 4 days. Cells were then fixed in PBS 4% paraformaldehyde for 30 min on ice, immuno-fluorescence labelling was performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036909#s4" target="_blank">Materials and Methods</a> and sample were analyzed using confocal microscope (Leica TCS SP5).</p