Skp2 and Slug Are Coexpressed in Aggressive Prostate Cancer and Inhibited by Neddylation Blockade.

Prostate cancer (PCa) is the second leading cause of cancer-related deaths in men in Western countries, and there is still an urgent need for a better understanding of PCa progression to inspire new treatment strategies. Skp2 is a substrate-recruiting component of the E3 ubiquitin ligase complex, whose activity is regulated through neddylation. Slug is a transcriptional repressor involved in the epithelial-to-mesenchymal transition, which may contribute to therapy resistance. Although Skp2 has previously been associated with a mesenchymal phenotype and prostate cancer progression, the relationship with Slug deserves further elucidation. We have previously shown that a high Gleason score (≥8) is associated with higher Skp2 and lower E-cadherin expression. In this study, significantly increased expression of Skp2, AR, and Slug, along with E-cadherin downregulation, was observed in primary prostate cancer in patients who already had lymph node metastases. Skp2 was slightly correlated with Slug and AR in the whole cohort (Rs 0.32 and 0.37, respectively), which was enhanced for both proteins in patients with high Gleason scores (Rs 0.56 and 0.53, respectively) and, in the case of Slug, also in patients with metastasis to lymph nodes (Rs 0.56). Coexpression of Skp2 and Slug was confirmed in prostate cancer tissues by multiplex immunohistochemistry and confocal microscopy. The same relationship between these two proteins was observed in three sets of prostate epithelial cell lines (PC3, DU145, and E2) and their mesenchymal counterparts. Chemical inhibition of Skp2, but not RNA interference, modestly decreased Slug protein in PC3 and its docetaxel-resistant subline PC3 DR12. Importantly, chemical inhibition of Skp2 by MLN4924 upregulated p27 and decreased Slug expression in PC3, PC3 DR12, and LAPC4 cells. Novel treatment strategies targeting Skp2 and Slug by the neddylation blockade may be promising in advanced prostate cancer, as recently documented for other aggressive solid tumors.

Aspartate-ß-hydroxylase and hypoxia marker expression in head and neck carcinomas: implications for HPV-associated tumors.

BACKGROUND: A proportion of head and neck carcinomas (HNSCCs) are induced by high-risk human papillomaviruses (HPVs) and are associated with better patient outcomes compared to patients with HNSCCs related to tobacco and alcohol abuse. In the microenvironment of solid tumors, including HNSCCs, oxygen levels are often reduced, and a hypoxic state is induced. This can lead to a poor treatment response and a worse patient prognosis. One of the hypoxia-responsive genes is aspartate-ß-hydroxylase (ASPH), whose activity promotes the growth, invasiveness, and metastasis of many types of solid tumors. METHODS: In our study, HNSCC samples were analyzed for the expression of ASPH and selected endogenous hypoxia markers by real-time PCR and/or multiplex fluorescence immunohistochemistry. RESULTS: Except for the EPAS1 gene, which had higher mRNA expression in the HPV-negative group of HNSCC (p < 0.05), we found no other differences in the expression of the tested genes that were related to HPV status. On the contrary, a statistically significantly higher number of cells producing ASPH (p < 0.0001), HIF1A (p < 0.0001), GLUT1 (p < 0.0001), and MMP13 (p < 0.05) proteins were detected in the HPV-positive tumor group than in the HPV-negative sample group. All the evaluated markers, except for MMP9/13, were more abundant in the tumor parenchyma than in the tumor stroma. The Cox proportional hazard models showed that increased numbers of cells with GLUT1 and HIF1A protein expression were positive prognostic markers for overall and disease-specific survival in patients independent of HPV tumor status. CONCLUSION: The study examined HNSCC samples and found that elevated ASPH and hypoxia marker proteins, typically associated with poor prognosis, may actually indicate active HPV infection, the strongest prognostic factor in HNSCC patients. In cases where HPV status is uncertain, increased expression of HIF1A and GLUT1 can serve as positive prognostic factors.

PD1+CD8+ Cells Are an Independent Prognostic Marker in Patients with Head and Neck Cancer.

Head and neck squamous cell carcinomas (HNSCCs) belong to a group of diverse tumors, which can be induced by infection with human papillomavirus (HPV) or tobacco and alcohol consumption. The viral etiology of HNSCC relates to better clinical outcomes reflecting a different immune system response. Here, we retrospectively analyzed 97 tissue samples from oral and oropharyngeal carcinomas associated and non-associated with HPV infection using multispectral fluorescent immunohistochemistry. To evaluate the immune cell infiltration in tumor and stroma compartments, we designed four panels of four to five antibodies. We detected more T lymphocytes in the stroma, compared to the tumor parenchyma. In HPV positive (HPV+) in comparison to HPV negative (HPV-) tumors, higher counts of CD3+CD4+, CD3+CD8+, PD1+CD4+, PD1+CD8+ T cells, and ICOS- Treg cells were detected while more ICOS+ Treg cells and CTLA4+CD4+ T cells were observed in HPV- than in HPV+ tumors. The results of the univariate and multivariate analyses confirmed the predominant impact of HPV status on prognosis. More importantly, the number of CD8+PD-1+ T cells was identified as an independent factor, influencing the overall and/or disease-specific survival of patients with oral cavity or oropharyngeal carcinomas.

ARG1 mRNA Level Is a Promising Prognostic Marker in Head and Neck Squamous Cell Carcinomas.

Head and neck squamous cell carcinomas (HNSCC) can be induced by smoking or alcohol consumption, but a growing part of cases relate to a persistent high-risk papillomavirus (HPV) infection. Viral etiology has a beneficial impact on the prognosis, which may be explained by a specific immune response. Tumor associated macrophages (TAMs) represent the main immune population of the tumor microenvironment with a controversial influence on the prognosis. In this study, the level, phenotype, and spatial distribution of TAMs were evaluated, and the expression of TAM-associated markers was compared in HPV positive (HPV+) and HPV negative (HPV-) tumors. Seventy-three formalin and embedded in paraffin (FFPE) tumor specimens were examined using multispectral immunohistochemistry for the detection of TAM subpopulations in the tumor parenchyma and stroma. Moreover, the mRNA expression of TAM markers was evaluated using RT-qPCR. Results were compared with respect to tumor etiology, and the prognostic significance was evaluated. In HPV- tumors, we observed more pro-tumorigenic M2 in the stroma and a non-macrophage arginase 1 (ARG1)-expressing population in both compartments. Moreover, higher mRNA expression of M2 markers-cluster of differentiation 163 (CD163), ARG1, and prostaglandin-endoperoxide synthase 2 (PTGS2)-was detected in HPV- patients, and of M1 marker nitric oxide synthase 2 (NOS2) in HPV+ group. The expression of ARG1 mRNA was revealed as a negative prognostic factor for overall survival of HNSCC patients.

Detailed Characteristics of Tonsillar Tumors with Extrachromosomal or Integrated Form of Human Papillomavirus.

The human papillomavirus (HPV) integration, the critical step in viral carcinogenesis, most frequently occurs in the E2 gene, which results in its inactivation and in an increase of E6/E7 transcription. However, in a substantial number of tumors, the virus is present in an extrachromosomal form. For those tumors, the transformation mechanisms are not fully elucidated. Here we evaluated the possible mechanism of inactivating the E2 without interruption of the gene, methylation or mutation of the E2 binding sites (E2BSs) in HPV16-positive tonsillar tumors by next-generation and Sanger sequencing. Viral genome status was analyzed by the amplification of papillomavirus oncogene transcripts assay (APOT) and mRNA mapping, and expression of viral oncogenes was performed by qPCR. The methylation of E2BSs was significantly higher in tumors with an integrated, in comparison to extrachromosomal, form of the viral genome. No mutations were detected in the E2BSs. The viral oncogenes were equally expressed in samples with an integrated and extrachromosomal form of the virus. Only the nucleotide variants were identified in the E2 gene. No proposed mechanism of E2 inactivation was confirmed in tonsillar tumors with an extrachromosomal form of the HPV genome. The expression of E6/E7 genes seems to be sufficient to initiate and maintain the carcinogenic process.

Implementation of Mass Cytometry for Immunoprofiling of Patients with Solid Tumors.

Monitoring immune responses to solid cancers may be a better prognostic tool than conventional staging criteria, and it can also serve as an important criterion for the selection of individualized therapy. Multiparametric phenotyping by mass cytometry extended possibilities for immunoprofiling. However, careful optimization of each step of such method is necessary for obtaining reliable results. Also, with respect to procedure length and costs, sample preparation, staining, and storage should be optimized. Here, we designed a panel of 31 antibodies which allows for identification of several subpopulations of lymphoid and myeloid cells in a solid tumor and peripheral blood simultaneously. For sample preparation, disaggregation of tumor tissue with two different collagenases combined with DNase I was compared, and removal of dead or tumor cells by magnetic separation was evaluated. Two possible procedures of barcoding for single-tube staining of several samples were examined. While the palladium-based barcoding affected the stability of several antigens, the staining with two differently labeled CD45 antibodies was suitable for cells isolated from a patient's blood and tumor. The storage of samples in the intercalation solution for up to two weeks did not influence results of the analysis, which allowed the measurement of samples collected within this interval on the same day. This procedure optimized on samples from patients with head and neck squamous cell carcinoma enabled identification of various immune cells including rare subpopulations.