In modulated electrohyperthermia (mEHT) the enrichment of electric field and the concomitant heat can selectively induce cell death in malignant tumors as a result of elevated glycolysis, lactate production (Warburg effect), and reduced electric impedance in cancer compared to normal tissues. Earlier, we showed in HT29 colorectal cancer xenografts that the mEHTprovoked programmed cell death was dominantly caspase independent and driven by apoptosis inducing factor activation. Using this model here, we studied the mEHT-related cell stress 0-, 1-, 4-, 8-, 14-, 24-, 48-, 72-, 120-, 168-and 216-h post-treatment by focusing on damage-associated molecular pattern (DAMP) signals.

Modulated EHT treatment can induce programmed cell death-related tumor destruction in HT29 colorectal adenocarcinoma xenografts, which dominantly follows a caspase-independent subroutine.

Background and AimsConnexins and their cell membrane channels contribute to the control of cell proliferation and compartmental functions in breast glands and their deregulation is linked to breast carcinogenesis. Our aim was to correlate connexin expression with tumor progression and prognosis in primary breast cancers.Materials and MethodsMeta-analysis of connexin isotype expression data of 1809 and 1899 breast cancers from the Affymetrix and Illumina array platforms, respectively, was performed. Expressed connexins were also monitored at the protein level in tissue microarrays of 127 patients equally representing all tumor grades, using immunofluorescence and multilayer, multichannel digital microscopy. Prognostic correlations were plotted in Kaplan-Meier curves and tested using the log-rank test and cox-regression analysis in univariate and multivariate models.ResultsThe expression of GJA1/Cx43, GJA3/Cx46 and GJB2/Cx26 and, for the first time, GJA6/Cx30 and GJB1/Cx32 was revealed both in normal human mammary glands and breast carcinomas. Within their subfamilies these connexins can form homo- and heterocellular epithelial channels. In cancer, the array datasets cross-validated each other’s prognostic results. In line with the significant correlations found at mRNA level, elevated Cx43 protein levels were linked with significantly improved breast cancer outcome, offering Cx43 protein detection as an independent prognostic marker stronger than vascular invasion or necrosis. As a contrary, elevated Cx30 mRNA and protein levels were associated with a reduced disease outcome offering Cx30 protein detection as an independent prognostic marker outperforming mitotic index and necrosis. Elevated versus low Cx43 protein levels allowed the stratification of grade 2 tumors into good and poor relapse free survival subgroups, respectively. Also, elevated versus low Cx30 levels stratified grade 3 patients into poor and good overall survival subgroups, respectively.ConclusionDifferential expression of Cx43 and Cx30 may serve as potential positive and negative prognostic markers, respectively, for a clinically relevant stratification of breast cancers.

The 180 kDa transmembrane collagen XVII is known to anchor undifferentiated keratinocytes to the basement membrane in hemidesmosomes while constitutively shedding a 120 kDa ectodomain. Inherited mutations or auto-antibodies targeting collagen XVII cause blistering skin disease. Collagen XVII is down-regulated in mature keratinocytes but re-expressed in skin cancer. By recently detecting collagen XVII in melanocyte hyperplasia, here we tested its expression in benign and malignant melanocytic tumors using endodomain and ectodomain selective antibodies. We found the full-length collagen XVII protein in proliferating tissue melanocytes, basal keratinocytes and squamous cell carcinoma whereas resting melanocytes were negative. Furthermore, the cell-residual 60 kDa endodomain was exclusively detected in 62/79 primary and 15/18 metastatic melanomas, 8/9 melanoma cell lines, HT199 metastatic melanoma xenografts and atypical nests in 8/63 dysplastic nevi. The rest of 19 nevi including common, blue and Spitz subtypes were also negative. In line with the defective ectodomain, sequencing of COL17A1 gene revealed aberrations in the ectodomain coding region including point mutations. Collagen XVII immunoreaction-stained spindle cell melanomas, showed partly overlapping profiles with those of S100B, Melan A and HMB45. It was concentrated at vertical melanoma fronts and statistically associated with invasive phenotype. Antibody targeting the extracellular aa507-529 terminus of collagen XVII endodomain promoted apoptosis and cell adhesion, while inhibiting proliferation in HT199 cells. These results suggest that the accumulation of collagen XVII endodomain in melanocytic tumors is associated with malignant transformation to be a potential marker of malignancy and a target for antibody-induced melanoma apoptosis.

BackgroundSeveral classification systems are available to assess pathological response to neoadjuvant chemotherapy in breast cancer, but reliable biomarkers to predict the efficiency of primary systemic therapy (PST) are still missing. Deregulation of gap junction channel forming connexins (Cx) has been implicated in carcinogenesis and tumour progression through loss of cell cycle control. In this study we correlated Cx expression and cell proliferation with disease survival and pathological response to neoadjuvant chemotherapy in breast cancers using existing classification systems.MethodsThe expression of Cx26, Cx32, Cx43, Cx46 and Ki67 was evaluated in 96 breast cancer patients prior to and after neoadjuvant chemotherapy using duplicate cores in tissue microarrays (TMA). Cx plaques of <1μm were detected with multilayer, multichannel fluorescence digital microscopy. Current classifications to assess residual tumour burden after primary systemic therapy included the EWGBSP, CPS-EG, Miller-Payne, Sataloff and NSABP systems.ResultsIn our cohort dominated by hormone receptor (ER/PR) positive and HER2 negative cases, only the CPS-EG classification showed prognostic relevance: cases with scores 1–2 had significantly better overall survival (p=0.015) than cases with scores 3–5. Pre-chemotherapy Cx43 expression correlated positively with hormone receptor status both before and after chemotherapy and had a negative correlation with HER2 expression pre-chemotherapy. There was a positive correlation between Cx32 and HER2 expression pre-chemotherapy and between Cx32 and Ki67 expression post-chemotherapy. A negative correlation was found between post-chemotherapy Cx46 and Ki67 expression. Decreased post-chemotherapy Cx26 expression (<5%) statistically correlated with better overall survival (p=0.011). Moderate or higher Cx46 expression (>20%) pre- and post-chemotherapy correlated with significantly better survival in the intermediate prognostic subgroups of EWGBSP TR2b (ppre-chemo=0.006; Sataloff TB (ppre-chemo=0.005; ppost-chemo=0.029) and in Miller-Payne G3 (ppre-chemo=0.002; ppost-chemo=0.012) classifications. Pre-chemotherapy, Cx46 expression was the only marker that correlated with overall survival within these subgroups.ConclusionOur results suggest that Cx46 and Cx26 expression in breast cancer may improve the assessment of pathological response and refine intermediate prognostic subgroups of residual tumour classifications used after neoadjuvant chemotherapy.

BackgroundExpression of transcription-factors as Slug and Sox9 was recently described to determine mammary stem-cell state. Sox10 was previously shown to be present also in breast cancer. Protein overexpression of Slug, Sox9 and Sox10 were associated with poor overall survival and with triple-negative phenotype in breast cancer. In this study we tested the stability of Slug, Sox9 and Sox10 expression during chemotherapy and addressed their prognostic role of in neoadjuvant treated primary breast-cancer and their correlation to pathological-response and overall survival.MethodsWe analyzed immunohistochemical expression of Slug, Sox9 and Sox10 in tissue microarrays of 96 breast cancers prior to and after neoadjuvant chemotherapy. Expression was evaluated in invasive tumor cells and in tumor stroma and scored as 0, 1+, 2+ 3+. Expression-profile prior to and after chemotherapy was correlated to overall survival (Kaplan Meier) and with established clinico-pathological parameter.ResultsSox9, Sox10 and Slug were expressed in 82–96% of the tumor cells prior to chemotherapy. Slug was expressed in 97% of the cases in tumor stroma before therapy. Change in expression-profile after chemotherapy occurred only in Slug expression in tumor-cells (decreased from 82 to 51%, p = 0.0001, Fisher’s exact test). The other markers showed no significant change after chemotherapy. Stromal Sox9 expression (0 to 2+) correlated to better overall survival after chemotherapy (p = 0.004) and reached almost statistical significance prior to chemotherapy (p = 0.065). There was no correlation between Sox9 and hormone-receptor expression. In multivariate-analysis, the stromal Sox9 expression after chemotherapy proved to be an independent and better prognostic marker than hormone-receptor status. Other clinico-pathological parameter (as HER2-status or pathological-stage) showed no correlation to the analyzed markers.ConclusionStrong stromal Sox9 expression in breast cancer after chemotherapy was found to bear negative prognostic information and was associated with shortened overall survival. Slug expression was significantly changed (reduced) in samples after neoadjuvant chemotherapy.

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Follicular dendritic cells (FDC) show homo- and heterocellular metabolic coupling through connexin 43 (Cx43) gap junctions and support B cell selection and maturation in germinal centers. In follicular lymphomas B cells escape apoptosis while FDC develop abnormally. Here we tested Cx43 channels in reactive FDC development and follicular lymphomas. In culture, the treatment of FDC-B cell clusters (resembling to “ex vivo” germinal centers) with Gap27 peptide, mimicking the 2nd extracellular loop of Cx43 protein, significantly impaired FDC-B cell cluster formation and cell survival. In untreated cultures of intact clusters, cell proliferation showed a moderate reduction. In tissues, Cx43 protein levels run parallel with the density of FDC both in reactive germinal centers and in malformed follicles of follicular lymphomas and showed strong upregulation in newly generated and/or degrading bi-/multinuclear FDC of rudimentary processes. However, the inverse correlation between Cx43 expression and B cell proliferation seen in reactive germinal centers was not detected in follicular lymphomas. Furthermore, Cx43 levels were not associated with either lymphoma grade or bone marrow involvement. Our results suggest that Cx43 channels are critical in FDC and “ex vivo” germinal center development and in the persistence of FDC in follicular lymphomas but do not affect tumor progression.