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Pterygial pathology is characterized by abnormal corneal epithelial proliferation, stromal modulation, matrix degradation and a strong tendency for otherwise absent corneal vascularization. As the proto-oncogene Ets-1 is known to play a key role in angiogenesis and matrix degradation in other tissues, its involvement in corneal vascularization was investigated. Fifteen pterygia representing two groups were studied. Group 1 consisted of five clinically active pterygia, and group 2 consisted of 10 samples of clinically non-active pterygia. 35S-labelled ets-1 antisense and sense riboprobes were used for in-situ hybridization of Ets-1 transcription factor in all pterygia. The cytoplasm of blood vessel endothelial cells showed strong expression of ets-1 mRNA in all group 1 pterygia. In contrast, no expression of ets-1 was found in group 2 pterygia. Proto-oncogene ets-1 expression has been shown for the first time in the metaplastic pterygium, an eye tissue of unknown pathogenesis. [ABSTRACT FROM AUTHOR]

Abstract Background HDGF is a growth factor which is overexpressed in a wide range of tumors. Importantly, expression levels were identified as a prognostic marker in some types of cancer such as melanoma. Methods To investigate the presumed oncogenic/transforming capacity of HDGF, we generated transgenic mice overexpressing HDGF in melanocytes. These mice were bred with mice heterozygous for a defective copy of the Ink4a tumor suppressor gene and were exposed to UV light to increase the risk for tumor development both genetically and physiochemically. Mice were analyzed by immunohistochemistry and Western blotting. Furthermore, primary melanocytes were isolated from different strains created. Results Transgenic animals overexpressed HDGF in hair follicle melanocytes. Interestingly, primary melanocytes isolated from transgenic animals were not able to differentiate in vitro whereas cells isolated from wild type and HDGF-deficient animals were. Although, HDGF-/-/Ink4a+/- mice displayed an increased number of epidermoid cysts after exposure to UV light, no melanomas or premelanocytic alterations could be detected in this mouse model. Conclusions The results therefore provide no evidence that HDGF has a transforming capacity in tumor development. Our results in combination with previous findings point to a possible role in cell differentiation and suggest that HDGF promotes tumor progression after secondary upregulation and may represent another protein fitting into the concept of non-oncogene addiction of tumor tissue.

The co-expression of miRNAs and their target proteins was studied on tissue microarrays from different prostate cancer (PCa) patients. PCa of primary Gleason pattern 4 (GP4), lymph node metastases of GP4, distant metastases, and normal tissue from the transitional and peripheral zones were co-stained by fluorescent miRNA in situ hybridization (miRisH) and protein immunohistofluorescence (IHF). The miRNAs and corresponding target proteins include the pairs miR-145/ERG, miR-143/uPAR, and miR-375/SEC23A. The fluorescence-stained and scanned tissue microarrays (TMAs) were evaluated by experienced uropathologists. The pair miR-145/ERG showed an exclusive staining for miR-145 in the nuclei of stromal cells, both in tumor and normal tissue, and for ERG in the cytoplasm with/without co-expression in the nucleus of tumor cells. The pair miR-143/uPAR revealed a clear distinction between miR-143 in the nuclei of stromal cells and uPAR staining in the cytoplasm of tumor cells. Metastases (lymph node and distant) however, showed tumor cells with cytoplasmic staining for miR-143/uPAR. In normal tissues, beside the nuclei of the stroma cells, gland cells could also express miR-143 and uPAR in the cytoplasm. miR-375 showed particular staining in the nucleoli of GP4 and metastatic samples, suggesting that nucleoli play a special role in sequestering proteins and miRNAs. Combined miRisH/IHF allows for the study of miRNA expression patterns and their target proteins at the single-cell level.

Ets-1 transcription factor overexpression in breast cancers is associated with invasive features and is associated with a poor prognosis. Beyond its role in driving carcinoma cell invasion, in this study, we wished to determine whether Ets-1 overexpression in cancer cells promotes angiogenesis by creating a paracrine pro-invasive environment for endothelial cells as well. To address this question, we set up different co-culture models of cancer cells with endothelial cells. Conditioned media from cancer cells induced endothelial cell proliferation, migration and morphogenesis in matrix models. Of note, co-culture assays in three-dimensional matrix models also revealed the reciprocal induction of cancer cell morphogenesis by endothelial cells, in support of an angiocrine action on tumor cells. Ets-1 emerged as a key regulator of the angiogenic potential of breast cancer cells, favoring their ability to induce, in a paracrine manner, the morphogenesis of endothelial cells and also to physically interact with the latter. Nevertheless, Ets-1 overexpression in cancer cells also restrained their chemoattractive potential for endothelial cells both in Boyden chambers and in ex vivo 3D co-cultures. Finally, Ets-1 modulation in breast cancer cells qualitatively altered the angiogenic pattern of experimental in vivo tumors, with a balance between vessel recruitment and intratumoral small capillaries sprouting. Taken together, our data highlight a critical and intriguing role for Ets-1 in the angiogenic potential of breast cancer cells, and reveal another facet of Ets-1 oncogenic activities.

Melanoma represents 2-3% of all cancers, 95% of them arise from skin, while only 5% are non-cutaneous melanoma. Despite an optimal surgery management, the risk of a local and systemic relapse remains high, particularly in high-risk patients (node-positive or node-negative T3b, T4 a/b). We conducted a systematic review of the main published and ongoing phase I/II/III trials between 2000 and June 2015 on the adjuvant treatment of cutaneous melanoma. The IFN remains the only option currently available for this aim. Ipilimumab represents a possible breakthrough in this setting, considering the positive results of the EORTC 18701 trials in terms of disease free survival (DFS), while data regarding OS are pending. Recent advances in the understanding of the biology of melanoma result in the identification of MAPK pathway role in the melanoma development. Based on these features, B-RAF inhibitors and their combination with immunotherapy could represent the upcoming therapeutic strategy., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.)

The main characteristic of senescence is its stability which relies on the persistence of DNA damage. We show that unlike fibroblasts, senescent epithelial cells do not activate an ATM-or ATR-dependent DNA damage response (DDR), but accumulate oxidative-stress-induced DNA single-strand breaks (SSBs). These breaks remain unrepaired because of a decrease in PARP1 expression and activity. This leads to the formation of abnormally large and persistent XRCC1 foci that engage a signalling cascade involving the p38MAPK and leading to p16 upregulation and cell cycle arrest. Importantly, the default in SSB repair also leads to the emergence of post-senescent transformed and mutated precancerous cells. In human-aged skin, XRCC1 foci accumulate in the epidermal cells in correlation with a decline of PARP1, whereas DDR foci accumulate mainly in dermal fibroblasts. These findings point SSBs as a DNA damage encountered by epithelial cells with aging which could fuel the very first steps of carcinogenesis.

We evaluated the prognostic value of the mitosis-associated marker phosphorylated histone H3 (PHH3) and Ki-67 in prostate cancer with respect to ERG status and androgen receptor (AR) expression.PHH3 and Ki-67 expression was immunohistochemically detected and digitally quantitated in a radical prostatectomy cohort (n = 640). The results were correlated to clinicopathological parameters including biochemical recurrence times. Prognostic values of PHH3 and Ki-67 were analysed by Cox regression and Kaplan-Meier statistics.In prostate cancer, mean Ki-67 and PHH3 rates were 3.40% (95%CI 3.16-3.63%) and 0.0152% (95%CI 0.0112-0.0191%), respectively.Ki-67 showed a significant correlation with Gleason scores, pT status, margin status, and AR expression, while PHH3 showed a significant correlation with Gleason scores and pT status. Univariate analyses for biochemical recurrence times demonstrated a significant prognostic value for median Ki-67 rate and for the PHH3 rate of the 90th percentile. Of importance, in patient subgroups stratified according to AR expression and ERG translocation, the prognostic power of proliferation markers PHH3 and Ki-67 was markedly enhanced in ERG translocation negative and high-level AR expressing ERG translocation positive prostate cancers.As expected, the proliferation markers PHH3 and Ki-67 predict adverse outcome of prostate cancer and have a particularly pronounced prognostic value in specific molecular subsets of prostate cancer (ERG- or AR+).

Ets-1 overexpression in human breast cancers is associated with invasiveness and poor prognosis. By overexpressing Ets-1 or a dominant negative mutant in MMT breast cancer cells, we previously highlighted the key role of Ets-1 in coordinating multiple invasive features of these cells. Interestingly, we also noticed that Ets-1 decreased the density of breast cancer cells cultured in three-dimensional extracellular matrix gels. The 3D context was instrumental to this phenomenon, as such downregulation was not observed in cells grown on two-dimensional plastic or matrix-coated dishes. Ets-1 overexpression was deleterious to anchorage-independent growth of MMT cells in soft agar, a standard model for in vitro tumorigenicity. The relevance of this mechanism was confirmed in vivo, during primary tumor growth and in a metastatic assay of lung colonization. In these models, Ets-1 was associated with epithelial-to-mesenchymal transition features and modulated the ratio of Ki67-positive cells, while hardly affecting in vivo apoptotic cell death. Finally, siRNA-mediated knockdown of Ets-1 in human breast cancer cell lines also decreased colony growth, both in anchorage-independent assays and 3D extracellular matrix cultures. These in vitro and in vivo observations shed light on an unsuspected facet of Ets-1 in breast tumorigenesis. They show that while promoting malignancy through the acquisition of invasive features, Ets-1 also attenuates breast tumor cell growth and could therefore repress the growth of primary tumors and metastases. This work also demonstrates that 3D models may reveal mechanisms of tumor biology that are cryptic in standard 2D models., (© 2014 UICC.)

The mediator complex is an evolutionary conserved key regulator of transcription of protein-coding genes and an integrative hub for diverse signaling pathways. In this study, we investigated whether the mediator subunit MED15 is implicated in castration-resistant prostate cancer (CRPC). MED15 expression and copy number/rearrangement status were assessed by immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH), respectively on 718 prostate cancer (PCa) specimens and sequenced by Sanger on a subset. Furthermore, SMAD3 phosphorylation, androgen receptor (AR) and proliferation markers were evaluated by IHC. In PCa cells, siRNA/shRNA knockdown of MED15 was followed by proliferation assays with/without dihydrotestosterone (DHT), and treatments with recombinant TGF-β3. Our results show that MED15 is overexpressed in 76% of distant metastatic CRPC (CRPC(MET) ) and 70% of local-recurrent CRPC (CRPC(LOC) ), in contrast to low frequencies in androgen-sensitive PCa, and no expression in benign prostatic tissue. Furthermore, MED15 overexpression correlates with worse clinical outcome thus defining a highly lethal phenotype. Moreover, TGF-β signaling activation associates with MED15 overexpression in PCa tissues, and leads to increased expression of MED15 in PCa cells. MED15 knockdown effects phosphorylation and shuttling of p-SMAD3 to the nucleus as well as TGF-β-enhanced proliferation. In PCa tissues, MED15 overexpression associates with AR overexpression/amplification and correlates with high proliferative activity. MED15 knockdown decreases both androgen-dependent and -independent proliferation in PCa cells. Taken together, these findings implicate MED15 in CRPC, and as MED15 is evolutionary conserved, it is likely to emerge as a lethal phenotype in other therapeutic-resistant diseases, and not restricted to our disease model., (© 2013 UICC.)

Purpose: Prostate cancer is the second most common cancer in men and the sixth most common cause of death from cancer in men worldwide. Currently, a sufficient pathological distinction between patients requiring further treatment and those for which active surveillance remains an option is still lacking, which leads to the problem of overtreatment. Cell proliferation is routinely assessed by detecting Ki-67 antigen. While Ki-67 is expressed throughout the interphase of proliferating cells, phosphorylation of the chromatin constituent histone H3 occurs only during the late G2 phase and mitosis thus providing a more strict assessment of the mitotic activity. We undertook this study to test whether expression of the recently introduced proliferation marker phospho-histone H3 (pHH3) in prostate carcinoma tissue sections exhibits prognostic significance in comparison with Ki-67., Methods: Protein expression of pHH3 and Ki-67 was assessed on TMA consisting of paraffin-embedded tissue from men that had undergone radical prostatectomy. The analysis included triplicate tissue cores of a total of 339 tumor foci. Immunohistochemical staining of pHH3 and Ki-67 was performed and analyzed using Definiens imaging software., Results: Prostate cancer tissue exhibited a significantly higher frequency of pHH3-positive cells compared to benign prostate tissue. pHH3 expression was significantly correlated with Ki-67 expression. Furthermore, statistical analysis revealed positive correlation between pHH3 expression and PSA levels at diagnosis and in addition negatively correlated with overall survival. In contrast to Ki-67 staining, pHH3 expression did not correlate with Gleason grade., Conclusion: Our data point to a conceivable role of pHH3 as prognostic biomarker in prostate carcinoma.

Castration-resistant prostate cancer (CRPC) is the most aggressive form of prostate cancer (PCa) and remains a significant therapeutic challenge. The key to the development of novel therapeutic targets for CRPC is to decipher the molecular alterations underlying this lethal disease. The aim of our study was to identify therapeutic targets for CRPC by assessing somatic copy number alterations (SCNAs) by whole-exome sequencing on five CRPC/normal paired formalin-fixed paraffin-embedded (FFPE) samples, using the SOLiD4 next-generation sequencing (NGS) platform. Data were validated using fluorescence in situ hybridization (FISH) on a PCa progression cohort. PTK2 and YWHAZ amplification, mRNA and protein expression were determined in selected PCa cell lines. Effects of PTK2 inhibition using TAE226 inhibitor and YWHAZ knock-down on cell proliferation and migration were tested in PC3 cells in vitro. In a larger validation cohort, the amplification frequency of YWHAZ was 3% in localized PCa and 48% in CRPC, whereas PTK2 was amplified in 1% of localized PCa and 35% in CRPC. YWHAZ knock-down and PTK2 inhibition significantly affected cell proliferation and migration in the PC3 cells. Our findings suggest that inhibition of YWHAZ and PTK2 could delay the progression of the disease in CRPC patients harbouring amplification of the latter genes. Furthermore, our validated whole-exome sequencing data show that FFPE tissue could be a promising alternative for SCNA screening using next-generation sequencing technologies., (Copyright © 2013 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.)

Purpose: Both genetic instability resulting in aneuploidy and increased proliferative activity are common features of tumor development and progression. Cytometric evaluation of tumor ploidy status was recently suggested as a prognostic marker. However, in prostate cancer (PCa), a chromosome-specific evaluation is lacking. With the present study, we sought to identify distinct chromosomal changes to complement cytometric results concerning the diagnosis and prognosis of PCa patients., Methods: We assessed a cohort of 428 PCa specimens (186 localized PCa, 75 lymph node metastasized PCa, 125 lymph node metastases, 42 hormone-refractory distant metastases) for numerical alterations of all 24 chromosomes by using fluorescence in situ hybridization (FISH). Conducting immunohistochemistry with phosphorylated histone H3 (PHH3) and Ki-67, we quantified the proliferation rate. FISH results were fit in a linear model and tested for predictive power., Results: As expected, we observed a significant increase in aneuploidy with advancing tumor stage. Similarly, an increased expression of the mitotic marker PHH3 was significantly associated with aneuploidy and higher pT-stage. We found aneusomy of chromosomes 4, 6, 20, and X to be indicative of lymph node metastasized PCa. However, with an AUC of 65%, this set of chromosomal changes was poorly suited to distinguish non-metastasized and lymph node metastasized primary tumors., Conclusion: Our results provide thorough insight into the so far incompletely elucidated chromosomal landscape of PCa. While overall ploidy status and PHH3 expression in primary tumors indicate advanced disease, a FISH-based test for distinct alterations does not seem to be beneficial for diagnostic or prognostic decisions.

Aims: p63 is one of the standard markers for basal cells of the prostate gland. Recently, it has been suggested that the p63 isoform p40 might be more specific as a basal cell marker. In this study we compare the staining characteristics of p63 and p40 in normal and malignant prostate tissues., Methods and Results: A prostatectomy cohort (n = 640) in tissue microarray format was evaluated for p63 (clone 4A4) and for p40 (rabbit polyclonal) immunoreactivity in malignant and normal tissues. Immunoreactivity of basal and secretory cells was evaluated in a semiquantitative manner and compared case-wise. In normal tissues, p40 showed highly similar immunoreactivity compared to p63. The staining patterns were absolutely identical in 88% of cases. Additional cytoplasmic p40 staining in tumour cells occurred in 59.6% of cancer cases. Differences were seen in nuclear staining of carcinomas: 1.4% of carcinomas were p63-positive, whereas 0.6% were p40-positive., Conclusions: In most cases, p40 stains prostatic basal cells as reliably as p63, with only minor differences. Aberrant staining of tumour cells is seen more rarely than with p63 (clone 4A4), establishing its higher specificity. However, additional cytoplasmic immunoreactivity narrows its eligibility for antibody cocktails (e.g. with alpha-methylacyl-CoA racemase)., (© 2013 John Wiley & Sons Ltd.)

The incidence of carcinoma increases greatly with aging, but the cellular and molecular mechanisms underlying this correlation are only partly known. It is established that senescent fibroblasts promote the malignant progression of already-transformed cells through secretion of inflammatory mediators. We investigated here whether the senescent fibroblast secretome might have an impact on the very first stages of carcinogenesis. We chose the cultured normal primary human epidermal keratinocyte model, because after these cells reach the senescence plateau, cells with transformed and tumorigenic properties systematically and spontaneously emerge from the plateau. In the presence of medium conditioned by autologous senescent dermal fibroblasts, a higher frequency of post-senescence emergence was observed and the post-senescence emergent cells showed enhanced migratory properties and a more marked epithelial-mesenchymal transition. Using pharmacological inhibitors, siRNAs, and blocking antibodies, we demonstrated that the MMP-1 and MMP-2 matrix metalloproteinases, known to participate in late stages of cancer invasion and metastasis, are responsible for this enhancement of early migratory capacity. We present evidence that MMPs act by activating the protease-activated receptor 1 (PAR-1), whose expression is specifically increased in post-senescence emergent keratinocytes. The physiopathological relevance of these results was tested by analyzing MMP activity and PAR-1 expression in skin sections. Both were higher in skin sections from aged subjects than in ones from young subjects. Altogether, our results suggest that during aging, the dermal and epidermal skin compartments might be activated coordinately for initiation of skin carcinoma, via a paracrine axis in which MMPs secreted by senescent fibroblasts promote very early epithelial-mesenchymal transition of keratinocytes undergoing transformation and oversynthesizing the MMP-activatable receptor PAR-1.

Quantification of protein expression based on immunohistochemistry (IHC) is an important step for translational research and clinical routine. Several manual ('eyeballing') scoring systems are used in order to semi-quantify protein expression based on chromogenic intensities and distribution patterns. However, manual scoring systems are time-consuming and subject to significant intra- and interobserver variability. The aim of our study was to explore, whether new image analysis software proves to be sufficient as an alternative tool to quantify protein expression. For IHC experiments, one nucleus specific marker (i.e., ERG antibody), one cytoplasmic specific marker (i.e., SLC45A3 antibody), and one marker expressed in both compartments (i.e., TMPRSS2 antibody) were chosen. Stainings were applied on TMAs, containing tumor material of 630 prostate cancer patients. A pathologist visually quantified all IHC stainings in a blinded manner, applying a four-step scoring system. For digital quantification, image analysis software (Tissue Studio v.2.1, Definiens AG, Munich, Germany) was applied to obtain a continuous spectrum of average staining intensity. For each of the three antibodies we found a strong correlation of the manual protein expression score and the score of the image analysis software. Spearman's rank correlation coefficient was 0.94, 0.92, and 0.90 for ERG, SLC45A3, and TMPRSS2, respectively (p⟨0.01). Our data suggest that the image analysis software Tissue Studio is a powerful tool for quantification of protein expression in IHC stainings. Further, since the digital analysis is precise and reproducible, computer supported protein quantification might help to overcome intra- and interobserver variability and increase objectivity of IHC based protein assessment.