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The tumor extracellular matrix (ECM) critically regulates cancer progression and treatment response. Expression of the basement membrane component collagen XVIII (ColXVIII) is induced in solid tumors, but its involvement in tumorigenesis has remained elusive. We show here that ColXVIII was markedly upregulated in human breast cancer (BC) and was closely associated with a poor prognosis in high-grade BCs. We discovered a role for ColXVIII as a modulator of epidermal growth factor receptor tyrosine kinase (ErbB) signaling and show that it forms a complex with ErbB1 and -2 (also known as EGFR and human epidermal growth factor receptor 2 [HER2]) and α6-integrin to promote cancer cell proliferation in a pathway involving its N-terminal portion and the MAPK/ERK1/2 and PI3K/AKT cascades. Studies using Col18a1 mouse models crossed with the mouse mammary tumor virus-polyoma virus middle T antigen (MMTV-PyMT) mammary carcinogenesis model showed that ColXVIII promoted BC growth and metastasis in a tumor cell-autonomous manner. Moreover, the number of mammary cancer stem cells was significantly reduced in the MMTV-PyMT and human cell models upon ColXVIII inhibition. Finally, ablation of ColXVIII substantially improved the efficacy of ErbB-targeting therapies in both preclinical models. In summary, ColXVIII was found to sustain the stemness properties of BC cells and tumor progression and metastasis through ErbB signaling, suggesting that targeting ColXVIII in the tumor milieu may have important therapeutic potential.

As the second most common skin malignancy, cutaneous squamous cell carcinoma (cSCC) is an increasing health concern, while its pathogenesis at molecular level remains largely unknown. We studied the expression and localisation of two homologous basement membrane (BM) collagens, types XV and XVIII, at different stages of cSCC. These collagens are involved in angiogenesis and tumorigenesis, but their role in cancer development is incompletely understood. Quantitative RT-PCR analysis revealed upregulation of collagen XVIII, but not collagen XV, in primary cSCC cells in comparison with normal human epidermal keratinocytes. In addition, the Ha-ras-transformed invasive cell line II-4 expressed high levels of collagen XVIII mRNA, indicating upregulation in the course of malignant transformation. Immunohistochemical analyses of a large human tissue microarray material showed that collagen XVIII is expressed by tumor cells from grade 1 onwards, while keratinocytes in normal skin and in premalignant lesions showed negative staining for it. Collagen XV appeared instead as deposits in the tumor stroma. Our findings in human cSCCs and in mouse cSCCs from the DMBA-TPA skin carcinogenesis model showed that collagen XVIII, but not collagen XV or the BM markers collagen IV or laminin, was selectively reduced in the tumor vasculature, and this decrease associated significantly with cancer progression. Our results demonstrate that collagens XV and XVIII are expressed in different sites of cSCC and may contribute in a distinct manner to processes related to cSCC tumorigenesis, identifying these collagens as potential biomarkers in the disease., (© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Genome-wide association studies have identified thousands of SNPs associated with predisposition to various diseases, including prostate cancer. However, the mechanistic roles of these SNPs remain poorly defined, particularly for noncoding polymorphisms. Here we find that the prostate cancer risk-associated SNP rs339331 at 6q22 lies within a functional HOXB13-binding site. The risk-associated T allele at rs339331 increases binding of HOXB13 to a transcriptional enhancer, conferring allele-specific upregulation of the rs339331-associated gene RFX6. Suppression of RFX6 diminishes prostate cancer cell proliferation, migration and invasion. Clinical data indicate that RFX6 upregulation in human prostate cancers correlates with tumor progression, metastasis and risk of biochemical relapse. Finally, we observe a significant association between the risk-associated T allele at rs339331 and increased RFX6 mRNA levels in human prostate tumors. Together, our results suggest that rs339331 affects prostate cancer risk by altering RFX6 expression through a functional interaction with the prostate cancer susceptibility gene HOXB13.

Toll-like receptor-9 (TLR9) is a member of the innate immune system and recognizes bacterial and vertebrate DNA in cells. In addition to being expressed in cells of the immune system, it is widely expressed in various types of human cancer, including prostate cancer. We have previously demonstrated that synthetic TLR9 ligands induce invasion in TLR9-expressing prostate cancer cells in vitro . However, the role of TLR9 in the pathophysiology of prostate cancer is unclear. The expression of TLR9 in radical prostatectomy samples (n=186) was studied using immunohistochemistry. TLR9 staining scores were compared with tumor stage, Gleason score and prostate-specific antigen (PSA) concentration prior to treatment and progression-free survival. Results revealed that 124 (66.7%) of the tumors were strongly positive, 59 (31.7%) were weakly positive and 3 (1.6%) were negative, for cytoplasmic TLR9 immunostaining in cancer cells. There was no significant association between cytoplasmic TLR9 expression and distributions of pT-class, prostatectomy sample margin status, Gleason score and preoperative PSA value. Prostate cancer-specific progression-free survival was significantly longer for patients whose tumors were graded as negative for cytoplasmic TLR9 expression, as compared with patients whose tumors were strongly immunopositive for cytoplasmic TLR9 (P=0.009). In the Cox regression analysis, high TLR9 expression was an independent marker of poor prognosis in prostate cancer. Expression of TLR9 is associated with poor progression-free survival in prostate cancer patients who were treated by radical prostatectomy with curative intent.

Background: The CIP2A protein is a recently characterized oncoprotein which inhibits protein phosphatase 2A activity. Expression of CIP2A has been detected in several carcinomas, but its expression and significance in prostate cancer has not been examined so far., Methods: Expression of the CIP2A protein was studied using immunohistochemistry in prostate cancer (n = 59) and in benign prostatic hyperplasia (n = 20) specimens. The CIP2A staining scores were compared with several clinicopathological parameters., Results: Expression of CIP2A was increased in prostate cancer epithelium as compared with the benign hyperplastic epithelium (p < 0.001). The expression of CIP2A was associated with high Gleason scores (p < 0.001) and among patients treated with radical prostatectomy, CIP2A expression was associated with pre-treatment risk stratification (p = 0.011) and pathological T-class (p = 0.031). No statistically significant association was detected between CIP2A expression and prostate specific antigen concentrations., Conclusions: Expression of the CIP2A protein is increased in prostate cancer specimens and its expression is associated with poorly differentiated and high-risk tumors.

Background: Toll-like receptor-9 (TLR9) is a cellular receptor for bacterial and vertebrate DNA. In addition to cells of the immune system, it is also expressed in various human cancer cell lines, including prostate cancer. We demonstrated previously that synthetic TLR9 ligands induce matrix metalloproteinase-13-mediated invasion in TLR9-expressing prostate cancer cells in vitro. Other studies have suggested possible sex steroid regulation of the function of the various TLRs. The role of TLR9 in the pathophysiology of prostate or any cancer is, however, unknown., Methods: Expression of TLR9, androgen receptor (AR), or the estrogen receptors alpha (ERalpha) and beta (ERbeta) were studied with immunohistochemistry in prostate cancer (n = 62) and benign prostatic hyperplasia (n = 45) specimens. TLR9 staining scores were compared with tumor stage, Gleason score, prostate-specific antigen (PSA) concentrations before tissue sampling and with the staining scores of AR, ERalpha, and ERbeta., Results: TLR9 expression was statistically significantly increased in prostate cancer epithelium and stroma, as compared with the same cellular compartments in benign hyperplasia. Significantly increased (P = 0.04) TLR9 expression was detected in cancers with high Gleason score (>7, n = 23), as compared with lower Gleason scores (< or =7, n = 39). No statistically significant associations were detected between TLR9 expression scores and PSA concentrations or tumor staging. Prostate adenocarcinoma cells were all positive for TLR9, AR, and ERbeta but negative for ERalpha expression. In cancer stroma cells, increased TLR9 expression was associated with increased ERalpha expression., Conclusions: Expression of TLR9 is increased in prostate cancer specimens, especially in the most poorly differentiated forms.

The angiogenesis inhibitor histidine-rich glycoprotein (HRG) constitutes one of several examples of molecules regulating both angiogenesis and hemostasis. The antiangiogenic properties of HRG are mediated via its proteolytically released histidine- and proline-rich (His/Pro-rich) domain. Using a combination of immunohistochemistry and mass spectrometry, we here provide biochemical evidence for the presence of a proteolytic peptide, corresponding to the antiangiogenic domain of HRG, in vivo in human tissue. This finding supports a role for HRG as an endogenous regulator of angiogenesis. Interestingly, the His/Pro-rich peptide bound to the vessel wall in tissue from cancer patients but not to the vasculature in tissue from healthy persons. Moreover, the His/Pro-rich peptide was found in close association with platelets. Relesate from in vitro-activated platelets promoted binding of the His/Pro-rich domain of HRG to endothelial cells, an effect mediated by Zn(2+). Previous studies have shown that zinc-dependent binding of the His/Pro-rich domain of HRG to heparan sulfate on endothelial cells is required for inhibition of angiogenesis. We describe a novel mechanism to increase the local concentration and activity of an angiogenesis inhibitor, which may reflect a host response to counteract angiogenesis during pathologic conditions. Our finding that tumor angiogenesis is elevated in HRG-deficient mice supports this conclusion.

Type XIII collagen is a transmembrane protein that also exists as a soluble extracellular variant because of ectodomain shedding by proprotein convertases. Because ectodomain shedding in a growing number of transmembrane proteins has recently been shown to be dependent on their localization in cholesterol-enriched detergent-resistant membrane microdomains, this work aimed at analyzing this aspect of type XIII collagen ectodomain processing. In HT-1080 cells type XIII collagen and its cleaving proprotein convertase furin localized partially in detergent-resistant cholesterol-containing membrane microdomains. Disruption of these domains by lowering either the level or availability of the cellular cholesterol reduced ectodomain shedding, implying that, in such membrane domains correct cholesterol level is important for the regulation of type XIII collagen ectodomain processing. In addition, we show here that ectodomain of type XIII collagen is also shed intracellularly. HT-1080 cells released vesicles from the Golgi apparatus, which contained only the cleaved variant. Intracellular processing and the subsequent entry of the cleaved ectodomain into the vesicles was totally blocked by inhibition of the proprotein convertase function by cell-permeable chloromethylketone, but not with cell-impermeable alpha1-antitrypsin Portland. This supports the hypothesis of type XIII collagen ectodomain also being cleaved intracellularly in the Golgi and suggests that the intracellular cleavage may act as a gating event in the vesicle-mediated ectodomain secretion.

Purpose: Type XIII collagen (ColXIII) is a transmembrane protein thought to be involved in cell-cell and cell-matrix interactions. We report here on its presence in the normal human cornea and compare the results for keratoconus and scarred corneas., Methods: Immunohistochemistry and in situ hybridization were applied to human corneal samples obtained by penetrating keratoplasty., Results: In the normal human cornea, ColXIII was immunolocalized to the corneal epithelial cells, and to a lesser degree to the stromal keratocytes. The keratoconus cases showed otherwise similar results, but in areas containing Bowman membrane disruptions showed thinned epithelial cells reduced immunostaining for ColXIII, whereas occasionally pronounced immunoreactivity was seen in the stromal keratocytes. The corneal scar samples contained highly increased ColXIII immunostaining by stromal cells in the fibrotic foci, whereas the peripheral areas showed less intense immunostaining. In situ hybridization confirmed that the corneal epithelium and keratocytes actively synthesize the transcript. Immunostaining with alphaSMA revealed that a substantial proportion of the ColXIII mRNA-expressing cells in the stromal scar tissues was myofibroblasts and that these areas lack CD34 immunoreactivity., Conclusions: The results indicate that ColXIII, which is predominantly confined to the basal corneal cells in the normal cornea, may have a role in the adhesion of corneal epithelial cells to each other and to the underlying basement membrane. Additionally, highly increased expression in scarred corneas suggests that it participates in the corneal wound healing process.

Type XIII collagen is a transmembrane collagen, which is known to exist also as a soluble variant due to ectodomain shedding. Earlier studies with the recombinant ectodomain have shown it to interact in vitro with a number of extracellular matrix proteins, e.g. Fn (fibronectin). In view of its strong binding to Fn, we examined in the present study whether the released soluble ectodomain can bind to the fibrillar Fn matrix under cell-culture conditions and, if so, influence its assembly. In this study, we demonstrate that the type XIII collagen ectodomain of mammalian cells can associate with Fn fibres and may eventually hamper incorporation of the fibrillar Fn meshwork. The association between type XIII collagen and Fn was implicated to be mediated by the C-terminal end of type XIII collagen and the N-terminal end of Fn. The results presented here imply that the shedding of the type XIII collagen ectodomain results in a biologically active molecule capable of remodelling the structure of the pericellular matrix.

Little information is available on the expression of transmembrane type XIII collagen in human diseases. The present study has investigated the expression of this collagen in cancer, in particular during malignant transformation. By combining the tissue microarray technique with in situ hybridization, a consistent pattern of clearly increased type XIII collagen mRNA expression was found in the stromal compartment of epithelial tumours and throughout mesenchymal tumours. Slightly elevated mRNA expression was observed in dysplastic samples and in malignant epithelial cells. It is also demonstrated that factors secreted into the culture medium by tumour cells, in particular the growth factor TGF-beta, contribute to the induction of type XIII collagen expression, and trigger concomitantly a profound phenotypic and morphological transition of cultured primary fibroblasts. Reciprocally, type XIII collagen may alter the growth milieu of malignant cells as the soluble type XIII collagen ectodomain influenced the adherence and spreading of cells cultured on vitronectin-rich matrix. It is proposed that malignant transformation stimulates the expression of type XIII collagen, particularly in the tumour stroma and to a lesser extent in the epithelium, and that this high type XIII collagen expression may contribute to tumour progression and behaviour by modulating cell-matrix interactions., (Copyright (c) 2005 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.)

Transmembrane type XIII collagen resides in adhesive structures of cells and tissues, and has therefore been implicated in cell adhesion and in adhesion-dependent cell functions. This collagen also exists as a soluble protein in the pericellular matrix, as the ectodomain is released from the plasma membrane by proteolytic cleavage. Analysis with various protease inhibitors led to confirmation of the furin family of proprotein convertases as the protease group responsible for the shedding of the ectodomain, cleaving at a site conforming to the consensus sequence for the proprotein convertases at the stem of the ectodomain. Both the trans -Golgi network and the plasma membrane were used as cleavage locations. Mammalian cells employed various intracellular mechanisms to modulate shedding of the ectodomain, all resulting in a similar cleavage event. Cell detachment from the underlying substratum was also found to augment the excision. The released ectodomain rendered the pericellular surroundings less supportive of cell adhesion, migration and proliferation, as seen specifically on a vitronectin substratum. Type XIII collagen ectodomain shedding thus resulted in the formation of a soluble, biologically active molecule, which eventually modulated cell behaviour in a reciprocal and substratum-specific manner. The dual existence of membrane-bound and soluble variants widens our biological understanding of type XIII collagen.