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PDF file - 28K, Supplementary Figure 2. A, the effects of the six most potent siRNA screen hits on IL-11 secretion in MDA-MB-231(SA) cells (n=3). B, the effects of heparan sulfate modifying enzymes on IL-11 secretion in MDA-MB-231(SA) cells (n=4). *, P < 0.05; **, P

XLS file - 49K, Supplementary Table 2. Effects of 196 siRNAs (including 2 siRNAs targeting IL11) on TGF-�-induced IL-11 production in MDA-MB-231(SA) cells. The relative IL-11 concentration values were obtained by dividing the IL-11 concentration by the amount of viable cells in the well (as determined by the CellTiter-Blue assay). The values are shown relative to the average values for the negative control siRNA.

TGF-β regulates several steps in cancer metastasis, including the establishment of bone metastatic lesions. TGF-β is released from bone during osteoclastic bone resorption and it stimulates breast cancer cells to produce osteolytic factors such as interleukin 11 (IL-11). We conducted a cell-based siRNA screen and identified heparan sulfate 6-O-sulfotransferase 2 (HS6ST2) as a critical gene for TGF-β–induced IL-11 production in highly bone metastatic MDA-MB-231(SA) breast cancer cells. HS6ST2 attaches sulfate groups to glucosamine residues in heparan sulfate glycosaminoglycans. We subsequently showed how heparin and a high-molecular-weight Escherichia coli K5-derived heparin-like polysaccharide (K5-NSOS) inhibited TGF-β–induced IL-11 production in MDA-MB-231(SA) cells. In addition, K5-NSOS inhibited bone resorption activity of human osteoclasts in vitro. We evaluated the therapeutic potential of K5-NSOS and fragmin in a mouse model of breast cancer bone metastasis. MDA-MB-231(SA) cells were inoculated into the left cardiac ventricle of athymic nude mice which were treated with fragmin, K5-NSOS, or vehicle once a day for four weeks. Both heparin-like glycosaminoglycans inhibited weight reduction, decreased osteolytic lesion area, and reduced tumor burden in bone. In conclusion, our data imply novel mechanisms involved in TGF-β induction and support the critical role of heparan sulfate glycosaminoglycans in cancer metastasis as well as indicate that K5-NSOS is a potential antimetastatic and antiresorptive agent for cancer therapy. This study illustrates the potential to translate in vitro siRNA screening results toward in vivo therapeutic concepts. Mol Cancer Res; 10(5); 597–604. ©2012 AACR.

PDF file - 6K, Supplementary Table 1. Primers and probes used for quantitative RT-PCR.

PDF file - 38K, Supplementary Figure 3. A, expression of HS6ST2 mRNA in MDA-MB-231(SA) versus parental MDA-MB-231 cells. B, validation of the knockdown efficacies of siRNAs by quantitative RT-PCR.

PDF file - 25K, Supplementary Figure 1. Basal and TGF-b-induced IL-11 production in parental MDA-MB-231 and highly metastatic MDA-MB-231(SA) cells. ***, P < 0.001 (t test).

Long non-coding RNAs (lncRNAs) have emerged as important regulators of cancer progression. Super enhancers (SE) play a role in tumorigenesis and regulate the expression of specific lncRNAs. We examined the role of BRD3OS, also named LINC00094, in cutaneous squamous cell carcinoma (cSCC). Elevated BRD3OS (LINC00094) expression was detected in cSCC cells, and expression was downregulated by SE inhibitors THZ1 and JQ1 and via the MEK1/ERK1/2 pathway. Increased expression of BRD3OS (LINC00094) was noted in tumor cells in cSCCs and their metastases compared to normal skin, actinic keratoses, and cSCCs in situ. Higher BRD3OS (LINC00094) expression was noted in metastatic cSCCs than in non-metastatic cSCCs. RNA-seq analysis after BRD3OS (LINC00094) knockdown revealed significantly regulated GO terms Cell-matrix adhesion, Basement membrane, Metalloendopeptidase activity, and KEGG pathway Extracellular matrix–receptor interaction. Among the top-regulated genes were MMP1, MMP10, and MMP13. Knockdown of BRD3OS (LINC00094) resulted in decreased production of MMP-1 and MMP-13 by cSCC cells, suppressed invasion of cSCC cells through collagen I, and growth of human cSCC xenografts in vivo. Based on these observations, BRD3OS (LINC00094) was named SERLOC (super enhancer and ERK1/2-Regulated Long Intergenic non-protein coding transcript Overexpressed in Carcinomas). These results reveal the role of SERLOC in cSCC invasion and identify it as a potential therapeutic target in advanced cSCC.

Long non-coding RNAs (lncRNAs) have emerged as important regulators of cancer progression. Super enhancers (SE) play a role in tumorigenesis and regulate the expression of specific lncRNAs. We examined the role of

Little is known about the signaling pathways involved in the differentiation of human osteoclasts. The present study evaluated the roles of the Ras/PI3K/Akt/mTOR, Ras/Raf/MEK1/2/ERK1/2, calcium-PKC, and p38 signaling pathways in human osteoclast differentiation. Mononuclear cells were isolated from the peripheral blood of control persons and patients with neurofibromatosis 1 (NF1), and the cells were differentiated into osteoclasts in the presence of signaling pathway inhibitors. Osteoclast differentiation was assessed using tartrate-resistant acid phosphatase 5B. Inhibition of most signaling pathways with chemical inhibitors decreased the number of human osteoclasts and disrupted F-actin ring formation, while the inhibition of p38 resulted in an increased number of osteoclasts, which is a finding contradictory to previous murine studies. However, the p38 inhibition did not increase the bone resorption capacity of the cells. Ras-inhibitor FTS increased osteoclastogenesis in samples from control persons, but an inhibitory effect was observed in NF1 samples. Inhibition of MEK, PI3K, and mTOR reduced markedly the number of NF1-deficient osteoclasts, but no effect was observed in control samples. Western blot analyses showed that the changes in the phosphorylation of ERK1/2 correlated with the number of osteoclasts. Our results highlight the fact that osteoclastogenesis is regulated by multiple interacting signaling pathways and emphasize that murine and human findings related to osteoclastogenesis are not necessarily equivalent.

Background Cutaneous squamous cell carcinoma (cSCC) is a common cancer that invades the dermis through the basement membrane. The role of the basement membrane in poorly differentiated cSCC is not well understood. Objectives To study the effect that loss of the laminin subunit alpha-3 (α3) chain from the tumour microenvironment has on tumour invasion and inflammatory cell recruitment. Methods We examined the role of the basement membrane proteins laminin subunits α3, β3 and γ2 in SCC invasion and inflammatory cell recruitment using immunohistochemistry, short hairpin RNA knockdown, RNA-Seq, mouse xenograft models and patient tumour samples. Results Analysis of SCC tumours and cell lines using antibodies specific to laminin chains α3, β3 and γ2 identified a link between poorly differentiated SCC and reduced expression of laminin α3 but not the other laminin subunits investigated. Knockdown of laminin α3 increased tumour invasion both in vitro and in vivo. Western blot and immunohistochemical staining identified increased phosphorylated myosin light chain with loss of laminin α3. Inhibition of ROCK (rho-associated protein kinase) but not Rac1 significantly reduced the invasive potential of laminin α3 knockdown cells. Knockdown of laminin subunits α3 and γ2 increased monocyte recruitment to the tumour microenvironment. However, only the loss of laminin α3 correlated with increased tumour-associated macrophages both in xenografted tumours and in patient tumour samples. Conclusions These data provide evidence that loss of the laminin α3 chain in cSCC has an effect on both the epithelial and immune components of cSCC, resulting in an aggressive tumour microenvironment.

Long non-coding RNAs (lncRNAs) are a largely uncharacterized group of non-coding RNAs with diverse regulatory roles in various biological processes. Recent observations have elucidated the functional roles of lncRNAs in cutaneous biology, e.g. in proliferation and differentiation of epidermal keratinocytes and in cutaneous wound repair. Furthermore, the role of lncRNAs in keratinocyte-derived skin cancers is emerging, especially in cutaneous squamous cell carcinoma (cSCC), which presents a significant burden to health care services worldwide and causes high mortality as metastatic disease. Elucidation of the functions of keratinocyte-specific lncRNAs will improve understanding of the molecular pathogenesis of epidermal disorders and skin cancers and can be exploited in development of new diagnostic and therapeutic applications for keratinocyte carcinomas. In this review, we summarize the current evidence of functionally important lncRNAs in cutaneous biology and in keratinocyte carcinomas.

Summary Background The incidence of epidermal keratinocyte‐derived cutaneous squamous cell carcinoma (cSCC) is increasing worldwide. Objectives To study the role of the complement classical pathway components C1q, C1r and C1s in the progression of cSCC. Methods The mRNA levels of C1Q subunits and C1R and C1S in cSCC cell lines, normal human epidermal keratinocytes, cSCC tumours in vivo and normal skin were analysed with quantitative real‐time polymerase chain reaction. The production of C1r and C1s was determined with Western blotting. The expression of C1r and C1s in tissue samples in vivo was analysed with immunohistochemistry and further investigated in human cSCC xenografts by knocking down C1r and C1s. Results Significantly elevated C1R and C1S mRNA levels and production of C1r and C1s were detected in cSCC cells, compared with normal human epidermal keratinocytes. The mRNA levels of C1R and C1S were markedly elevated in cSCC tumours in vivo compared with normal skin. Abundant expression of C1r and C1s by tumour cells was detected in invasive sporadic cSCCs and recessive dystrophic epidermolysis bullosa‐associated cSCCs, whereas the expression of C1r and C1s was lower in cSCC in situ, actinic keratosis and normal skin. Knockdown of C1r and C1s expression in cSCC cells inhibited activation of extracellular signal‐related kinase 1/2 and Akt, promoted apoptosis of cSCC cells and significantly suppressed growth and vascularization of human cSCC xenograft tumours in vivo. Conclusions These results provide evidence for the role of tumour‐cell‐derived C1r and C1s in the progression of cSCC and identify them as biomarkers and putative therapeutic targets in cSCC. What's already known about this topic? The incidences of actinic keratosis, cutaneous squamous cell carcinoma (cSCC) in situ and invasive cSCC are increasing globally.Few specific biomarkers for progression of cSCC have been identified, and no biological markers are in clinical use to predict the aggressiveness of actinic keratosis, cSCC in situ and invasive cSCC. What does this study add? Our results provide novel evidence for the role of complement classical pathway components C1r and C1s in the progression of cSCC. What is the translational message? Our results identify complement classical pathway components C1r and C1s as biomarkers and putative therapeutic targets in cSCC., Linked Comment: https://doi.org/10.1111/bjd.18419. https://doi.org/10.1111/bjd.18821 available online

Cutaneous squamous cell carcinoma (cSCC) is the most common metastatic skin cancer, with increasing incidence worldwide. Previous studies have shown the role of the complement system in cSCC progression. In this study, we have investigated the mechanistic role of serine proteinase C1r, a component of the classical pathway of the complement system, in cSCC. Knockout of C1r in cSCC cells using CRISPR/Cas9 resulted in a significant decrease in their proliferation, migration, and invasion through collagen type I compared with that of wild-type cSCC cells. Knockout of C1r suppressed the growth and vascularization of cSCC xenograft tumors and promoted apoptosis of tumor cells in vivo. mRNA-sequencing analysis after C1r knockdown revealed significantly regulated Gene Ontology terms cell-matrix adhesion, extracellular matrix component, basement membrane, and metalloendopeptidase activity and Kyoto Encyclopedia of Genes and Genomes pathway extracellular matrix-receptor interaction. Among the significantly regulated genes were invasion-associated matrix metalloproteinases (MMPs) MMP1, MMP13, MMP10, and MMP12. Knockout of C1r resulted in decreased production of MMP-1, MMP-13, MMP-10, and MMP-12 by cSCC cells in culture. Knockout of C1r inhibited the expression of MMP-13 by tumor cells, suppressed invasion, and reduced the amount of degraded collagen in vivo in xenografts. These results provide evidence for the role of C1r in promoting the invasion of cSCC cells by increasing MMP production.

Background: Cutaneous squamous cell carcinoma (cSCC) possesses metastatic potential and causes significant mortality. However, clinical assessment of metastasis risk is challenging. We approached this challenge by harnessing artificial intelligence (AI) algorithm to identify metastatic primary cSCCs. Methods: Residual neural network-architectures were trained and fine-tuned to identify metastatic cSCCs on tumor tiles extracted from clinician annotated, hematoxylin and eosin-stained whole slide images representing metastatic (n=45) and non-metastatic (n=59) primary cSCCs. Findings: Several models were trained and tested with best area under the receiver operating characteristic curve (AUROC) of 0·689 when all primary tumors (n=104) regardless of time to metastasis were included. Metastatic primary tumors were further divided into two groups that metastasize rapidly (≤180d) (n=22) or slowly (>180d) after primary tumor detection (n=23). The algorithm was able to predict whether primary tumor was non-metastatic or classified as rapidly metastatic with slide-level AUROC of 0·747. This prediction was superior to blind assessment by pathologist and did not appear to rely on classical clinicopathological features or staging systems. Furthermore, risk factor (RF) model taking into account prediction by AI, Clark’s level and tumor diameter provided higher AUROC (0·917) than other tested RF models and predicted high 5-year disease specific survival (DSS) for patients with cSCC with 0 or 1 RFs (100% and 95·7%) and poor 5-year DSS for patients with cSCCs with 2 or 3 RFs (41·7% and 40·0%). Interpretation: These results provide evidence, that AI recognizes unknown features associated with metastasis and provides added value to challenging clinical risk assessment of metastasis and prognosis of primary cSCC. Funding: Sigrid Juselius Foundation, Finnish Cancer Research Foundation, Jane and Aatos Erkko Foundation, Cancer Foundation of the Southwest Finland, Finnish Dermatological Society, The Maud Kuistila Memorial Foundation, and Turku University Hospital. JSK is a doctoral candidate in the Doctoral Program for Clinical Investigation of the University of Turku. Declaration of Interest: None to declare. Ethical Approval: The study was approved by The Ethics Committee of the Hospital District of Southwest Finland (187/2006) and Auria Biobank’s Scientific Steering Committee (AB15-9721).

The incidence of cutaneous keratinocyte‐derived cancers is increasing globally. Basal cell carcinoma (BCC) is the most common malignancy worldwide, and cutaneous squamous cell carcinoma (cSCC) is the most common metastatic skin cancer. BCC can be classified into subtypes based on the histology, and these subtypes are classified further into low‐ and high‐risk tumors. There is an increasing need to identify new therapeutic strategies for the treatment of unresectable and metastatic cSCC, and for aggressive BCC variants such as infiltrating, basosquamous or morpheaform BCCs. The most important risk factor for BCC and cSCC is solar UV radiation, which causes genetic and epigenetic alterations in keratinocytes. Similar gene mutations are noted already in sun‐exposed normal skin emphasizing the role of the alterations in the tumor microenvironment in the progression of cSCC. Early events in cSCC progression are alterations in the composition of basement membrane and dermal extracellular matrix induced by influx of microbes, inflammatory cells and activated stromal fibroblasts. Activated fibroblasts promote inflammation and produce growth factors and proteolytic enzymes, including matrix metalloproteinases (MMPs). Transforming growth factor‐β produced by tumor cells and fibroblasts induces the expression of MMPs by cSCC cells and promotes their invasion. Fibroblast‐derived keratinocyte growth factor suppresses the malignant phenotype of cSCC cells by inhibiting the expression of several MMPs. These findings emphasize the importance of interplay of tumor and stromal cells in the progression of cSCC and BCC and suggest tumor microenvironment as a therapeutic target in cSCC and aggressive subtypes of BCC.

Simple Summary The incidence of the most common metastatic skin malignancy, cutaneous squamous cell carcinoma (cSCC), is growing worldwide, and the prognosis of the metastatic disease is poor. Presently, there are no biomarkers or therapeutic targets for high-risk cSCCs. Recent studies have demonstrated the essential role of autocrine complement synthesis in the progression of cSCC. Here, we have evaluated the role of complement Factor D (FD), the rate-limiting enzyme of the alternative complement pathway, in cSCC development. The results identify FD as a novel biomarker and putative therapeutic target for cSCC and propose the small-molecule FD inhibitor Danicopan as a highly specific drug candidate in the therapy of advanced cSCC. It is expected that the discovery of complement-associated molecular markers for cSCC progression would improve diagnosis, classification, prognostication, and targeted therapy of cSCC and its precursors in the future. Cutaneous squamous cell carcinoma (cSCC) is the most prevalent metastatic skin cancer. Previous studies have demonstrated the autocrine role of complement components in cSCC progression. We have investigated factor D (FD), the key enzyme of the alternative complement pathway, in the development of cSCC. RT-qPCR analysis of cSCC cell lines and normal human epidermal keratinocytes (NHEKs) demonstrated significant up-regulation of FD mRNA in cSCC cells compared to NHEKs. Western blot analysis also showed more abundant FD production by cSCC cell lines. Significantly higher FD mRNA levels were noted in cSCC tumors than in normal skin. Strong tumor cell-associated FD immunolabeling was detected in the invasive margin of human cSCC xenografts. More intense tumor cell-specific immunostaining for FD was seen in the tumor edge in primary and metastatic cSCCs, in metastases, and in recessive dystrophic epidermolysis bullosa-associated cSCCs, compared with cSCC in situ, actinic keratosis and normal skin. FD production by cSCC cells was dependent on p38 mitogen-activated protein kinase activity, and it was induced by interferon-gamma and interleukin-1 beta. Blocking FD activity by Danicopan inhibited activation of extracellular signal-regulated kinase 1/2 and attenuated proliferation of cSCC cells. These results identify FD as a novel putative biomarker and therapeutic target for cSCC progression.

Long noncoding RNAs (lncRNAs) have emerged as putative biomarkers and therapeutic targets in cancer. The role of lncRNA LINC00346 in cutaneous squamous carcinoma (cSCC) was examined. The expression of LINC00346 was up-regulated in cSCC cells compared with normal human epidermal keratinocytes. Elevated expression of LINC00346 was noted in tumor cells in cSCC tissue sections in vivo, as compared with cSCC in situ, and actinic keratosis by RNA in situ hybridization; and the expression in seborrheic keratosis and normal skin was very low. Immunohistochemical analysis of cSCC tissue sections and functional assays of cSCC cells in culture showed that LINC00346 expression is down-regulated by p53. Knockdown of LINC00346 inhibited invasion of cSCC cells in culture and suppressed growth of human cSCC xenografts in vivo. Knockdown of LINC00346 inhibited expression of activated STAT3 and resulted in down-regulation of the expression of matrix metalloproteinase (MMP)-1, MMP-3, MMP-10, and MMP-13. Based on these observations LINC00346 was named p53 regulated carcinoma-associated STAT3-activating long intergenic non-protein coding transcript (PRECSIT). These results identify PRECSIT as a new p53-regulated lncRNA, which promotes progression of cSCC via STAT3 signaling.

Cutaneous squamous cell carcinoma (cSCC) is the most common metastatic skin cancer, with increasing incidence worldwide. The molecular basis of cSCC progression to invasive and metastatic disease is still incompletely understood. Here, we show that fibroblasts and transforming growth factor-β (TGF-β) signaling promote laminin-332 synthesis in cancer cells in an activated H-Ras-dependent manner, which in turn promotes cancer cell invasion. Immunohistochemical analysis of sporadic UV-induced invasive human cSCCs (n = 208) revealed prominent cSCC cell specific immunostaining for laminin-332 γ2 chain, located in the majority of cases (90%, n = 173) in the invasive edge of the tumors. To mimic the progression of cSCC we established 3D spheroid cocultures using primary skin fibroblasts and HaCaT/ras-HaCaT human keratinocytes. Our results indicate that in 3D spheroids, unlike in monolayer cultures, TGF-β upregulates laminin-332 production, but only in cells that harbour oncogenic H-Ras. Accumulation of laminin-332 was prevented by both H-Ras knock down and inhibition of TGF-β signaling by SB431542 or RAdKD-ALK5 kinase-defective adenovirus. Furthermore, fibroblasts accelerated the invasion of ras-HaCaT cells through collagen I gels in a Ras/TGF-β signaling dependent manner. In conclusion, we demonstrate the presence of laminin-332 in the invasive front of cSCC tumors and report a new Ras/TGF-β-dependent mechanism that promotes laminin-332 accumulation and cancer cell invasion.

Purpose The two main etiological factors for vulvar squamous cell carcinoma (vSCC) are the vulvar dermatosis lichen sclerosus (LS) and high-risk human papillomavirus (hrHPV). Serpin A1 (α1-antitrypsin) is a serine protease inhibitor, which plays a role in the tumorigenesis of various cancer types. The aim of the study was to evaluate the expressions of Serpin A1 in LS, premalignant vulvar lesions, and vSCC using immunohistochemistry (IHC) and serum analysis, and to compare Serpin A1 stainings to the tumor markers p53 and p16. Methods In total, 120 samples from 74 patients were studied with IHC for Serpin A1, p53 and p16: 18 normal vulvar skin, 53 LS, 9 premalignant vulvar lesions (dVIN/HSIL) and 40 vSCC samples. Serum concentrations of Serpin A1 were analyzed from 30 LS, 44 vSCC and 10 control patients. Expressions were compared to clinical data. Results Tumor cell-specific Serpin A1 overexpression was detected in 88% of vSCC samples, independent of the etiology. The intensity of Serpin A1 expression was significantly higher in vSCC than in healthy vulvar skin, LS, or premalignant vulvar lesions. Serpin A1 showed an association with p53 positivity. No difference in overall survival was found between Serpin A1-, p53-, or p16-positive vSCC patients. Serum concentrations of Serpin A1 were equal in the LS, vSCC, and control groups. Conclusion Tumor cell-specific Serpin A1 overexpression is a potential biomarker in vSCC.

Keratinocyte-derived skin cancer, cutaneous squamous cell carcinoma (cSCC), is the most common metastatic skin cancer. We have examined the role of Eph/ephrin signaling in the progression of cSCC. Analysis of the expression of EPH and EFN families in cSCC cells and normal epidermal keratinocytes revealed overexpression of EPHB2 mRNA in cSCC cells and cSCC tumors in vivo . Tumor cell–specific overexpression of EphB2 was detected in human cSCCs and in chemically induced mouse cSCCs with immunohistochemistry, whereas the expression of EphB2 was low in premalignant lesions and normal skin. Knockdown of EphB2 expression in cSCC cells suppressed growth and vascularization of cSCC xenografts in vivo and inhibited proliferation, migration, and invasion of cSCC cells in culture. EphB2 knockdown downregulated expression of genes associated with biofunctions cell viability , migration of tumor cells , and invasion of tumor cells . Among the genes most downregulated by EphB2 knockdown were MMP1 and MMP13 . Moreover, activation of EphB2 signaling by ephrin-B2-Fc enhanced production of invasion proteinases matrix metalloproteinase-13 (MMP13) and MMP1, and invasion of cSCC cells. These findings provide mechanistic evidence for the role of EphB2 in the early progression of cSCC to the invasive stage and identify EphB2 as a putative therapeutic target in this invasive skin cancer.

The incidence of cutaneous squamous cell carcinoma (cSCC) is rising worldwide. We have examined the role of complement components in the progression of cSCC. Analysis of cSCC cell lines (n=8) and normal human epidermal keratinocytes (n=11) with whole transcriptome profiling (SOLiD), quantitative real-time reverse transcriptase–PCR, and western blotting revealed marked overexpression of complement factor I (CFI) in cSCC cells. Immunohistochemical analysis for CFI in vivo showed stronger tumor cell–specific labeling intensity in invasive sporadic cSCCs (n=83) and recessive dystrophic epidermolysis bullosa–associated cSCCs (n=7) than in cSCC in situ (n=65), premalignant epidermal lesions (actinic keratoses, n=64), benign epidermal papillomas (seborrheic keratoses, n=39), and normal skin (n=9). The expression of CFI was higher in the aggressive Ha-ras-transformed cell line (RT3) than in less tumorigenic HaCaT cell lines (HaCaT, A5, and II-4). The expression of CFI by cSCC cells was upregulated by IFN-γ and IL-1β. Knockdown of CFI expression inhibited proliferation and migration of cSCC cells and resulted in inhibition of basal extracellular signal–regulated kinase (ERK) 1/2 activation. Knockdown of CFI expression potently inhibited growth of human cSCC xenograft tumors in vivo. These results provide evidence for the role of CFI in the progression of cSCC and identify it as a potential therapeutic target in this nonmelanoma skin cancer.

High expression of the collagen receptor, α2β1 integrin, on platelets of ITGA2 807T-allele carriers has been identified as a risk factor for thromboembolic conditions, and α2β1 inhibitors are considered to be potential therapeutic agents. In 59 genotyped individuals, we measured α2 expression levels on platelets and analyzed platelet adhesion to collagen under flow conditions. A sulfonamide-type small-molecule inhibitor of α2β1 integrin decreased average platelet adhesion in individuals with the C/T807T genotype but not in those harboring C807C. Thus, genotype can be used to select a human subpopulation that has the highest probability of showing a positive response to α2β1 inhibitors.

The incidence of cutaneous squamous cell carcinoma (cSCC) is rapidly increasing, and the prognosis of patients with metastatic disease is poor. There is an emerging need to identify molecular markers for predicting aggressive behaviour of cSCC. Here, we have examined the role of tight junction (TJ) components in the progression of cSCC. The expression pattern of mRNAs for TJ components was determined with RNA sequencing and oligonucleotide array-based expression analysis from cSCC cell lines (n=8) and normal human epidermal keratinocytes (NHEK, n=5). The expression of CLDN11 was specifically elevated in primary cSCC cell lines (n=5), but low or absent in metastatic cSCC cell lines (n=3) and NHEKs. Claudin-11 was detected in cell-cell contacts of primary cSCC cells in culture by indirect immunofluorescence analysis. Analysis of a large panel of tissue samples from sporadic UV-induced cSCC (n=65), cSCC in situ (n=56), actinic keratoses (n=31), seborrhoeic keratoses (n=7) and normal skin (n=16) by immunohistochemistry showed specific staining for claudin-11 in intercellular junctions of keratinizing tumor cells in well and moderately differentiated cSCCs, whereas no staining for claudin-11 was detected in poorly differentiated tumors. The expression of claudin-11 in cSCC cells was dependent on the activity of p38δ MAPK and knock-down of claudin-11 enhanced cSCC cell invasion. These findings provide evidence for the role of claudin-11 in regulation of cSCC invasion and suggest loss of claudin-11 expression in tumor cells as a biomarker for advanced stage of cSCC.

In this issue, Rohani et al. (2015) report on the role of macrophage-derived stromelysin-2 (matrix metalloproteinase (MMP)-10) in promoting the turnover of extracellular matrix (ECM) during cutaneous wound repair. They provide evidence that MMP-10 specifically enhances collagenolytic activity of murine MMP-13 produced by M2-like macrophages. These results emphasize the important role of macrophage-derived MMP-10 in regulating tissue remodeling and scar formation during wound healing.

Cutaneous squamous cell carcinoma (cSCC) is one of the most common metastatic skin cancers with increasing incidence. We examined the roles of complement component C3 and complement factor B (CFB) in the growth of cSCC. Analysis of cSCC cell lines (n = 8) and normal human epidermal keratinocytes (n = 11) with real-time quantitative PCR and Western blotting revealed up-regulation of C3 and CFB expression in cSCC cells. Immunohistochemical staining revealed stronger tumor cell-specific labeling for C3 and CFB in invasive cSCCs (n = 71) and recessive dystrophic epidermolysis bullosa-associated cSCCs (n = 11) than in cSCC in situ (n = 69), actinic keratoses (n = 63), and normal skin (n = 5). Significant up-regulation of C3 and CFB mRNA expression was noted in chemically induced mouse cSCCs, compared to benign papillomas. Knockdown of C3 and CFB expression inhibited migration and proliferation of cSCC cells and resulted in potent inhibition of extracellular signal-regulated kinase 1/2 activation. Knockdown of C3 and CFB markedly inhibited growth of human cSCC xenograft tumors in vivo. These results provide evidence for the roles of C3 and CFB in the development of cSCC and identify them as biomarkers and potential therapeutic targets in this metastatic skin cancer.

Epidermal keratinocyte-derived cutaneous squamous cell carcinoma (cSCC) is the most common metastatic skin cancer, and its incidence is increasing worldwide. Solar UV radiation is an important risk factor for cSCC and leads to genetic and epigenetic changes both in epidermal keratinocytes and dermal cells. Tumor cells in cutaneous cSCCs typically harbor several driver gene mutations, but epidermal keratinocytes in sun-exposed normal skin also contain mutations in these same genes. Therefore, alterations in the microenvironment of premalignant lesions are evidently required for their progression to invasive and metastatic cSCC. For example, alterations in the composition of basement membrane and dermal extracellular matrix are early events in cSCC progression. The presence of microbial structures and the influx of inflammatory cells promote the secretion of proteases, which in turn regulate the availability of growth factors, cytokines, and chemokines and thus influence the growth and invasion of cSCC. Together, these observations emphasize the role of the tumor microenvironment in the progression of cSCC and identify it as a novel therapeutic target in cSCC and other malignant tumors. Graphical abstract Tumor-stroma interactions in the progression of cutaneous squamous cell carcinoma (cSCC). Epidermal layer is separated by a well-organized basement membrane (BM) from the dermal layer. UV radiation, other environmental insults, and aging target both epidermal keratinocytes and dermal fibroblasts and lead to genetic and epigenetic changes in these cells. In addition, epidermal keratinocytes in normal sun-exposed skin harbor several mutations in the cSCC driver genes. During transition to premalignant actinic keratosis (AK), the differentiation of keratinocytes is disturbed resulting in a neoplastic epithelium with hyperplastic cells. Expression of proteinases, such as matrix metalloproteinases (MMP) by neoplastic cells and activated stromal fibroblasts and macrophages is induced in AK, and collagen XV and XVIII are lost from the dermal BM. Furthermore, inflammatory cells accumulate at the site of the hyperplastic epithelium. During a later stage of cSCC progression, the number of inflammatory cells increases, and the expression of complement components and inhibitors by tumor cells is induced (CFI complement factor I, CFH complement factor H, FHL-1 Factor H-like protein 1). In addition to MMPs, activated fibroblasts also produce growth factors and promote inflammation, growth, and invasion of tumor cells.

Cutaneous squamous cell carcinoma (cSCC) is the second most common cancer and a leading cause of cancer mortality among solid organ transplant recipients. MicroRNAs (miR) are short RNAs that regulate gene expression and cellular functions. Here, we show a negative correlation between miR-203 expression and the differentiation grade of cSCC. Functionally, miR-203 suppressed cell proliferation, cell motility, and the angiogenesis-inducing capacity of cSCC cells in vitro and reduced xenograft tumor volume and angiogenesis in vivo. Transcriptomic analysis of cSCC cells with ectopic overexpression of miR-203 showed dramatic changes in gene networks related to cell cycle and proliferation. Transcription factor enrichment analysis identified c-MYC as a hub of miR-203–induced transcriptomic changes in squamous cell carcinoma. We identified c-MYC as a direct target of miR-203. Overexpression of c-MYC in rescue experiments reversed miR-203–induced growth arrest in cSCC, which highlights the importance of c-MYC within the miR-203–regulated gene network. Together, miR-203 acts as a tumor suppressor in cSCC, and its low expression can be a marker for poorly differentiated tumors. Restoration of miR-203 expression may provide a therapeutic benefit, particularly in poorly differentiated cSCC.

Keratinocyte-derived cutaneous squamous cell carcinoma (cSCC) is the most common metastatic skin cancer, and its incidence is increasing globally. Long noncoding RNAs (lncRNA) are involved in various biological processes, and their role in cancer progression is emerging. Whole transcriptome analysis of cSCC cells (n = 8) and normal human epidermal keratinocytes (n = 4) revealed overexpression of long intergenic ncRNA (LINC00162) in cSCC cells. The expression of LINC00162 in cSCC cells was upregulated by inhibition of the p38α and p38δ mitogen-activated protein kinases. Analysis of tissue sections by RNA in situ hybridization showed that LINC00162 is specifically expressed by tumor cells in cSCCs but not by keratinocytes in normal skin in vivo. Knockdown of LINC00162 inhibited proliferation and migration of cSCC cells, and suppressed the growth of human cSCC xenografts in vivo. Furthermore, knockdown of LINC00162 inhibited extracellular signal-regulated kinase 1/2 activity and upregulated expression of dual specificity phosphatase 6 (DUSP6) in cSCC cells. Based on these observations, LINC00162 was named p38 inhibited cutaneous squamous cell carcinoma associated lincRNA (PICSAR). Our results provide mechanistic evidence for the role of PICSAR in promoting cSCC progression via activation of extracellular signal-regulated kinase 1/2 signaling pathway by downregulating DUSP6 expression. These results also identify PICSAR as a biomarker and putative therapeutic target in cSCC.

TGF-β regulates several steps in cancer metastasis, including the establishment of bone metastatic lesions. TGF-β is released from bone during osteoclastic bone resorption and it stimulates breast cancer cells to produce osteolytic factors such as interleukin 11 (IL-11). We conducted a cell-based siRNA screen and identified heparan sulfate 6-O-sulfotransferase 2 (HS6ST2) as a critical gene for TGF-β–induced IL-11 production in highly bone metastatic MDA-MB-231(SA) breast cancer cells. HS6ST2 attaches sulfate groups to glucosamine residues in heparan sulfate glycosaminoglycans. We subsequently showed how heparin and a high-molecular-weight Escherichia coli K5-derived heparin-like polysaccharide (K5-NSOS) inhibited TGF-β–induced IL-11 production in MDA-MB-231(SA) cells. In addition, K5-NSOS inhibited bone resorption activity of human osteoclasts in vitro. We evaluated the therapeutic potential of K5-NSOS and fragmin in a mouse model of breast cancer bone metastasis. MDA-MB-231(SA) cells were inoculated into the left cardiac ventricle of athymic nude mice which were treated with fragmin, K5-NSOS, or vehicle once a day for four weeks. Both heparin-like glycosaminoglycans inhibited weight reduction, decreased osteolytic lesion area, and reduced tumor burden in bone. In conclusion, our data imply novel mechanisms involved in TGF-β induction and support the critical role of heparan sulfate glycosaminoglycans in cancer metastasis as well as indicate that K5-NSOS is a potential antimetastatic and antiresorptive agent for cancer therapy. This study illustrates the potential to translate in vitro siRNA screening results toward in vivo therapeutic concepts. Mol Cancer Res; 10(5); 597–604. ©2012 AACR.

A novel virus- and integrin clustering–specific pathway diverts integrin from its normal endo/exocytic traffic to a nonrecycling degradative endosomal route. Clustering of α2β1 integrin causes redistribution of the integrin to perinuclear endosomes, leading to enhanced integrin turnover promoted by calpains., Collagen receptor integrins recycle between the plasma membrane and endosomes and facilitate formation and turnover of focal adhesions. In contrast, clustering of α2β1 integrin with antibodies or the human pathogen echovirus 1 (EV1) causes redistribution of α2 integrin to perinuclear multivesicular bodies, α2-MVBs. We show here that the internalized clustered α2 integrin remains in α2-MVBs and is not recycled back to the plasma membrane. Instead, receptor clustering and internalization lead to an accelerated down-regulation of α2β1 integrin compared to the slow turnover of unclustered α2 integrin. EV1 infection or integrin degradation is not associated with proteasomal or autophagosomal processes and shows no significant association with lysosomal pathway. In contrast, degradation is dependent on calpains, such that it is blocked by calpain inhibitors. We show that active calpain is present in α2-MVBs, internalized clustered α2β1 integrin coprecipitates with calpain-1, and calpain enzymes can degrade α2β1 integrin. In conclusion, we identified a novel virus- and clustering-specific pathway that diverts α2β1 integrin from its normal endo/exocytic traffic to a nonrecycling, calpain-dependent degradative endosomal route.

Collagen binding integrins are an important family of cell surface receptors that mediate bidirectionally signals between the interior of the cell and the extracellular matrix. The protein-protein interactions between cells and collagen are necessary for many physiological functions, but also promote diseases. For example, the interaction of α2β1 integrin and collagen has been shown to have an important role in thrombus formation and cancer spread. The fact that the discovery of small molecules that can block such protein-protein interactions is highly challenging has significantly hindered the discovery of pharmaceutical agents to treat these diseases. Here, we present a rationally designed novel fluorescent molecule that can be synthesized in just a few minutes from commercially available starting materials. This molecule blocks the protein-protein interaction between α2β1 integrin and collagen, and due to its fluorescent properties, it can be employed in wide variety of biological applications.

The incidence of cutaneous squamous cell carcinoma (cSCC) is increasing worldwide. We have analyzed the role of complement components C1r and C1s in the progression of cSCC. Analysis of cSCC cell lines (n=8) and normal human epidermal keratinocytes (NHEK)(n=11) with oligonucleotide arrays, RNA-seq and quantitative RT-PCR revealed significantly elevated C1r and C1s mRNA levels in cSCC cell lines. Western blot analysis showed increased production of C1r and C1s in cSCC cells compared to NHEKs. The mRNA levels for C1r and C1s were markedly elevated in cSCC tumors (n=6) compared to normal skin (n=11) in vivo. Immunohistochemical analysis for C1r and C1s revealed strong tumor cell specific expression of C1r and C1s in invasive sporadic cSCCs (n=164) and recessive dystrophic epidermolysis bullosa-associated cSCCs (n=16), whereas the expression of both was clearly lower in cSCC in situ (n=63), in premalignant epidermal lesions (actinic keratoses, n=61), and in normal skin (n=9). Knockdown of C1r and C1s expression with two specific siRNAs inhibited proliferation of cSCC cells and growth of human cSCC xenograft tumors in vivo. These results provide evidence for the role of tumor cell-derived C1r and C1s in the progression of cSCC independently of C1q and identify these serine proteinases as putative therapeutic targets in unresectable and metastatic cSCC. Citation Format: Pilvi Riihilä, Liisa Nissinen, Mehdi Farshchian, Markku Kallajoki, Atte Kivisaari, Seppo Meri, Reidar Grénman, Sirkku Peltonen, Juha Peltonen, Veli-Matti Kähäri. Tumor cell-derived complement components C1r and C1s promote growth of cutaneous squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5214.