Osteopontin promotes the invasive growth of melanoma cells by activating integrin αvß3 and down-regulating tetraspanin CD9.

Overexpression of osteopontin (OPN) is strongly associated with the invasiveness/metastasis of many cancers, including melanomas. However, the molecular mechanisms of OPN in these processes remain poorly understood. We found that forced expression of OPN in early vertical-growth-phase melanoma cells dramatically increased their migration/invasion and growth/survival in a three-dimensional collagen I gel. Neutralizing antibodies to OPN, integrin ß1, and integrin αvß3, but not to CD44, negated the effects of OPN. Conversely, knocking down OPN in metastatic melanoma cells abrogated the invasive growth. OPN overexpression activated and OPN knockdown inactivated αvß3 and αvß5 integrins, negligibly affecting their expression. We further found OPN expression to inversely correlate with tetraspanin CD9 expression. Early-stage melanoma cells displayed low OPN and high CD9 expression, and conversely, metastatic cells displayed high OPN and low CD9 expression. Overexpression of OPN in vertical-growth-phase melanoma cells induced down-regulation of CD9, and knockdown of OPN in metastatic melanoma cells up-regulated CD9. Reversion of these CD9 changes abolished the effects of OPN. Furthermore, knockdown of CD9 in early-stage melanoma cells stimulated their invasive capacity in three-dimensional collagen. Similarly, microarray analyses of benign nevi and primary melanomas from different stages revealed an inverse correlation between OPN and CD9. These data suggest that OPN promotes melanoma cell invasion by activating integrin αvß3 and down-regulating CD9, a putative metastasis suppressor.

Toll-like receptors -4 and -5 in oral and cutaneous squamous cell carcinomas.

BACKGROUND: Oral squamous cell carcinoma (OSCC) has a worse prognosis than cutaneous squamous cell carcinoma (CSCC). Toll-like receptor- 4 (TLR-4) and TLR-5 are transmembrane proteins that recognize endogenous and microbial agents. Their activation has been connected to cancer invasion. OBJECTIVE: The aim was to study the expression of TLR-4 and TLR-5 in OSCC and CSCC samples, and the effects of TLR-5 ligand flagellin on the proliferation, migration, and invasion of different mucocutaneous cell lines in vitro. METHODS: Samples of early-stage tumors (T1-T2N0M0) from 63 patients with OSCC and CSCC were obtained, in addition to eight normal mucosa and skin tissues from healthy subjects. Oral-cavity-derived highly aggressive HSC-3, less invasive SAS, and HPV-transformed benign IHGK as well as C-ha-ras-transformed (HaCat) skin carcinoma II-4 and non-invasive A5 cell lines were used. Flagellin-induced mucocutaneous cell lines were compared by using BrdU-proliferation, scratch migration, and myoma organotypic invasion assays. RESULTS: TLR-4 expression was similar in OSCC and CSCC tumors. TLR-5 was more abundant in OSCC than in CSCC samples. Flagellin induced the proliferation of SAS, II-4 and A5, migration of IHGK, II-4 and A5, and the invasion of II-4 cells. It had no effect on HSC-3 cells. CONCLUSIONS: Flagellin, a TLR-5 agonist, induced the migration and invasion of less aggressive mucocutaneous cell lines, but it had no effect on the most invasive oral carcinoma cells. The more aggressive clinical behavior of OSCC compared to CSCC may partially be related to the differences in the expression of TLR-5 in these malignancies.

TGF-ß signaling, activated stromal fibroblasts, and cysteine cathepsins B and L drive the invasive growth of human melanoma cells.

Accumulating evidence indicates that interactions between cancer cells and stromal cells are important for the development/progression of many cancers. Herein, we found that the invasive growth of melanoma cells in three-dimensional-Matrigel/collagen-I matrices is dramatically increased on their co-culture with embryonic or adult skin fibroblasts. Studies with fluorescent-labeled cells revealed that the melanoma cells first activate the fibroblasts, which then take the lead in invasion. To identify the physiologically relevant invasion-related proteases involved, we performed genome-wide microarray analyses of invasive human melanomas and benign nevi; we found up-regulation of cysteine cathepsins B and L, matrix metalloproteinase (MMP)-1 and -9, and urokinase- and tissue-type plasminogen activators. The mRNA levels of cathepsins B/L and plasminogen activators, but not MMPs, correlated with metastasis. The invasiveness/growth of the melanoma cells with fibroblasts was inhibited by cell membrane-permeable inhibitors of cathepsins B/L, but not by wide-spectrum inhibitors of MMPs. The IHC analysis of primary melanomas and benign nevi revealed cathepsin B to be predominantly expressed by melanoma cells and cathepsin L to be predominantly expressed by the tumor-associated fibroblasts surrounding the invading melanoma cells. Finally, cathepsin B regulated TGF-ß production/signaling, which was required for the activation of fibroblasts and their promotion of the invasive growth of melanoma cells. These data provide a basis for testing inhibitors of TGF-ß signaling and cathepsins B/L in the therapy of invasive/metastatic melanomas.

Epithelial and stromal syndecan-1 and -2 are distinctly expressed in oral- and cutaneous squamous cell carcinomas.

BACKGROUND: Oral squamous cell carcinoma (OSCC) and cutaneous squamous cell carcinoma (CSCC) are epithelial neoplasms of which OSCC has worse survival and higher risk of metastasis than CSCC. The aim of this study was to explore the differences of immunoexpressions between syndecan-1 and -2 in OSCC and head and neck CSCC. METHODS: A total of 35 patients diagnosed with OSCC and 25 with CSCC, presented T1 and T2 tumors and treated at Helsinki University Central Hospital between years 2001 and 2009, were selected into this study. The levels and locations of syndecan-1 and -2 immunostainings were analyzed using formalin-fixed and paraffin-embedded tissue samples of OSCC and CSCC cases together with clinical data. RESULTS: Cell membrane epithelial syndecan-1 expression decreased significantly compared to normal tissue in both cancer types. Cell membrane syndecan-1 expression in the invasive front had negative correlation with invasion depth of both tumors (OSCC, r = -0.339, P = 0.025; CSCC, r = -0.469, P = 0.004). In cancers over 4-mm invasion depth, the number of stromal syndecan-1-positive collagen fibers and inflammatory cells were higher in OSCC than in CSCC. Syndecan-2 expression in non-malignant stroma was higher in CSCC than in OSCC tumors. In addition, unlike syndecan-1, syndecan-2 was more often and more intensively expressed in the tumor inflammatory cells in CSCC than in OSCC. CONCLUSION: Our results suggest that variable stromal expression of syndecan-1 and -2 in OSCC compared to CSCC may at least partially explain the differences in their clinical behavior.

[Cutaneous leukocytoclastic vasculitis in a patient with an adenocarcinoma of the colon]. / Peräsuolisyöpää sairastavan potilaan ihon leukosytoklastinen vaskuliitti--seurausta syövästä?

Association of cutaneous leukocytoclastic vasculitis with colon carcinoma has occasionally been reported. We report a case of acute cutaneous leucocytoclastic vasculitis that developed over two weeks after liver resection due to metastatic rectal adenocarcinoma. The primary tumor had earlier been resected and treated with neoadjuvant chemotherapy. No actual infection was found and the medication was not changed recently except for the prophylaxis cephalosporin for five days at the time of liver resection. The patient received corticosteroid therapy and had remission of vasculitis at one month control but still ongoing haematuria. Eight months after liver resection colonoscopy, contrast enhanced whole-body computed tomography, tumour markers and urine red cell count showed no significant findings.

[Punch biopsy in the diagnosis of skin tumors]. / Stanssibiopsia ihokasvainten diagnostiikassa.

Punch biopsy is a feasible method in the early diagnosis of nonmelanotic skin cancer in primary health care, but should not be applied to lesions that appear clinically as melanomas. Small skin lesions suspected to be malignant can often be completely excised in primary health care. If the skin tumor is large, a biopsy from the tumor area is worth taking so that the edge of the tumor remains intact. A sufficient specimen for histological examination is usually obtained with a punch having a diameter of 3 to 4 millimeters.

Metastatic outgrowth encompasses COL-I, FN1, and POSTN up-regulation and assembly to fibrillar networks regulating cell adhesion, migration, and growth.

Although the outgrowth of micrometastases into macrometastases is the rate-limiting step in metastatic progression and the main determinant of cancer fatality, the molecular mechanisms involved have been little studied. Here, we compared the gene expression profiles of melanoma lymph node micro- and macrometastases and unexpectedly found no common up-regulation of any single growth factor/cytokine, except for the cytokine-like SPP1. Importantly, metastatic outgrowth was found to be consistently associated with activation of the transforming growth factor-beta signaling pathway (confirmed by phospho-SMAD2 staining) and concerted up-regulation of POSTN, FN1, COL-I, and VCAN genes-all inducible by transforming growth factor-beta. The encoded extracellular matrix proteins were found to together form intricate fibrillar networks around tumor cell nests in melanoma and breast cancer metastases from various organs. Functional analyses suggested that these newly synthesized protein networks regulate adhesion, migration, and growth of tumor cells, fibroblasts, and endothelial cells. POSTN acted as an anti-adhesive molecule counteracting the adhesive functions of FN1 and COL-I. Further, cellular FN and POSTN were specifically overexpressed in the newly forming/formed tumor blood vessels. Transforming growth factor-beta receptors and the metastasis-related matrix proteins, POSTN and FN1, in particular, may thus provide attractive targets for development of new therapies against disseminated melanoma, breast cancer, and possibly other tumors, by affecting key processes of metastasis: tumor/stromal cell migration, growth, and angiogenesis.

Tumor burden of sentinel lymph node metastasis in Merkel cell carcinoma.

BACKGROUND: The applicability of sentinel lymph node biopsy (SLNB) for staging has been recognized in association with Merkel cell carcinoma (MCC). However, the concept of tumor burden with respect to MCC involving sentinel lymph nodes has not been analyzed. Our aim was to assess tumor burden in the sentinel lymph nodes of MCC patients. METHODS AND RESULTS: This retrospective study analyzes 15 consecutive patients and 57 sentinel lymph nodes. Immunohistochemical re-evaluation of the sentinel lymph nodes was performed. Positive sentinel node metastases were classified according to type of invasion, size and tumor burden. Occult metastases were observed in 36% of the patients. Maximal sizes of the metastatic foci and tumor burden in the lymph nodes were larger than what has been reported in melanoma and breast cancer sentinel lymph nodes. The distribution of metastatic cells in the involved lymph nodes was mostly combination type with only one marginal sinus type. False-negative lymph nodes were found in 30% of the patients. Immunohistochemical re-evaluation decreased this figure to 22%. CONCLUSIONS: Metastatic foci and tumor burden in positive sentinel lymph nodes seem to be larger in MCC than in melanoma and breast cancer sentinel nodes. Statistical analysis showed no correlation between tumor burden and survival. In the context of MCC, false-negative sentinel lymph nodes can and should be limited by using immunohistochemistry.

MMP-10 (Stromelysin-2) and MMP-21 in human and murine squamous cell cancer.

The squamous cell cancers (SCC) of renal transplant recipients are more aggressive and metastasize earlier than those of the non-immunocompromised population. Matrix metalloproteinases (MMPs) have a central role in tumor initiation, invasion and metastasis. Our aim was to compare the expression of MMPs-10, -12 and -21 in SCCs from immunosuppressed (IS) and control patients and the contribution of MMPs-10 and -21 to SCC development in the FVB/N-Tg(KRT5-Nfkbia)3Rto mouse line. Immunohistochemical analysis of 25 matched pairs of SCCs, nine of Bowen's disease and timed back skin biopsies of mice with selective inhibition of Rel/NF-kappaB signalling were performed. Semiquantitatively assessed stromal MMP-10 expression was higher (P = 0.009) in the control group when compared with IS patients. Tumor cell-derived MMP-10, -12 and -21 expression did not differ between the groups but stromal fibroblasts of the control SCCs tended to express MMP-21 more abundantly. MMP-10 expression was observed already in Bowen's disease while MMP-21 was absent. MMP-10 and -21 were present in inflammatory or stromal cells in ageing mice while dysplastic keratinocytes and invasive cancer were negative. Our results suggest that MMP-10 may be important in the initial stages of SCC progression and induced in the stroma relating to the general host-response reaction to skin cancer. MMP-21 does not associate with invasion of SCC but may be involved in keratinocyte differentiation.

Cutaneous T-cell lymphoma-associated lung cancers show chromosomal aberrations differing from primary lung cancer.

Cutaneous T-cell lymphoma (CTCL) patients have an increased risk of certain secondary cancers, the most common of which are lung cancers, especially small cell lung cancer. To reveal the molecular pathogenesis underlying CTCL-associated lung cancer, we analyzed genomic aberrations in CTCL-associated and reference lung cancer samples. DNA derived from microdissected lung cancer cells of five CTCL-associated lung cancers and five reference lung cancers without CTCL association was analyzed by comparative genomic hybridization (CGH). Fluorescent in situ hybridization (FISH), immunohistochemistry (IHC), and loss of heterozygosity (LOH) analysis were performed for selected genes. In CTCL-associated lung cancer, CGH revealed chromosomal aberrations characterizing both lung cancer and CTCL, but also losses of 1p, and 19, and gains of 4q and 7, hallmarks of CTCL. LOH for the CTCL-associated NAV3 gene was detected in two of the four informative primary lung cancers. FISH revealed increased copy number of the KIT gene in 3/4 of CTCL-associated lung cancers and 1/5 of primary lung cancers. PDGFRA and VEGFR2 copy numbers were also increased. IHC showed moderate KIT expression when the gene copy number was increased. CTCL-associated lung cancer shows chromosomal aberrations different from primary lung cancer, especially amplifications of 4q, a chromosome arm frequently deleted in the latter tumor type. Copy numbers and expression of selected genes in chromosome 4 differed between CTCL-associated and reference lung cancers. These preliminary observations warrant further prospective studies to identify the common underlying factors between CTCL and CTCL-associated lung cancer.

Differential expression of stromal MMP-1, MMP-9 and TIMP-1 in basal cell carcinomas of immunosuppressed patients and controls.

Matrix metalloproteinases (MMPs) have an important role in the initiation, growth, and invasion of malignant tumors. Basal cell cancer (BCC) is the most common human malignancy. The risk of BCC is 10-16 times higher among organ transplant recipients compared with the nontransplanted population. The aim of this study was to compare the expression of several MMPs and their tissue inhibitors (TIMPs) in BCCs from kidney transplant recipients and controls. Expression of MMPs-1, -7, -8, -9, -10, -13, -26, and TIMPs-1 and -3 was evaluated by immunohistochemistry in 25 samples of BCC of kidney transplant recipients and 25 matched controls representing superficial and nodular subtypes. No significant differences were detected in MMP expression of BCC tumor cells between immunocompetent and immunodeficient patients. However, MMPs-1 and -9 and TIMP-1 were expressed more frequently in stromal macrophages in the BCCs of immunocompetent patients. When tumor subtypes were compared irrespective of the patient group, more MMP-1-positive fibroblasts and MMP-9-positive neutrophils were detected in the superficial subtype, while stromal MMP-10 expression was more abundant in nodular tumors. Our results suggest that abundant peritumoral expression of TIMP-1 in non-immunocompromised patients limits ECM degradation permissive for cancer cell migration.

Melkersson-Rosenthal syndrome.

OBJECTIVES: To study characteristics of Melkersson-Rosenthal syndrome (MRS) patients with facial palsy (FP) and differences in patients treated at the Departments of Otorhinolaryngology and Dermatology. METHODS: Clinical picture of MRS was studied from patient charts at two departments. Patients with FP received a questionnaire and were examined. Tissue biopsies were searched for non-necrotizing granulomatous infiltrations typical of MRS and blood DNA for UNC-93B1 gene mutations predisposing to herpesvirus infection. RESULTS: At the Department of Otorhinolaryngology, all 18 MRS patients had FP, 9 the triad form. Two patients revealed non-necrotizing granulomatous infiltrations during acute edema episodes; another two had association with uveitis. Edema was rarely persistent and did not dominate the clinical picture. No UNC-93B1 mutations were found. At the Department of Dermatology, 2 patients had triad MRS and 15 had monosymptomatic granulomatous cheilitis with persistent edema and typical MRS histology. CONCLUSION: The clinical picture of MRS with FP differed from the current knowledge of edema-dominated MRS. More studies focusing on MRS with FP would broaden our understanding of the syndrome.

Apocrine mixed tumor of the skin ("mixed tumor of the folliculosebaceous-apocrine complex"). Spectrum of differentiations and metaplastic changes in the epithelial, myoepithelial, and stromal components based on a histopathologic study of 244 cases.

BACKGROUND: A systematic analysis of the entire spectrum of various forms of differentiation and metaplastic epiphenomena in cutaneous apocrine mixed tumor (AMT) has never been performed. OBJECTIVE: The purpose of our study was to study a large number of cutaneous mixed tumors so as to fully characterize the entire spectrum of differentiations and metaplastic changes that may occur in the epithelial, myoepithelial, and stromal components of AMT. METHODS: This article reports a light-microscopic study of 244 cases of cutaneous AMT, complemented by a literature review. RESULTS: All types of differentiation along the lines of the folliculosebaceous-apocrine unit can be seen in AMT. The spectrum of metaplastic changes in the epithelial components includes squamous metaplasia, mucinous metaplasia, oxyphilic metaplasia, clear cell change, columnar metaplasia, hobnail metaplasia, and cytoplasmic vacuolization. The following changes in the myoepithelial component were documented: clear cell change, hyaline cells, plasmacytoid cells, spindling, and collagenous spherulosis. Stromal alterations included chondroid metaplasia, osseous metaplasia, and adipose metaplasia. LIMITATIONS: This study utilizes tissue specimens that mainly came as consultations; therefore some inherent selection bias exists. CONCLUSIONS: AMT displays a wide range of differentiation and metaplastic changes in its epithelial, myoepithelial, and stromal components. These phenomena are not mutually exclusive. When unduly prominent, they may present diagnostic pitfalls. Our findings corroborate those of previous publications, stressing the remarkable diversity of differentiation and metaplasias that may be found in cutaneous AMT. We propose that the most appropriate name for these lesions is "mixed tumor of the folliculosebaceous-apocrine complex."

Gene expression analyses of primary melanomas reveal CTHRC1 as an important player in melanoma progression.

Melanoma is notorious for its high tendency to metastasize and its refractoriness to conventional treatments after metastasis, and the responses to most targeted therapies are short-lived. A better understanding of the molecular mechanisms behind melanoma development and progression is needed to develop more effective therapies and to identify new markers to predict disease behavior. Here, we compared the gene expression profiles of benign nevi, and non-metastatic and metastatic primary melanomas to identify any common changes in disease progression. We identified several genes associated with inflammation, angiogenesis, and extracellular matrix modification to be upregulated in metastatic melanomas. We selected one of these genes, collagen triple helix repeat containing 1 (CTHRC1), for detailed analysis, and found that CTHRC1 was expressed in both melanoma cells and the associated fibroblasts, as well as in the endothelium of tumor blood vessels. Knockdown of CTHRC1 expression by shRNAs in melanoma cells inhibited their migration in Transwell assays and their invasion in three-dimensional collagen and Matrigel matrices. We also elucidated the possible down-stream effectors of CTHRC1 by gene expression profiling of the CTHRC1-knockdown cells. Our analyses showed that CTHRC1 is regulated coordinately with fibronectin and integrin ß3 by the pro-invasive and -angiogenic transcription factor NFATC2. We also found CTHRC1 to be a target of TFGß and BRAF. These data highlight the importance of tumor stroma in melanoma progression. Furthermore, CTHRC1 was recognized as an important mediator of melanoma cell migration and invasion, providing together with its regulators-NFATC2, TGFß, and BRAF-attractive therapeutic targets against metastatic melanomas.

MMP-21 is upregulated at early stages of melanoma progression but disappears with more aggressive phenotype.

The expression of matrix metalloproteinases (MMPs) is frequently altered during malignant transformation. We examined the profile of three recently cloned MMPs, MMP-21, MMP-26, and MMP-28, in melanomas in vivo and in culture. Immunohistochemistry for MMPs-21, -26, -28, and -13 in melanoma specimens (27 nonmetastatic, 26 with nodal micrometastases, and 10 in situ melanomas) from 63 patients was performed. MMP-21 was expressed in melanoma cells in 29/53 cases, being more frequent in melanoma samples without micrometastases. Six out of ten in situ melanomas were positive, while five nevus samples were negative. MMP-26 and -28 were not generally expressed in melanoma cells. MMP-13 was detected in melanoma cells in 36/53 samples. MMP-21 was not found in sentinel nodes with metastases, while MMP-13 was seen in all of them. MMP-21 messenger RNA was variably expressed in all five melanoma cell lines investigated using reverse transcriptase-polymerase chain reaction. Our results suggest that expression of MMP-21 may serve as a marker of malignant transformation of melanocytes and does not associate with the presence of micrometastases.

Prospective immunohistochemical analysis of BRAF V600E mutation in melanoma.

The v-raf murine sarcoma viral oncogene homolog B1 (BRAF) V600E mutation is the most common activating genetic alteration of this oncogene and a predictive marker for the therapeutic use of BRAF inhibitors in melanoma. Our aim was to evaluate the performance of BRAF V600E mutation-specific monoclonal antibody (VE1) in a prospective diagnostic setting of melanoma patients (n = 102). All 41 cases (40.2%) that showed a V600E mutation in the cyclic minisequencing analysis of the DNA were also initially scored immunopositive. Two cases that were scored as BRAF V600E mutation positive by immunohistochemistry were negative in the DNA-based mutation analysis and determined to be immunonegative in a repeated staining with more representative specimens. Thus, BRAF V600E mutation detection using immunohistochemistry was 100% sensitive and 96.8% specific, when compared with the analysis of the DNA. None of the BRAF V600K mutations was detected by the VE1 antibody (n = 7). However, the VE1 antibody detected a rare V600E2 mutation. We also studied the role of BRAF V600E mutation in a set of melanoma patients who had been investigated for sentinel node metastasis. Melanoma lymph node metastases were diagnosed in 21.8% (12/55) of the sentinel nodes, and BRAF V600E immunopositivity was detected in 34.5% (19/55) of the cases. BRAF V600E mutation status did not correlate with any clinicopathological parameters. In conclusion, analysis of BRAF V600E mutation in melanoma by immunohistochemistry is a sensitive and specific method, which can be used to identify BRAF inhibitor-sensitive melanoma patients as a first-line method due to its rapid and affordable nature.

MMP-7, MMP-8, and MMP-9 in oral and cutaneous squamous cell carcinomas.

OBJECTIVES: Oral squamous cell carcinoma (OSCC) and cutaneous squamous cell carcinoma (CSCC) are epithelial neoplasms, of which OSCC has a worse prognosis. Matrix metalloproteinases (MMPs) are involved in the initiation, invasion, metastasis, and defense of cancer. This study aimed to compare differences in MMP expression in these cancers. STUDY DESIGN: Sixty-one patients with early-stage (T1-T2 N0 M0) cancers, of which 36 were OSCC and 25 CSCC, were enrolled into this study. Immunohistochemical staining was performed with MMP-7, MMP-8, and MMP-9 antibodies. RESULTS: MMP-7 expression was stronger in OSCC than in CSCC, mainly in the invasive front. MMP-8 was absent and MMP-9 was mildly expressed in OSCC and CSCC cells. However, MMP-8 and MMP-9 were positive in peritumoral inflammatory cells in both cancers. In addition, MMP-7, MMP-8, and MMP-9 were not associated with the overall survival of patients with OSCC and CSCC patients. CONCLUSIONS: The increased expression of MMP-7 in the invasive front may partly explain the aggressiveness of OSCC.