Immunohistochemical analysis of TIMP-3 and MMP-9 in actinic keratosis,squamous cell carcinoma of the skin,and basal cell carcinoma

The expression of metalloproteinases and their inhibitors has been related to different invasive and metastatic potentials in cancer. This study aims to investigate the immunohistochemical expression of TIMP-3 and MMP-9 in samples of basal cell carcinoma (BCC), squamous cell carcinoma of the skin (SCC), and actinic keratosis (AK). Immunohistochemistry was performed to evaluate the expression of TIMP-3 and MMP-9 in samples of BCC (n = 22), SCC (n = 10), and AK (n = 15). Ten fields of both tumor parenchyma and tumor stroma were photographed and counted in image software. The ratio of positive cells to total cells was used to quantify the staining. A higher expression of MMP-9 was found in tumor stroma of SCC compared to BCC and AK. No significant differences in TIMP-3 expression were observed among the groups. Considering the well-described differences between these neoplasms, these results provide additional evidence of the role of MMP-9 in tumor invasiveness of keratinocyte-derived tumors.     

Differential expression patterns of MMPs and their role in the invasion of epithelial premalignant tumors and invasive cutaneous squamous cell carcinoma

Co-expression of several members of the matrix metalloproteinase (MMP) family is characteristic of human malignant tumors. MMP-2, MMP-9, TIMP-2, and MT1-MMP are thought to be involved in the process of destruction of basement membranes and stromal invasion by neoplastic epithelial cells. In this study, we investigated the expression and role of MMPs in cutaneous oncogenesis. Tissue microarray consisting of 62 squamous cell carcinomas (SCC), 32 Bowen's disease (BD) samples, 25 normal epidermis samples were obtained for the study. MMP-2,-9, MT1-MMP and TIMP-2 proteins were examined by immunohistochemical staining and mRNA level was detected by quantitative RT-PCR in fresh tissues consisting of 5 cutaneous SCCs and paired normal epidermis samples. Gelatinase activity of MMP-2 and MMP-9 was investigated by gelatin zymography and protein levels of MT1-MMP and TIMP-2 were measured by western blot in 2 human SCC cell lines. The invasive property was evaluated with invasion assays using Transwell filters. SCC exhibited significantly increased MMP-2, MT1-MMP and decreased TIMP-2 mRNA and protein expression compared to that of the normal epithelium. Immunohistochemical staining revealed that MT1-MMP was strongly expressed on the invasive front of SCCs, whereas BD exhibited higher expression around the dyskeratotic cells in the epithelium. In comparison with the expression observed in BD, SCC exhibited significantly increased MMP-2 expression. In addition, high MMP-2 and MT1-MMP expression and low TIMP-2 expression had a significant positive correlation with the invasiveness of SCC cell lines in vitro. Our results revealed significantly increased MT1-MMP and MMP-2 expression and decreased TIMP-2 expression in cutaneous SCC, and the expression correlated with the invasiveness of SCC cell lines. Therefore, the expression of these factors in cutaneous tumors may serve as an indicator of tumor aggressiveness and invasion.     

COX-2 expression correlates with microvessel density in non-melanoma skin cancer from renal transplant recipients and immunocompetent individuals

Angiogenesis, the generation of a new vascular network, is regulated in part by inducers of endothelial cell migration and proliferation, such as cyclooxygenase-2 (COX-2). Microvessel density (MVD) measurement is widely used to quantify angiogenesis in tissue sections of tumors, including cutaneous malignancies. The increasing number of successful renal transplantations worldwide is producing a progressive increase in patients at risk for non-melanoma skin cancers, such as squamous cell carcinoma (SCC), basal cell carcinoma (BCC), and Bowen's disease (BD), and at significantly increased risk for metastatic SCC. The aim of this study was to investigate whether there was any difference in angiogenesis between these tumor types in renal transplant recipients (RTRs) and immunocompetent individuals (ICIs) and whether angiogenesis in these tumors was related to COX-2 expression. The study measured angiogenesis and COX-2 expression in BD, SCC, BCC, and normal skin from both RTRs and ICIs. Vessel counts were performed, and COX-2 immunoexpression was assessed semiquantitatively. The MVD counts differed significantly between normal skin and all tumor types. Significant differences in MVD density were found between all SCCs and BCCs. BCCs from RTRs had significantly greater MVD at the invasive front of the tumor than BCCs from ICIs. Increased COX-2 expression correlated with increased MVD in all tumors examined. These findings indicate a difference in vascular profiles between RTRs and ICIs in BCCs and suggest a relationship between COX-2 and angiogenesis that may provide a possible treatment target for skin tumors in these 2 patient populations.     

Immunohistochemical galectin-3 expression in non-melanoma skin cancers

Galectin-3 is a ss-galactoside-binding lectin. It participates in a variety of normal and pathologic processes, including cancer progression. In this study, we evaluated the pattern of expression of galectin-3 in cutaneous squamous cell carcinoma (SCC) and basal cell carcinoma (BCC), and its correlation with the grade of differentiation in SCC and tumor size. Galectin-3 expression was evaluated by immunohistochemistry in 31 SCCs, 30 BCCs, and 29 non-tumoral skin samples. Galectin-3 expression was higher in normal epidermis than in non-melanoma skin cancers, except for cytoplasmic immunoreactivity in SCC. Cytoplasmic galectin-3 immunoreactivity was significantly higher than nuclear immunoreactivity in non-melanoma skin cancers. Cytoplasmic galectin-3 immunoreactivity was significantly higher in SCC than in both circumscribed and infiltrative BCCs, but no difference was detected between these two types of BCC. Cytoplasmic galectin-3 immunoreactivity predominated within SCCs (p=0.000), and a positive correlation was detected between tumor size and cytoplasmic immunoreactivity (r=0.385, p=0.043). There was no correlation between galectin-3 staining and tumor differentiation and lymph node metastasis. Decreased nuclear galectin-3 expression and cytoplasmic immunoreactivity in tumors are important factors in the progression from the normal to the cancerous state in non-melanoma skin cancers. We speculate that cytoplasmic galectin-3 expression may be one of the factors that contribute to tumor aggressiveness in SCC.     

MMP-3和TIMP-3在卵巢癌中的表达及其意义

目的探讨MMP-3和TIMP-3在卵巢癌的表达及其意义。方法本文采用光镜、透射电镜和免疫组化方法对27例卵巢癌组织中的MMP-3及TIMP-3表达进行检测。结果结果表明,临床分期为晚期的卵巢癌组织中MMP-3阳性细胞的数密度和面密度明显高于早期,而TIMP-3则低于早期。电镜下,早期淋巴细胞和树突状细胞浸润较多,癌细胞没有穿过基底膜;晚期淋巴细胞和树突状细胞浸润较少,可见癌细胞穿基底膜。结论MMP-3和TIMP-3的表达程度及MMP-3/TIMP-3的比值,可作为判断卵巢癌病程的指标。     

Association between matrix metalloproteinase (MMP)-2 polymorphisms and MMP-2 levels in hypertensive disorders of pregnancy

We examined whether two functional polymorphisms (g.-1306C>T and g.-735C>T) in matrix metalloproteinase (MMP)-2 gene are associated with preeclampsia (PE) or gestational hypertension (GH), and whether they modify MMP-2 or tissue inhibitor of metalloproteinase (TIMP)-2 plasma concentrations in these hypertensive disorders of pregnancy. We studied 130 healthy pregnant (HP), 130 pregnant with GH, and 133 pregnant with PE. Genomic DNA was extracted from whole blood and genotypes for g.-1306C>T and g.-735C>T polymorphisms were determined by Real Time-PCR, using Taqman allele discrimination assays. Haplotypes were inferred using the PHASE program. Plasma MMP-2 and TIMP-2 concentrations were measured by ELISA. The main findings were that pregnant with PE have higher plasma MMP-2 and TIMP-2 concentrations than HP (P<0.05), although the MMP-2/TIMP-2 ratios were similar (P>0.05). Moreover, pregnant with GH have elevated plasma MMP-2 levels and MMP-2/TIMP-2 ratios compared to HP (P<0.05). While MMP-2 genotypes and haplotypes are not linked with hypertensive disorders of pregnancy, MMP-2 genotypes and haplotypes are associated with significant alterations in plasma MMP-2 and TIMP-2 concentrations in preeclampsia (P<0.05). Our findings may help to understand the relevance of MMP-2 and its genetic polymorphisms to the pathophysiology of hypertensive disorders of pregnancy. It is possible that patients with PE and the MMP-2 haplotype combining the C and T alleles for the g.-1306C>T and g.-735C>T polymorphisms may benefit from the use of MMPs inhibitors such as doxycycline. However, this possibility remains to be determined.     

Expression of MMP-2 and MMP-9, their inhibitors, and the activator MT1-MMP in primary breast carcinomas.

Enhanced expression of the type IV collagenases MMP-2 and MMP-9, or lack of their inhibitors TIMP-1 and TIMP-2, has been associated with tumour invasion and metastatic potential in several experimental models. Regulation of enzyme activity is clearly a key step in tumour invasion, and recently a potent activator of MMP-2, the membrane-associated MT1-MMP, has been described and characterized. Using an immunohistochemical approach, this study has examined the expression and distribution of the type IV collagenases, their inhibitors, and the activator MT1-MMP, in a series of 79 infiltrating ductal carcinomas (IDCs), 8 tubular carcinomas, and 27 infiltrating lobular carcinomas (ILCs). MMP-2 and MT1-MMP were expressed in more than 90 per cent of all carcinomas, with predominantly stromal and tumour cell cytoplasmic staining. However, reactivity localized on tumour cell membranes was recorded for MMP-2 in 34 per cent of cases with a monoclonal antibody and 55 per cent of cases with a polyclonal antibody, and for MT1-MMP in 68 per cent of tumours. In each case, this pattern of staining was significantly associated with the presence of lymph node metastasis (p=0.001, p=0. 008, and p=0.1, respectively). Both tumour cell and stromal staining was observed for TIMP-2, but there was no correlation with metastatic status. The 92 kD gelatinase MMP-9 was expressed by 68 per cent of carcinomas, either in the stromal compartment or by tumour cells. There was a highly significant correlation between the expression pattern of MMP-9 and tumour type, with ILCs displaying greater frequency and more homogeneous cytoplasmic staining than IDCs (p=0.0004). Staining for TIMP-1 was seen in the stroma and also in relation to small blood vessels, with more than 90 per cent of tumours showing this staining pattern using a polyclonal antibody. This study indicates distinct patterns of expression for different MMPs and demonstrates the potential importance of the MMP-2/MT1-MMP system in breast tumour progression. The association of MMP-9 with the infiltrating lobular phenotype may reveal novel mechanisms of control for this metalloproteinase.     

Matrix metalloproteinase 2 (MMP-2) and their tissue inhibitor 2 (TIMP-2) in gastric cancer patients

BackgroundMatrix metalloproteinase 2 (MMP-2) is able to degrade type IV collagen and its activity is mostly regulated by tissue inhibitor of matrix metalloproteinase 2 (TIMP-2). These proteins might play a role in tumor progression, including gastric cancer (GC).MethodsThe study included 108 individuals, GC patients and healthy subjects. Serum levels of all analyzed markers were evaluated by the immunological methods, while immunohistochemistry was used to assess the expression of these proteins in GC, interstitial inflammatory cells and normal tissues.ResultsThe percentage of positive reactions of MMP-2 and TIMP-2 was higher in GC and inflammatory cells compared to normal tissue, while serum levels of these proteins were statistically lower in GC patients in comparison to healthy subjects. There was a significant positive correlation between TIMP-2 immunoreactivity in inflammatory cells and the presence of lymph node metastasis. Area under ROC curve (AUC) for TIMP-2 was higher than MMP-2, while serum MMP-2 was an independent prognostic factor of GC patients' survival.ConclusionOur findings suggest that TIMP-2 seems to be a predictor of tumor progression, especially for nodal involvement, whereas serum MMP-2 might be useful as an independent prognostic factor of patients' survival.     

Metastatic and non-metastatic colorectal cancer (CRC) cells induce host metalloproteinase production in vivo

Numerous studies have demonstrated the persistent localization of matrix metalloproteinase (MMP) expression to the interface between invading human colorectal cancer (CRC) cells and surrounding stroma supporting a role for MMPs in CRC invasion and metastasis. The present study sought to determine whether CRC cells of varying metastatic potential would have differential effects on host MMP release. Subcutaneous CRC tumors were generated in BALB/c nude mice using three CRC cell lines: SW480, SW620, and the highly metastatic SW620S5 clone. Representative samples from the subcutaneous CRC were then orthotopically implanted on the cecum of recipient nude mice. Subcutaneous and cecal tumors were analyzed for MMP expression via zymography, western blot, and RT-PCR. In vitro, none of the three cell lines expressed MMP-2 nor MMP-9. In contradistinction, the subcutaneous tumors expressed limited amounts of MMP-2 and MMP-9 while the cecal tumors expressed significant amounts of MMP-2 and MMP-9 as well as other smaller members of the MMP family. MMP-9 mRNA and protein was confirmed as host in origin by RT-PCR with mouse specific primers and a mouse MMP-9 molecular weight of 105 kDa as determined by zymography and western blot analysis. In situ hybridization also localized the mRNA for MMP-9 to the host stromal cells. In conclusion, CRC cells appear incapable of producing MMP-2 and MMP-9 in vitro but are capable of up-regulating host MMP production in vivo. Enhanced host MMP-9 production in metastatic CRC cell-derived subcutaneous and cecal tumors suggests that metastatic colon cells may acquire the expression of important MMP regulating factor(s) in vivo.     

Eutopic endometrium and peritoneal,ovarian and bowel endometriotic tissues express a different profile of matrix metalloproteinases-2, -3 and -11, and of tissue inhibitor metalloproteinases-1 and -2

Endometriosis is subsequent to the ability of endometrial glands to invade normal tissues. Matrix metalloproteinases (MMPs)—enzymes that mediate normal tissue turnover, including endometrial breakdown during menstruation—appear to be involved in this invasive process. Here, we examined the immunohistochemical expression of MMP-2, MMP-3, MMP-11, tissue inhibitor metalloproteinase (TIMP)-1 and TIMP-2 in endometrium from women with (n=9) or without endometriosis (n=18) in comparison with peritoneal (n=20), ovarian (n=20) and colorectal endometriosis (n=20). Women with endometriosis showed decreased endometrial MMP-2 expression compared with women without endometriosis (mean±SD positive cells: 24.3±28.3% and 69.3±12.1%), together with loss of MMP-3 expression (0 versus 17.5%±20.2). MMP-11, TIMP-1 and TIMP-2 expression was similar in the two groups. Endometrial MMP-2, -3 and -11 expression and TIMP-1 and -2 expression were similar in women with endometriosis and in those with peritoneal endometriosis. MMP-2, -3 and -11 expression was higher in colorectal endometriosis than in ovarian and peritoneal endometriosis. TIMP-2 expression was lower in colorectal endometriosis (P=0.0002) and ovarian endometriotic cysts (P=0.003) than in peritoneal endometriosis. TIMP-1 expression did not vary according to the location of endometriotic lesions. These results suggest that MMP-2 and -3 and TIMP-2 may be involved in the pathogenesis of endometriosis. Interestingly, MMP-2 and -3 overexpression was related to the infiltrative nature of endometriotic lesions, with possible sequential expression from peritoneal to colorectal endometriosis.     

Reduced expression of tissue inhibitor of metalloproteinase (TIMP)-2 in gestational trophoblastic diseases

To elucidate the involvement of type IV collagenases [matrix metalloproteinase (MMP)-2 and MMP-9] and their tissue inhibitors (TIMP-1 and TIMP-2) in the development of gestational trophoblastic disease (GTD), we quantified their levels in hydatidiform mole and choriocarcinoma tissues using specific enzyme-linked immunosorbent assays, and the results were compared with those from normal first trimester placenta. Levels of pro-MMP-2 were increased in hydatidiform mole, and they were further elevated in choriocarcinoma. Levels of pro-MMP-9 in choriocarcinoma and those of TIMP-1 in both hydatidiform mole and choriocarcinoma were also increased. In contrast, TIMP-2 levels were markedly decreased in both hydatidiform mole and choriocarcinoma. Similar results were obtained by the tissue culture of first trimester placenta and hydatidiform mole. Gelatin zymography indicated that the levels of both pro- and activated forms of MMP-2 and MMP-9 were higher in hydatidiform mole and choriocarcinoma. The decreased expression of TIMP-2 in hydatidiform mole and choriocarcinoma was confirmed by Western blot, Northern blot and immunohistochemistry, with the decrease being more pronounced in choriocarcinoma. Taken together, the present study shows that both TIMP-2 mRNA and protein levels are markedly decreased in GTD and the imbalance of MMP-TIMP production, shifted toward greater MMP activity, may be involved in the pathogenesis of GTD.     

Upregulation of tissue inhibitor of metalloproteinases (TIMP)-2 promotes matrix metalloproteinase (MMP)-2 activation and cell invasion in a human glioblastoma cell line

Local invasiveness is a characteristic feature of glioblastoma that makes surgical resection nearly impossible and accounts in large part for its poor prognosis. To identify mechanisms underlying glioblastoma invasion and motility, we used Transwell invasion chambers to select for a more potently invasive subpopulation of U87MG human glioblastoma cells. The stable population of tumor cells (U87-C1) obtained through this in vitro selection process were three times more invasive than parental U87MG cells and demonstrated faster monolayer wound healing and enhanced radial motility from cell spheroids. This enhanced invasiveness was associated with an 80% increase in matrix metalloproteinase 2 (MMP-2) activation. No differences in expression levels of pro-MMP-2, membrane-type matrix metalloproteinase I (MT1-MMP), or integrin alphavbeta3 (mediators of MMP-2 activation) were detected. However, U87-C1 cells exhibited two-fold elevation of tissue inhibitor of metalloproteinases (TIMP)-2 mRNA and protein relative to parental cells. Exogenous addition of comparable levels of purified TIMP-2 to parental U87MG cells increased MMP-2 activation and invasion. Similarly, U87MG cells engineered to overexpress TIMP-2 at the same levels as U87-C1 cells also demonstrated increased MMP-2 activation, indicating that an increase in physiological levels of TIMP-2 can promote MMP-2 activation and invasion in glioblastoma cells. However, exogenous administration or recombinant overexpression of higher amounts of TIMP-2 in U87MG cells resulted in inhibition of MMP-2 activation. These results demonstrate that the complex balance between TIMP-2 and MMP-2 is a critical determinant of glioblastoma invasion, and indicate that increasing TIMP-2 in glioblastoma patients may potentially cause adverse effects, particularly in tumors containing high levels of MT1-MMP and MMP-2.     

Progesterone receptor modulator CDB-2914 induces extracellular matrix metalloproteinase inducer in cultured human uterine leiomyoma cells

Effects of progesterone receptor modulator CDB-2914 on the expression of the extracellular matrix (ECM) components were examined in cultured human uterine leiomyoma and myometrial cells. ECM metalloproteinase inducer (EMMPRIN), matrix metalloproteinases (MMPs), tissue inhibitors of MMP (TIMPs) and collagen levels were assessed by Western blot analysis, MMP activity assay and real-time RT-PCR. RNA interference (RNAi) of EMMPRIN was performed using small interfering mRNA. In cultured leiomyoma cells, CDB-2914 treatment at concentrations greater than or equal to 10(-8) M significantly increased EMMPRIN, MMP-1 and MMP-8 protein contents and MMP-1, MMP-2, MMP-3 and MMP-9 mRNA levels, and activity of MMP-1, MMP-2, MMP-3 and MMP-9 in the medium. TIMP-1 and TIMP-2 were significantly decreased at mRNA and protein levels by CDB-2914 treatment at concentrations > or =10(-7) M in these cells. CDB-2914 treatment decreased types I and III collagen protein contents. However, CDB-2914 treatment did not affect the ECM component expression in cultured myometrial cells. RNAi of EMMPRIN abrogated CDB-2914-mediated both induction of MMPs and reduction of TIMPs and collagens in cultured leiomyoma cells. These results suggest that CDB-2914 modulates the expression of EMMPRIN, MMPs, TIMPs and collagens in cultured leiomyoma cells without comparable effects on myometrial cells.     

Increased expression of matrix metalloproteinase-2, matrix metalloproteinase-9 and matrix metalloproteinase-13 in lesional skin of bullous pemphigoid

BACKGROUND: Matrix metalloproteinase (MMP)-2, MMP-9 and MMP-13 can degrade type IV collagen which is the major component of the basement membrane zone (BMZ). In bullous pemphigoid (BP), the separation occurs within the BMZ. OBJECTIVE: To evaluate the involvement of MMPs in the pathogenesis of BP, we examined the expression of MMP-2, MMP-9 and MMP-13 in the lesional skin of BP patients. METHODS: The expression of MMP-2, MMP-9, MMP-13, tissue inhibitors of metalloproteinase (TIMP)-1 and TIMP-2 was analyzed by immunohistochemistry in the lesional skin of BP patients in comparison with that in normal human skin. Next, the cellular sources of MMP-2, MMP-9 and MMP-13 were analyzed by double immunohistochemistry. Finally, the levels of these MMPs in the serum and blister fluid of BP patients were measured by ELISA. RESULTS: The number of cells expressing MMP-2, MMP-9 and MMP-13 were significantly increased in the lesional skin of BP patients as compared to that in normal skin. Although the number of cells expressing TIMP-1 and TIMP-2 were also increased in the lesional skin of BP patients as compared to that in normal skin, the ratio of MMPs to TIMPs in the lesional skin of BP patients was high (2.4:1). T cells comprised the major source of MMP-2, MMP-9 and MMP-13, while a proportion of mast cells and eosinophils also expressed these MMPs. Furthermore, marked expression of MMP-2 was detected in the epidermal keratinocytes. The levels of these MMPs in the blister fluid were significantly greater than those in the serum. CONCLUSION: These results suggest that MMP-2, MMP-9 and MMP-13 may be involved in the mechanism of blister formation in BP and that besides infiltrating inflammatory cells, structural cells like epidermal keratinocytes may also participate in the induction of blister formation in BP.     

Matrix metalloproteinases and E-cadherin immunoreactivity in different basal cell carcinoma histological types

The immunohistochemical staining of matrix metalloproteinases (MMPs) and E-cadherin in tumor epithelial and stromal cells was analyzed in a group of solid, superficial spreading and cystic tumors and in a group of morpheaform and recurrent basal cell carcinomas (BCC) in order to determine whether any of these factors possibly contribute to tumor therapy resistance. Tumor tissues of 64 patients were obtained by complete excisional or curettage biopsy of BCC and these were immunohistochemically stained for MMP-1, MMP-2, MMP-9, MMP-13 and E-cadherin. In the morpheaform and recurrent BCC, MMP-9 expression significantly increased in the stroma, while E-cadherin expression was negative in epithelial cells. Odds ratio for development of morpheaform and recurrent BCC was 6.2 for positive MMP-1 immunostaining in epithelial tumor cells, 5.8 for positive MMP-9 immunostaining in tumor stroma, 3.2 for positive MMP-13 immunostaining in tumor stroma, and 4.5 for negative E-cadherin in epithelial tumor cells. Our results suggest that MMP-1 immunostaining in tumor cells, MMP-9 expression in stromal cells, and absence of E-cadherin expression are associated with morpheaform and recurrent BCC.     

Prognostic value of MMP-2, -9 and TIMP-1,-2 immunoreactive protein at the invasive front in advanced head and neck squamous cell carcinomas

In head and neck cancer as well as in other carcinomas, tumor expansion and spread to distant sites require the secretion of destructive enzymes that degrade the extracellular matrix. A variety of proteases contribute to matrix destruction. Characteristics of the invasive tumor front may reflect tumor prognosis better than do other parts of the tumor. Therefore, it was the aim of the present study to (i) compare central and peripheral tumor zones for differences in the expression of matrix-metalloproteinases (MMP) -2 and -9 and their naturally occurring inhibitors (tissue inhibitor of matrix-metalloproteinases (TIMP) -1 and -2), (ii) examine the morphological potential of malignancy, and (iii) correlate these findings with clinicopathological parameters. The study population consisted of 106 surgical specimens of advanced head and neck squamous cell carcinomas. The invasive front was graded for malignancy, and immunohistochemical staining with MMP-2, MMP-9, TIMP-1 and TIMP-2 antibodies was performed. Both MMP-2 and MMP-9 were found to be significantly overexpressed at the tumor front. The MMP-2-positive invasive front exhibited diminished overall survival times. In multivariate analysis, MMP-2 expression retained its correlation with overall survival in addition to nodal status and total malignancy score. Expression of TIMP-2 correlated with local tumor invasion. We conclude that the expression of MMP-2 at the invasive front is a marker of poor survival and appears to be associated with early recurrence in initially lymph node-negative patients.