Expression of collagenase-3 (matrix metalloproteinase-13) in squamous cell carcinomas of the head and neck.

Squamous cell carcinomas (SCCs) of the head and neck are malignant tumors with high capacity to invade and metastasize. We have examined expression of the new collagenase, collagenase-3 (MMP-13), in SCCs of the head and neck. MMP-13 mRNAs were detected in 22 of 29 SCC cell lines: in 14 of 15 primary SCC cell lines and in 8 of 14 SCC cell lines from recurrent tumors or metastases. MMP-13 mRNAs were expressed by all 6 cell lines from highly invasive primary tumors and in all 4 cell lines from small aggressive tumors. Using in situ hybridization, MMP-13 mRNAs were detected in 15 of 17 SCC tumor samples. In most tumors, MMP-13 was expressed by tumor cells at the invading front of the tumors, but in a subset of SCCs, MMP-13 mRNA was also expressed by stromal fibroblasts. No MMP-13 expression was detected in intact skin or oral mucosa. MMP-13 mRNA levels in SCC cells were enhanced by transforming growth factor-beta, tumor necrosis factor-alpha, transforming growth factor-alpha, and keratinocyte growth factor. Specific expression of MMP-13 by SCC cells in vitro and in vivo strongly suggests a role for MMP-13 in the high invasion capacity of SCC cells.     

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.     

Serpin peptidase inhibitor clade A member 1 (SerpinA1) is a novel biomarker for progression of cutaneous squamous cell carcinoma

The incidence of keratinocyte-derived nonmelanoma skin cancers is increasing worldwide because of cumulative recreational exposure to sunlight. At present, no specific molecular markers are available for assessing the progression of premalignant actinic keratoses to invasive cutaneous squamous cell carcinoma (SCC). We examined the role of the Serpin family in skin SCCs. Expression profiling of cutaneous SCC cell lines (n = 8) revealed up-regulation of SerpinA1 compared with normal epidermal keratinocytes (n = 5). Analysis with quantitative RT-PCR showed that the mean level of SerpinA1 mRNA was markedly up-regulated in cutaneous SCC cell lines (n = 8) compared with in normal keratinocytes. SerpinA1 production by SCC cells was dependent on p38 mitogen-activated protein kinase activity and was up-regulated by epidermal growth factor, tumor necrosis factor-α, interferon-γ, and IL-1β. Immunostaining of tissue arrays with 148 human tissue samples revealed tumor cell-associated expression of SerpinA1 in 19 of 36 actinic keratoses, 22 of 29 Bowen's disease samples, 67 of 71 sporadic SCCs, and all 12 recessive dystrophic epidermolysis bullosa-associated SCCs examined. Moreover, tumor cell-associated SerpinA1 staining was detected in all chemically induced mouse skin SCCs studied (n = 17). Overexpression of SerpinA1 mRNA was also detected by quantitative RT-PCR in chemically induced mouse skin SCCs (n = 14) compared with control tissues (n = 14). These data identify SerpinA1 as a novel tumor cell-associated biomarker for progression of cutaneous SCCs.     

Metalloelastase (MMP-12) expression by tumour cells in squamous cell carcinoma of the vulva correlates with invasiveness,while that by macrophages predicts better outcome

Human metalloelastase (MMP-12) has been implicated in elastin degradation and macrophage migration in many pathological conditions. It also generates angiostatin, thus having a potential to prevent tumour angiogenesis. It has previously been shown that transformed epithelial cells express MMP-12 in skin cancer. The aim of this study was further to elucidate the role of metalloelastase in squamous cell cancer (SCC) progression. By in situ hybridization, expression of MMP-12 mRNA was detected in 28/33 vulvar SCC samples in CD-68-positive macrophages, while 10 samples had positive cancer cells. By immunohistochemistry, MMP-12 protein was seen in the same area as the mRNA. MMP-12 mRNA expression in tumour cells correlated with more aggressive histology (p = 0.0099). In contrast, macrophage-derived MMP-12 mRNA was more abundant in well-differentiated grade I than grade III tumours (p = 0.01). However, the level of MMP-12 mRNA, regardless of its origin, did not correlate with metastasis or patient survival. No significant correlation was found between macrophage-derived MMP-12 mRNA and a low amount of blood vessels, as quantitated after von Willebrand staining. In agreement with vulvar SCCs in vivo, MMP-12 was expressed in cultured SCC cells by northern and western blot analysis. In HaCaTs and epithelial MCF-10f cells, MMP-12 mRNA was induced by transforming growth factor-beta1 (TGF-beta1) and tumour necrosis factor-alpha (TNF-alpha) as measured by quantitative RT-PCR (TaqMan). Two MMPs capable of generating angiostatin in vivo, matrilysin (MMP-7) and gelatinase B (MMP-9), were also examined in these tumours. MMP-7 mRNA was mainly expressed by epithelial tumour cells, particularly in less differentiated tumours. MMP-9 was usually expressed by neutrophils and macrophages; epithelial protein was predominantly found in grade II/III tumours. These results suggest a dual role for MMP-12 in tumour progression.     

IκB kinase alpha and cancer

IκB kinase alpha (Ikk-α) gene mutations and IKK-α downregulation have been detected in various human squamous cell carcinomas (SCCs), which are malignancies derived from squamous epithelial cells. These squamous epithelial cells distribute to many organs in the body; however, the epidermis is the only organ mainly composed of stratified squamous epithelial cells, called keratinocytes. SCC is the second most common type of skin cancer. Reducing IKK-α expression promotes tumor initiation, and its loss greatly enhances tumor progression from benign papillomas to malignant carcinomas during chemical skin carcinogenesis in mice. Thus, IKK-α has emerged as a tumor suppressor for SCCs. Furthermore, inducible deletion of IKK-α in the keratinocytes of adult mice causes spontaneous skin papillomas and carcinomas, indicating that IKK-α deletion functions as a tumor initiator as well as a tumor promoter. This article discusses IKK-α biological activities and associated molecular events in skin tumor development, which may provide insight into the diagnosis, treatment, and prevention of human squamous cell carcinomas (SCCs) in the future.     

Increased detection of HPV 16 virus in invasive, but not in early cervical cancers.

Human papilloma virus type 16 (HPV 16) DNA is found in about 50% of cervical squamous cell carcinomas (SCCs), and this association has raised the possibility of a causal role for HPV 16 in cervical carcinogenesis. We have tested this hypothesis by assaying a series of biopsies (n = 119) ranging from normal mucosa to infiltrating SCC with the PCR-technique for the presence of HPV 16 DNA. While HPV 16 DNA was detected in 50% of our cases with invasive SCC, the incidence of HPV 16-positive samples was about 10% in all other biopsies ranging from normal mucosa to cases of carcinoma in situ. HPV 16 therefore appears to be involved in late tumor promotion but not in early tumor development.     

Differential expression of matrilysin-1 (MMP-7), 92 kD gelatinase (MMP-9), and metalloelastase (MMP-12) in oral verrucous and squamous cell cancer

Squamous cell carcinoma (SCC) of the oral cavity is a highly invasive tumour of stratified squamous epithelium that spreads through degradation of the basement membrane (BM) and extracellular matrix (ECM). There are currently no reliable tissue or serum markers to predict whether the tumour has metastasized at the time of diagnosis. Verrucous carcinoma (VC) of the oral cavity is a rare low-grade variant of oral SCC that penetrates into the subepithelial connective tissue. Many matrix metalloproteinases (MMPs), such as MMP-1, -2, -7, -9, -13, and -14, as well as integrin receptors have been implicated in cancer invasion. Integrin alphavbeta6 is induced in SCC and appears to be involved in up-regulation of MMP-9 expression by oral keratinocytes and promotion of their migration. The aim of this study was to investigate whether the pattern of MMP expression or that of alphavbeta6 integrin contributes to the differences in the biological behaviour of oral SCC and VC. The results show that the less aggressive nature of oral VC may be connected to its MMP expression profile. Typically, VCs were devoid of epithelial MMP-3, -7, -9, -12 and -13 expression, compared with SCCs. MMP-19 was expressed by epithelial keratinocytes in hyperproliferative areas of verrucous hyperplasia, VC, and SCC, but was absent in the invasive cancer cell nests of SCC. MMP-26 was expressed by hyperproliferative keratinocytes in VC as well as by invasive cancer cells in SCCs. MMP-10 was expressed widely in the epithelium of all SCC specimens. alphavbeta6 integrin expression was also detected in some cases of epithelial hyperplasia but was significantly more abundant in cancers at the invasive front. The absence of MMP-7, -9 and -12 from epithelial cells may serve as a good prognostic marker of non-invasive oral carcinoma. Blocking the activity of invasion-specific MMPs or alphavbeta6 integrin might offer novel therapeutic modalities in early-stage oral carcinoma.     

Expression of selectin ligands by cutaneous squamous cell carcinoma.

Recent evidence suggests that interactions between endothelial selectins and tumor surface selectin ligands may be of importance in cancer metastasis. To investigate the role of such mechanisms in cutaneous tumors, whole skin biopsies were examined immunohistochemically for a variety of selectin ligands including sialyl-Lewis-X, sialyl-Lewis-A (S-Le(a)), sulfatides, and CD15. In 12 of 12 squamous cell carcinomas (SCCs), there was expression of sialyl-Lewis-X and CD15, but no tumor expressed S-Le(a). Occasional keratinocytes in eight of 12 SCCs expressed sulfatides. All selectin ligands were absent on keratinocytes in basal cell carcinomas (BCCs, n = 8) and normal skin (n = 8), with the exception of one BCC that expressed S-Le(a). E-selectin was not present in normal skin, but was strongly expressed by dermal endothelium in both SCC and BCC. Keratinocyte cell lines A431, HaCaT, and SVK14 were investigated by flow cytometry, which demonstrated sialyl-Lewis-X and S-Le(a) expression by all three, whereas normal human keratinocytes did not express these molecules. These findings suggest a potential role for selectin-mediated events in early and late metastasis, and differential expression of these ligands by BCC and SCC may explain the relatively low metastatic potential of the former.     

Transformation-specific matrix metalloproteinases, MMP-7 and MMP-13, are present in epithelial cells of keratoacanthomas.

Keratoacanthomas are rapidly growing hyperproliferative skin tumors that may clinically or histologically be difficult to distinguish from well-differentiated squamous cell cancers (SCCs). UV light, trauma, and immune suppression represent their etiological factors. As matrix metalloproteinases (MMPs) are implicated at all stages of tumorigenesis, we investigated the expression profile of several cancer-related MMPs to find markers that would differentiate keratoacanthomas from SCCs and shed light to the pathobiology of keratoacanthoma. Samples from 31 keratoacanthomas and 15 grade I SCCs were studied using immunohistochemistry for MMP-2, -7, -8, -9, -10, -13, and -19 and p16 and laminin-5gamma2 chain. In situ hybridization for MMP-7, -10, and -13 was performed in a subset of tumors. Keratinocytes with atypia, presence of neovascularization, and composition of the inflammatory infiltrate were graded from hematoxylin-eosin stainings. MMP-7 was present in the epithelium of 4/31 keratoacanthomas and 9/15 SCCs, MMP-8 in 3/30 keratoacanthomas and 0/15 SCCs, but MMP-13 in 16/31 keratoacanthomas and 10/15 SCCs, and MMP-10 in 28/31 keratoacanthomas and all cancers. MMP-9 was detected in the epithelium in 5/31 keratoacanthomas and 8/15 SCCs, whereas MMP-2 was only present in fibroblasts in both tumors. MMP-19 was upregulated in proliferating epithelium of keratoacanthomas as was p16. Cytoplasmic laminin-5gamma2 was particularly abundant in keratinocytes at the pushing border of MMP-13-positive keratoacanthomas. We conclude that although some MMPs (MMP-10 and -13) are abundantly expressed in keratoacanthomas, the presence of MMP-7 and -9 in their epithelial pushing border is rare and should raise suspicion of SCC. Further, the loss of MMP-19 and p16 could aid in making the differential diagnosis between well-differentiated SCC and keratoacanthoma. Frequent expression of the transformation-specific MMP-13 in keratoacanthomas suggests that they are not benign tumors but incomplete SCCs.     

Analysis of clonality and HPV infection in benign, hyperplastic, premalignant, and malignant lesions of the vulvar mucosa

To elucidate the pathogenesis of vulvar carcinomas, we studied clonality and human papillomavirus (HPV) infection in vulvar epithelial diseases. Monoclonal composition was demonstrated in all 9 invasive tumors (squamous cell carcinoma [SCC], 6; basal cell carcinoma, 1; malignant melanoma, 2), 15 of 20 cases of vulvar intraepithelial neoplasia (VIN), 7 of 9 cases of Paget disease, 2 of 6 cases of lichen sclerosus (LS), and 2 of 3 cases of squamous cell hyperplasia (SCH); high-risk type HPV was revealed in 5 of 6 SCCs and 17 of 20 VINs. These observations might imply that a subset of cases of LS and SCH result from a neoplastic proliferation, similar to VINs but not related to infection with high-risk type HPV. In 1 case of SCC with concurrent VIN 3 in an adjacent lesion, both lesions showed the same pattern of X chromosome inactivation and the presence of HPV-16 in episomal and integrated forms, suggesting that monoclonal expansion triggered by high-risk type HPV integration is an early event for carcinogenesis of HPV-associated SCC.     

Maspin expression in normal skin and usual cutaneous carcinomas

Maspin is a serine protease inhibitor whose gene is located on 3q27. Several lines of evidence point towards its putative role as tumor suppressor gene and angiogenesis inhibitor; however, there are compelling data showing that maspin is also expressed in the nuclear compartment and might be associated with the differentiation of specific cell lineages. No systematic study of maspin expression in normal skin and usual skin carcinomas have been published so far. We semiquantitatively analyzed the distribution and immunoreactivity pattern of maspin in 14 squamous cell carcinomas (SCCs) and 16 basal cell carcinomas (BCCs) and in the adjacent normal epidermis of all cases. We also examined the correlation of maspin expression with histological type, grade, vascular invasion, perineural infiltration, and mitotic counting. Cytoplasmic expression of maspin was observed in suprabasal, prickle, and granular cell layers of normal epidermis; cells of the germinative hair matrix, Henle's and Huxley's layers, and cuticle of hair follicles; mature sebaceous cells and sweat gland's secretory cells. Nuclear expression was detected in some basal/myoepithelial cells of the sweat glands and scattered mature sebaceous cells. All SCCs but one grade IV SCC showed maspin expression, and it was correlated with the differentiation of these neoplasms. BCCs presented variable maspin expression, while metatypical carcinomas showed moderate to intense maspin expression, nodular BCCs variable contents of maspin and displayed a peculiar distribution, confined to the center of the neoplastic nodules. Two BCCs and one SCC showed maspin nuclear expression. No correlation with other clinical pathological features was observed. Our findings do not support the role of maspin as a tumor suppressor gene and suggest that this serpin is probably associated with specific lines of differentiation.     

Association of vascular endothelial growth factor and mast cells with angiogenesis in laryngeal squamous cell carcinoma

We investigated the expression of vascular endothelial growth factor (VEGF) and microvascular density in 54 cases of invasive laryngeal squamous cell carcinoma (SCC) and in ten samples of normal laryngeal tissue using immunohistochemistry methods. The study also focused on the distribution of mast cells in and around the SCCs. The microvascular density in laryngeal carcinoma tissue was higher than that in normal tissue (P=0.02). VEGF was localized in SCCs, stromal cells, endothelial cells, minor salivary glands, and non-cancer epithelium adjacent to the tumor. VEGF expression in the tumor cells was found in 13 of 54 cases (24.1%), whereas mast cells around the carcinomas were VEGF positive in all 54 cases. Staining of VEGF in SCCs was strong in the area of high microvascular density (P=0.0002). Using a multi-labeling subtraction immunostaining method, VEGF-positive stromal cells were classified mostly as mast cells and, in a few instances, as macrophages. VEGF staining in SCCs was associated with the mast cell count (P=0.0001). There was no distinct correlation between VEGF expression and pTNM stage of an SCC. In conclusion, the results suggest that VEGF might be an important angiogenic factor in cancer invasion. Laryngeal cancer cells and mast cells may control the angiogenic response by releasing VEGF. Received: 22 March 1999 / Accepted: 14 September 1999     

Chromosome 17 aneusomy detected by fluorescence in situ hybridization in vulvar squamous cell carcinomas and synchronous vulvar skin

Vulvar squamous cell carcinoma (SCC) affects a spectrum of women with granulomatous vulvar diseases, human papillomavirus (HPV) infections, and chronic inflammatory vulvar dermatoses. To determine whether there is evidence of chromosomal instability occurring in synchronous skin surrounding vulvar SCCs, we investigated abnormalities in chromosome 17 copy number. Samples of SCC, vulvar intraepithelial neoplasia (VIN), and surrounding vulvar skin were obtained from all vulvar excisions performed for squamous neoplasia at Albany Medical College from 1996 to 1997. Histological categorization, fluorescent in situ hybridization (FISH) for the alpha satellite region of chromosome 17, DNA content by image analysis, and Ki-67 labeling were evaluated. Controls of normal vulvar skin not associated with cancer were used for comparison. One hundred ten specimens were obtained from 33 patients with either SCC or VIN 3 and consisted of 49 neoplastic, 52 nonneoplastic, and 9 histologically normal vulvar skin samples. The majority of SCCs (88%) and a minority (18%) of VIN 3 excisions were associated with lichen sclerosus. Normal vulvar skin controls did not exhibit chromosome 17 polysomy (cells with more than four FISH signals), whereas 56% of normal vulvar skin associated with cancer did. Moreover, the frequency of polysomy significantly increased as the histological classification progressed from normal to inflammatory to neoplastic lesions. The largest mean value and variance for chromosome 17 copy number was identified in SCCs (2.4 +/- 1.0) with intermediate values identified, in decreasing order, for SCC in situ (2.1 +/- 1.0), VIN 2 (2.1 +/- 0.8), lichen sclerosus (2.0 +/- 0.5), lichen simplex chronicus (1.9 +/- 0.4), and normal skin associated with SCC (1.8 +/- 0.4) compared with control vulvar skin (1.5 +/- 0. 05). Concordance of chromosome 17 aneusomy between cancers and synchronous skin lesions was found in 48% of patients. Loss of chromosome 17 was identified 5% of all samples and was significantly associated with women with SCC in situ (HPV-related). Both DNA content and Ki-67 labeling positively and significantly correlated with mean chromosome 17 copy number (r = 0.1, P: = 0.007). A high degree of genetic instability (aneuploidy) occurs in the skin surrounding vulvar carcinomas. As these events could be detected in histologically normal skin and inflammatory lesions (lichen sclerosus), chromosomal abnormalities may be a driving force in the early stages of carcinogenesis. Differences in chromosomal patterns (loss or gain) support the concept of at least two pathways in vulvar carcinogenesis.     

Complexity of expression of intermediate filament proteins, including glial filament protein, in endometrial and ovarian adenocarcinomas.

The expression patterns of intermediate filament proteins of primary and metastatic endometrial (n = 18) and ovarian (n = 24) adenocarcinomas were analyzed by immunocytochemistry using a panel of specific antibodies and by gel electrophoresis of cytoskeletal preparations, followed by immunoblotting. All cells of all endometrial adenocarcinomas studied contained the "simple epithelial"-type cytokeratins (CKs) 8, 18, and (mostly) 19, with variable numbers of cells also positive for CK 7 and vimentin. In addition, most of these tumors contained individual cells or groups of cells that were positive for the stratification-related CKs 4, 5, 6, 13, 14, and 17. The latter CKs were often associated with squamous cell foci, but were also found in some single (nonsquamous) tumor cells, indicative of early stages of squamous cell differentiation. Ovarian carcinomas of various histologic types and grades contained predominantly CKs 7, 8, 18, and 19. Serous, endometrioid, and anaplastic tumors, but not mucinous and clear cell tumors, also contained minor amounts of stratification-related CKs in variable combinations, mostly including CK 4. In all tumor types except mucinous tumors, vimentin was consistently detected in variable proportions of tumor cells which, however, were rather low in anaplastic carcinomas. Surprisingly, glial filament protein was detected in a minor proportion (< or = 20%) of tumor cells in seven of 14 serous and endometrioid ovarian carcinomas and in three of 18 endometrial carcinomas. These different intermediate filament expression patterns of müllerian duct-type carcinomas, only partly related to the morphologic appearance of the specific type of tumor, might reflect the multipotentiality of differentiation of müllerian duct-derived epithelia. Cytoskeletal features of potential diagnostic value, especially in metastatic carcinomas, are discussed.     

Alterations in the expression of uvomorulin and Na+,K(+)-adenosine triphosphatase during mouse skin tumor progression.

Uvomorulin (E-cadherin), a cell adhesion molecule, and Na+,K(+)-adenosine triphosphatase (ATPase), a marker protein of the basal-lateral cell membrane domains of polarized epithelial cells, were investigated in a group of mouse skin tumors induced by a two-stage chemical carcinogenesis protocol and in cell lines derived from mouse skin papillomas and squamous cell carcinomas (SCC). Although these two markers were present in benign tumors and in nontumorigenic cell lines, the Na+,K(+)-ATPase showed an altered pattern of distribution that included the presence of enzyme not only in the basolateral domain but also on the apical domain of the cell membrane of basal and spinous cells in well-differentiated squamous cell carcinomas (SCC). In higher grade SCC, a loss of Na+,K(+)-ATPase immunoreactivity was simultaneously detected with a marginal or absent expression of uvomorulin. The more differentiated SCC and papillomas expressed less uvomorulin immunoreactivity than normal epidermal cells. Both markers were seen in tumor cell lines that produced well-differentiated SCC after subcutaneous inoculation into nude mice. Neither Na+,K(+)-ATPase nor uvomorulin could be detected in cell lines that produced high grade, poorly differentiated SCC. Northern blots confirmed the absence of uvomorulin mRNA in these highly malignant cell lines. These data indicate that progression from premalignant papilloma to low-grade SCC and subsequently to high-grade SCC is accompanied by loss of epithelial cell polarity as detected by changes in Na+,K(+)-ATPase and by decreased or absent expression of uvomorulin in tumors and cell lines characterized by an advanced malignant phenotype.