Adenoviral Gene Transfer of β3 Integrin Subunit Induces Conversion from Radial to Vertical Growth Phase in Primary Human Melanoma

Abstract

Expression of the β3 subunit of the αvβ3 vitronectin receptor on melanoma cells is associated with tumor thickness and the ability to invade and metastasize. To address the role of αvβ3 in the complex process of progression from the nontumorigenic radial to the tumorigenic vertical growth phase of primary melanoma, we examined the biological consequences of overexpressing αvβ3 in early-stage melanoma cells using an adenoviral vector for gene transfer. Overexpression of functional αvβ3 in radial growth phase primary melanoma cells 1) promotes both anchorage-dependent and -independent growth, 2) initiates invasive growth from the epidermis into the dermis in three-dimensional skin reconstructs, 3) prevents apoptosis of invading cells, and 4) increases tumor growth in vivo. Thus, αvβ3 serves diverse biological functions during the progression from the nontumorigenic radial growth phase to the tumorigenic and invasive vertical growth phase primary melanoma. Expression of the β3 subunit of the αvβ3 vitronectin receptor on melanoma cells is associated with tumor thickness and the ability to invade and metastasize. To address the role of αvβ3 in the complex process of progression from the nontumorigenic radial to the tumorigenic vertical growth phase of primary melanoma, we examined the biological consequences of overexpressing αvβ3 in early-stage melanoma cells using an adenoviral vector for gene transfer. Overexpression of functional αvβ3 in radial growth phase primary melanoma cells 1) promotes both anchorage-dependent and -independent growth, 2) initiates invasive growth from the epidermis into the dermis in three-dimensional skin reconstructs, 3) prevents apoptosis of invading cells, and 4) increases tumor growth in vivo. Thus, αvβ3 serves diverse biological functions during the progression from the nontumorigenic radial growth phase to the tumorigenic and invasive vertical growth phase primary melanoma. The easy accessibility and long-term clinical and histopathological observation of cutaneous melanoma has led to the definition of five major steps of tumor progression in the human melanocytic system. The lesions representing each step are common acquired nevus, dysplastic nevus, radial growth phase (RGP) primary melanoma, vertical growth phase (VGP) primary melanoma, and metastatic melanoma.1Clark Jr, WH Elder DE Guerry D Epstein MN Greene MH Van Horn M A study of tumor progression: the precursor lesions of superficial spreading and nodular melanoma.Hum Pathol. 1984; 15: 1147-1165Abstract Full Text PDF PubMed Scopus (782) Google Scholar, 2Clark Jr, WH Tumor progression and the nature of cancer.Br J Cancer. 1991; 64: 631-644Crossref PubMed Scopus (240) Google Scholar The common acquired nevus is composed of nests of mature melanocytes. Increasing levels of cytological and architectural atypia and aberrant cell growth are observed in dysplastic nevi. The RGP primary melanoma is the first recognizable malignant step, but the malignant cells grow only within or in close proximity to the epidermis and they do not have competence for metastasis.2Clark Jr, WH Tumor progression and the nature of cancer.Br J Cancer. 1991; 64: 631-644Crossref PubMed Scopus (240) Google Scholar Eventually, cells acquire the ability to invade deeply into the dermis (VGP), a stage that is associated with metastatic dissemination and dependent on tumor thickness, mitotic index, and degree of lymphocytic infiltration.2Clark Jr, WH Tumor progression and the nature of cancer.Br J Cancer. 1991; 64: 631-644Crossref PubMed Scopus (240) Google Scholar, 3Clark Jr, WH Elder DE Guerry IV, D Braitman LE Trock BJ Schultz D Synnestvedt M Halpern AC Model predicting survival in stage I melanoma based on tumor progression.J Natl Cancer Inst. 1989; 82: 626-627Google Scholar Thus, the conversion of primary melanomas from RGP to VGP is the most critical step in melanoma progression and ultimately in disease outcome. The successful isolation and in vitropropagation of cells derived from different stages of progression has provided an excellent experimental model for studying tumor progression.4Herlyn M Thurin J Balaban G Bennicelli JL Herlyn D Elder DE Bondi E Guerry D Nowell P Clark WH Koprowski H Characteristics of cultured human melanocytes isolated from different stages of tumor progression.Cancer Res. 1985; 45: 5670-5676PubMed Google Scholar, 5Herlyn M Balaban G Bennicelli J Guerry D Halaban R Herlyn D Elder DE Maul GG Steplewski Z Nowell PC Clark Jr, WH Koprowski H Primary melanoma cells of the vertical growth phase: similarities to metastatic cells.J Natl Cancer Inst. 1985; 74: 283-289PubMed Google Scholar, 6Herlyn M Clark WH Rodeck U Mancianti ML Jambrosic J Koprowski H Biology of tumor progression in human melanocytes.Lab Invest. 1987; 56: 461-474PubMed Google Scholar, 7Satyamoorthy K Dejesus E Linnenbach A Kraj B Kornreich DL Rendle S Elder DE Herlyn M Melanoma cell lines from different stages of progression and their biological and molecular analyses.Melanoma Res. 1997; 7: S35-S42Crossref PubMed Scopus (136) Google Scholar, 8Hsu M-Y, Elder DE, Herlyn M: The Wistar melanoma (WM) cell lines. Human Cell Culture, vol. 3: Solid Cancers. Edited by Masters JRW, Palsson B,. Kluwer Academic Publishers, Norwell, MA, 1998 (in press)Google Scholar Cultured cells from RGP and VGP progression stages have biological properties that reflect the stagein vivo. Cells with RGP-like phenotype 1) require complex media containing several growth factors for continuous proliferation in culture due to their limited autoexpression of growth factors,7Satyamoorthy K Dejesus E Linnenbach A Kraj B Kornreich DL Rendle S Elder DE Herlyn M Melanoma cell lines from different stages of progression and their biological and molecular analyses.Melanoma Res. 1997; 7: S35-S42Crossref PubMed Scopus (136) Google Scholar 2) do not grow in soft agar or form very few colonies,4Herlyn M Thurin J Balaban G Bennicelli JL Herlyn D Elder DE Bondi E Guerry D Nowell P Clark WH Koprowski H Characteristics of cultured human melanocytes isolated from different stages of tumor progression.Cancer Res. 1985; 45: 5670-5676PubMed Google Scholar, 7Satyamoorthy K Dejesus E Linnenbach A Kraj B Kornreich DL Rendle S Elder DE Herlyn M Melanoma cell lines from different stages of progression and their biological and molecular analyses.Melanoma Res. 1997; 7: S35-S42Crossref PubMed Scopus (136) Google Scholar 3) show a heterogeneous response to keratinocyte-mediated control of growth and regulation of cell surface receptor expression,9Shih I-M Elder DE Hsu M-Y Herlyn M Regulation of MelCAM/MUC18 expression on melanocytes of different stages of tumor progression by normal keratinocytes.Am J Pathol. 1994; 145: 837-845PubMed Google Scholar and 4) are nontumorigenic or grow very slowly in immunodeficient nude or SCID mice to a small tumor mass (<150 μg) over 3 to 4 months.7Satyamoorthy K Dejesus E Linnenbach A Kraj B Kornreich DL Rendle S Elder DE Herlyn M Melanoma cell lines from different stages of progression and their biological and molecular analyses.Melanoma Res. 1997; 7: S35-S42Crossref PubMed Scopus (136) Google Scholar These results are consistent with analyses from patients indicating that RGP primary melanoma cells have a nonmalignant phenotype relatively similar to precursor cells.3Clark Jr, WH Elder DE Guerry IV, D Braitman LE Trock BJ Schultz D Synnestvedt M Halpern AC Model predicting survival in stage I melanoma based on tumor progression.J Natl Cancer Inst. 1989; 82: 626-627Google Scholar However, RGP primary melanoma cells are clearly distinguished from normal melanocytes or nevus cells by prolonged survival in culture or, as we have demonstrated with seven cell lines, by indefinite growth.7Satyamoorthy K Dejesus E Linnenbach A Kraj B Kornreich DL Rendle S Elder DE Herlyn M Melanoma cell lines from different stages of progression and their biological and molecular analyses.Melanoma Res. 1997; 7: S35-S42Crossref PubMed Scopus (136) Google Scholar Most, but not all, cell lines are also independent of basic fibroblast growth factor (bFGF) and the phorbol ester TPA for growth and survival.7Satyamoorthy K Dejesus E Linnenbach A Kraj B Kornreich DL Rendle S Elder DE Herlyn M Melanoma cell lines from different stages of progression and their biological and molecular analyses.Melanoma Res. 1997; 7: S35-S42Crossref PubMed Scopus (136) Google Scholar In contrast to cells from RGP lesions, those with a VGP-like phenotype are readily adapted to growth in tissue culture8Hsu M-Y, Elder DE, Herlyn M: The Wistar melanoma (WM) cell lines. Human Cell Culture, vol. 3: Solid Cancers. Edited by Masters JRW, Palsson B,. Kluwer Academic Publishers, Norwell, MA, 1998 (in press)Google Scholar and need, at most, one growth factor (insulin-like growth factor (IGF)-1 or insulin) for proliferation.10Rodeck U Herlyn M Menssen HD Furlanetto RW Koprowski H Metastatic but not primary melanoma cells grow in vitro independently of exogenous growth factors.Int J Cancer. 1987; 40: 687-690Crossref PubMed Scopus (146) Google Scholar VGP cells express a variety of growth factors for either autocrine or paracrine stimulation11Rodeck U Melber K Kath R Menssen H-D Varello M Atkinson B Herlyn M Constitutive expression of multiple growth factor genes by melanoma cells but not normal melanocytes.J Invest Dermatol. 1991; 97: 20-26Abstract Full Text PDF PubMed Google Scholar and readily adapt to growth in growth-factor-free medium, leading to increased invasiveness through basement membranes in vitro and metastasis formationin vivo.12Kath R Jambrosic J Holland L Rodeck U Herlyn M Development of invasive and growth factor-independent cell variants from primary melanomas.Cancer Res. 1991; 51: 2205-2211PubMed Google Scholar VGP cells also form colonies in soft agar,4Herlyn M Thurin J Balaban G Bennicelli JL Herlyn D Elder DE Bondi E Guerry D Nowell P Clark WH Koprowski H Characteristics of cultured human melanocytes isolated from different stages of tumor progression.Cancer Res. 1985; 45: 5670-5676PubMed Google Scholar do not respond to growth control by keratinocytes,9Shih I-M Elder DE Hsu M-Y Herlyn M Regulation of MelCAM/MUC18 expression on melanocytes of different stages of tumor progression by normal keratinocytes.Am J Pathol. 1994; 145: 837-845PubMed Google Scholar and are tumorigenic in immunodeficient mice, with continuous local growth until the host dies.5Herlyn M Balaban G Bennicelli J Guerry D Halaban R Herlyn D Elder DE Maul GG Steplewski Z Nowell PC Clark Jr, WH Koprowski H Primary melanoma cells of the vertical growth phase: similarities to metastatic cells.J Natl Cancer Inst. 1985; 74: 283-289PubMed Google Scholar, 13Kath R Rodeck U Parmiter A Jambrosic J Herlyn M Growth factor independence in vitro of primary melanoma cells from advanced but not early or intermediate lesions.Cancer Ther Control. 1990; 1: 179-191Google Scholar Integrins constitute a family of membrane glycoproteins that are responsible for cell-extracellular matrix and cell-cell adhesion. 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In three-dimensional skin the is in β3 expression an invasive phenotype and prevents In β3 tumor Human melanoma cell has an RGP-like isolated as Jr, WH Elder DE Guerry IV, D Braitman LE Trock BJ Schultz D Synnestvedt M Halpern AC Model predicting survival in stage I melanoma based on tumor progression.J Natl Cancer Inst. 1989; 82: 626-627Google Scholar, P Integrin expression in human melanoma cells with invasive and metastatic 1992; PubMed Scopus Google Scholar, J SA P expression of the vitronectin receptor and the receptor in metastatic melanoma cells.J Invest. 1995; PubMed Scopus Google Scholar from a superficial spreading melanoma a primary melanoma cell with an RGP-like a from for Cancer is a VGP-like cell M-Y, Elder DE, Herlyn M: The Wistar melanoma (WM) cell lines. Human Cell Culture, vol. 3: Solid Cancers. Edited by Masters JRW, Palsson B,. 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PubMed Scopus Google Scholar, D M Biol. 1989; PubMed Scopus Google Scholar, B expression of and their in an skin Cell 1993; Google Scholar with isolated from and to in with and of the in tissue culture with of and at a surface that as a for the The and the in growth composed of 3 and with 4 and for at The and at for Melanoma cells and in with at a in to a cell of 3 and of cell the surface of and to 3 to of and with 4 and and to the to for with in and in The invasive of melanoma cells by using and Cell survival into using an in Melanoma cells at 3 in growth SCID mice mice in the with of cell Tumor as vector cell surface expression of β3 within of cell at a of of to as by the in cells from to and to in and cells, in using cells as in cells, express using a and of cells to with molecular to and β3 in with the a in αvβ3 expression in 3 and 4) and and 2) cells as with their control up-regulation of β3 in cells by limited and In of β3 using an of a functional association between β3 and in cells not To the role of the β3 integrin subunit in growth regulation of melanoma cells in cell proliferation in culture and in soft agar Growth of in cells but not in cells in soft agar in both cell lines, with a and in and cell growth, growth in soft agar of melanoma cells overexpressing Melanoma cells at a of of in on an and 3 to 4 of as the of cells colonies containing or Ten examined for each using P and for and cells, integrin is by the B The matrix as a cell survival 1993; PubMed Scopus Google Scholar, PC M G Integrin αvβ3 tumor by apoptosis of 1994; Full Text PDF PubMed Scopus Google Scholar, Z M of and apoptosis in cells by 1995; PubMed Scopus Google Scholar, Integrins and Opin Cell Biol. 1996; 8: PubMed Scopus Google Scholar, The is expression of αvβ3 and integrins during human melanoma cell Res. 1993; Google Scholar, PC S of matrix to the surface of invasive cells by with integrin 1996; Full Text Full Text PDF PubMed Scopus Google Scholar, L G expression of the β3 integrin and the in human melanoma cell in nude J Cancer. 1996; PubMed Scopus Google Scholar, Integrins and 1996; Google Scholar we examined melanoma by the cells into three-dimensional skin of skin from isolated cell and composed of a of and a of in and a basement membrane by skin B expression of and their in an skin Cell 1993; Google Scholar cells into the dermis and cell nests A and cells only B and cells the of including membrane and These cells their cell cells To study the biological consequences of β3 in nontumorigenic melanoma in of cells in SCID mice with 3 The of by cells at and their However, due to the nature of adenoviral analyses of integrin expression in different stages of melanoma have the of β3 integrin expression as one of the most markers of the from RGP to VGP primary and metastatic SM Mette SA Elder DE Stewart R Damjanovich L Herlyn M Buck CA Integrin distribution in malignant melanoma: association of the β3 subunit with tumor progression.Cancer Res. 1990; 50: 6757-6764PubMed Google Scholar, 22Danen EH Ten Berge Van Muijen GN Ruiter DJ of and αvβ3 expression in melanocytic 1994; 24: PubMed Scopus Google Scholar, EH Van Ruiter DJ Van Muijen GN in human melanoma: of αvβ3 and of are to tumor progression in but not to metastatic of cell lines in nude mice in J Cancer J Cancer. 1995; PubMed Scopus Google Scholar, A Expression of and integrin in melanocytic J Cancer. 1995; PubMed Scopus Google Scholar that expression of the β3 subunit results in a functional complex with that the malignant phenotype of the progression from RGP to cells a growth in and soft increased invasiveness and survival in skin in and in vivo. that of either subunit of the αvβ3 receptor into metastatic melanoma cell lines not cell proliferation in B CA of integrin gene expression in human melanoma Invest. 1992; PubMed Scopus Google Scholar, EH Ruiter DJ Muijen GN Integrin β3 into a metastatic human melanoma cell and experimental 1996; PubMed Scopus Google Scholar In we that of β3 in vitro growth of is not as between αvβ3 and growth factor as and K Ruoslahti E of receptor with 1994; PubMed Scopus Google Scholar as as growth factor M K Ruoslahti E αvβ3 integrin with and and the biological of 1997; PubMed Scopus Google Scholar has to control cell However, the not in cells, have a proliferation that of cells Thus, αvβ3 to in cell growth the of is that functions of αvβ3 are in by The is expression of αvβ3 and integrins during human melanoma cell Res. 1993; Google Scholar or H M G M M JL human melanoma variants with in tumor growth and metastatic do not express the β3 integrin J 1994; PubMed Scopus Google Scholar are in melanoma A of malignant is growth of the the β3 integrin we demonstrated an in of early-stage melanoma cells in soft The of growth β3 are not In melanoma, cell-cell through the adhesion receptor a of the gene to an also by melanoma I-M Elder DE Hsu M-Y Herlyn M Regulation of MelCAM/MUC18 expression on melanocytes of different stages of tumor progression by normal keratinocytes.Am J Pathol. 1994; 145: 837-845PubMed Google Scholar, I-M Hsu M-Y E Herlyn M Melanoma cell-cell are through Res. 1997; Google Scholar αvβ3 by melanoma cells to of the gene on melanoma Integrin αvβ3 melanoma cells from apoptosis in three-dimensional Natl 1994; PubMed Scopus Google Scholar is that cell-cell for survival and into the dermis is one of the most of VGP melanoma process of melanoma cells from and to and of basement membrane by and proliferation in the In skin not only for but also for factors from cells, control cells in a RGP lesions, cells a VGP growth The cells and in the dermis of control cells in the by apoptosis in the expression and of αvβ3 with that of matrix and The is expression of αvβ3 and integrins during human melanoma cell Res. 1993; Google Scholar, PC S of matrix to the surface of invasive cells by with integrin 1996; Full Text Full Text PDF PubMed Scopus Google Scholar a role in of the matrix during matrix to cell surface to the leading of tumor A cell Cell 1997; PubMed Google Scholar Integrin αvβ3 melanoma cells from apoptosis in three-dimensional Natl 1994; PubMed Scopus Google Scholar have provided evidence that survival and proliferation of melanoma cells in a three-dimensional matrix is through the of the to the of a role for the αvβ3 integrin in melanoma progression an In of early-stage melanoma cells increased of the β3 with that as the in an B CA of integrin gene expression in human melanoma Invest. 1992; PubMed Scopus Google Scholar or that metastatic potential on β3 EH Ruiter DJ Muijen GN Integrin β3 into a metastatic human melanoma cell and experimental 1996; PubMed Scopus Google Scholar The reflect a in the degree and of phenotype with the stage of the of αvβ3 expression on the biological properties of melanoma metastatic cells are nontumorigenic RGP primary melanoma cells to by αvβ3 Thus, the biological functions of αvβ3 in melanoma in on the cellular of a stage of tumor