Melanocyte Transformation Associated with Substrate Adhesion Impediment

Abstract

Exclude experimental models of malignant transformation employ chemical and physical carcinogens or genetic manipulations to study tumor progression. In this work, different melanoma cell lines were established after submitting a nontumorigenic melanocyte lineage (melan-a) to sequential cycles of forced anchorage impediment. The great majority of these cells underwent anoikis when maintained in suspension. After one deadhesion cycle, phenotypic alterations were noticeable in the few surviving cells, which became more numerous and showed progressive alterations after each adhesion impediment step. No significant differences in cell surface expression of integrins were detected, but a clear electrophoretic migration shift, compatible with an altered glycosylation pattern, was observed for beta1 chain in transformed cell lines. In parallel, a progressive enrichment of tri- and tetra-antennary N-glycans was apparent, suggesting increased N-acetylglucosaminyltransferase V activity. Alterations both in proteoglycan glycosylation pattern and core protein expression were detected during the transformation process. In conclusion, this model corroborates the role of adhesion state as a promoting agent in transformation process and demonstrates that cell adhesion disturbances may act as carcinogenic stimuli, at least for a nontumorigenic immortalized melanocyte lineage. These findings have intriguing implications for in vivo carcinogenesis, suggesting that anchorage independence may precede, and contribute to, neoplastic conversion.

Epigenetic reprogramming as a key contributor to melanocyte malignant transformation

“…Different cell lines (i.e., 4C, 4C11-and 4C11+) were obtained as a result of sequential cycles of anchorage blockade of melan-a melanocytes. 19 Melan-a cells for 96 h cells submitted to two, three and four de-adhesion cycles gave rise to three different cell lineages, named 2C, 3C and 4C, respectively. In this study we used 4C cell line that is incapable to form tumors in vivo, present structural chromosomal abnormalities and is more resistant to anoikis than melan-a cells.…”

“…5C and respectively) characterized the conversion of 4C pre-malignant melanocyte cell line to tumorigenic, non-metastatic, 4C11-melanoma cells. In melanoma cell line 4C11-, and metastatic melanoma cell line 4C11+, established after submitting melan-a cells to sequential cycles of anchorage blockade 19 were cultured as melan-a cells, but in the absence of PMA. For anchorage independent cycles, melan-a cell line was plated on 1% agarose coated plates and cultured in this condition for 96 h. Small spheroids were then collected by decantation and cultured on standard condition.…”

Signaling Pathways Altered During the Metastatic Progression of Melanoma

“…The acquisition of anoikis-resistant phenotype is one of the critical steps during tumor progression. In melanoma, we and others [17][18][19] demonstrated that malignant transformation is associated with resistance to anoikis [16]. One of the key molecules involved in anoikis are integrins.…”

“…Murine melan-a melanocytes surviving after 1, 2, 3 and 4 cycles of adhesion impediment (named respectively 1C, 2C, 3C and 4C cell lines) showed changes in morphology and growth independent of phorbol myristate acetate (PMA). Different melanoma cell lines (such as 4C3-, 4C3+, 4C11-, and 4C11+) were established after subjecting the surviving spheroids formed after blocking the adhesion of 4C cell line to a limiting dilution [17]. Previous data from our laboratory show a progressive increase in the TIMP1 expression along the melanoma genesis, and this increase is related to resistance to anoikis and to a more aggressive phenotype in vivo [76].…”

Relação entre estresse oxidativo, alterações epigenéticas e câncer

“…Diversos clones, entre eles os nomeados 4C11-e 4C11+, foram injetados em camundongos singenéicos e todos se mostraram tumorigênicos. Desta forma, obtivemos um modelo linear de progressão do melanoma, representado por melanócitos não tumorais (melan-a), pré-malignos (1C a 4C), tumorais não metastáticos (4C11-) e metastáticos (4C11+) (32 (20), importantes pesquisadores da área de epigenética. Os mecanismos pelos quais o estresse oxidativo regula as alterações epigenéticas encontram-se em investigação em nosso laboratório na Unifesp.…”