Multidrug Resistance Decreases with Mutations of Melanosomal Regulatory Genes

Abstract

Whereas resistance to chemotherapy has long impeded effective treatment of metastatic melanoma, the mechanistic basis of this resistance remains unknown. One possible mechanism of drug resistance is alteration of intracellular drug distribution either by drug efflux or sequestration into intracellular organelles. Melanomas, as well as primary melanocytes from which they arise, have intracellular organelles, called melanosomes, wherein the synthesis and storage of the pigment melanin takes place. In this study, comparisons of congenic cells with and without functional molecules regulating melanosome formation show that sensitivity to the chemotherapeutic agent cis-diaminedichloroplatinum II (cis-platin) significantly increases with the mutation of genes regulating melanosome formation, concomitant disruption of melanosome morphology, and loss of mature melanosomes. Absence of the melanosomal structural protein gp100/Pmel17 causes increased cis-platin sensitivity. Independent mutations in three separate genes that regulate melanosome biogenesis (Dtnbp1, Pldn, Vps33a) also result in increased cis-platin sensitivity. In addition, a mutation of the gene encoding the integral melanosomal protein tyrosinase, resulting in aberrant melanosome formation, also causes increased cis-platin sensitivity. Furthermore, sensitivity to agents in other chemotherapeutic classes (e.g., vinblastine and etoposide) also increased with the mutation of Pldn. In contrast, a mutation in another melanosomal regulatory gene, Hps1, minimally affects melanosome biogenesis, preserves the formation of mature melanosomes, and has no effect on cis-platin or vinblastine response. Together, these data provide the first direct evidence that melanosomal regulatory genes influence drug sensitivity and that the presence of mature melanosomes likely contributes to melanoma resistance to therapy. [Cancer Res 2009;69(3):992-9]

Targeting protein-trafficking pathways alters melanoma treatment sensitivity

“…The data here indicate that melanomas can be protected from apoptosis by melanosomal sequestration of treatment agents, consistent with our and other groups' previous findings that melanosome function plays a role in melanoma treatment resistance (13,(27)(28)(29)(30)(31). Gottesman and colleagues (29) have shown that chemosensitivity is independent of melanoma pigment content, consistent with a melanosome function alternative to that of melanin synthesis (e.g., sequestration) influencing chemosensitivity.…”

“…We previously demonstrated that primary mouse melanocytes with mutations in Vps33a or Cno had aberrant melanosome formation and increased sensitivity to cis-platin (cDDP) compared with melanocytes from the congenic parental mouse strains (13). To assess whether depletion of VPS33A or CNO proteins in human melanoma cells affects drug sensitivity, we established stable isogenic cell lines by selecting clones of the pigmented human melanoma cell line MNT-1 after transduction with shRNA targeted to either VPS33A or CNO.…”

“…Human melanoma cells MNT-1 and SK-MEL-24 were cultured as previously published (13). The mutant mouse melanocyte line melan-ru1-3 was obtained by crossing ruby-eye (Hps6 ru /Hps6 ru ) mice to C57BL/6 Cdkn2a (Ink4a-Arf) null mice, yielding cells that are immortal (33).…”

“…Drug sensitivity studies were performed as previously published (13). To test UV sensitivity, cells were drained of medium and exposed to ultraviolet C radiation (254 nm, 1.4 J·m −2 /s).…”

“…Mutations in VPS33A and CNO cause cell type-specific symptoms in patients and mice largely limited to melanocytes and platelets resulting in pigment dilution and nonlife-threatening prolonged bleeding times (8). Mutations in the Vps33a or Cno gene both resulted in increased sensitivity to cDDP, etoposide, and vinblastine (13) and blocked the normal process of eumelanosome maturation through four morphological stages (stages I-IV) (10,14), indicating a fundamental role for VPS33A and CNO in contributing to cellular resistance to multiple cytotoxic agents.…”