Inducibly decreased MITF levels do not affect proliferation and phenotype switching but reduce differentiation of melanoma cells

Abstract

Melanoma arises from neural crest‐derived melanocytes which reside mostly in the skin in an adult organism. Epithelial–mesenchymal transition (EMT) is a tumorigenic programme through which cells acquire mesenchymal, more pro‐oncogenic phenotype. The reversible phenotype switching is an event still not completely understood in melanoma. The EMT features and increased invasiveness are associated with lower levels of the pivotal lineage identity maintaining and melanoma‐specific transcription factor MITF (microphthalmia‐associated transcription factor), whereas increased proliferation is linked to higher MITF levels. However, the precise role of MITF in phenotype switching is still loosely characterized. To exclude the changes occurring upstream of MITF during MITF regulation in vivo, we employed a model whereby MITF expression was inducibly regulated by shRNA in melanoma cell lines. We found that the decrease in MITF caused only moderate attenuation of proliferation of the whole cell line population. Proliferation was decreased in five of 15 isolated clones, in three of them profoundly. Reduction in MITF levels alone did not generally produce EMT‐like characteristics. The stem cell marker levels also did not change appreciably, only a sharp increase in SOX2 accompanied MITF down‐regulation. Oppositely, the downstream differentiation markers and the MITF transcriptional targets melastatin and tyrosinase were profoundly decreased, as well as the downstream target livin. Surprisingly, after the MITF decline, invasiveness was not appreciably affected, independently of proliferation. The results suggest that low levels of MITF may still maintain relatively high proliferation and might reflect, rather than cause, the EMT‐like changes occurring in melanoma.

Dissecting Mechanisms of Melanoma Resistance to BRAF and MEK Inhibitors Revealed Genetic and Non-Genetic Patient- and Drug-Specific Alterations and Remarkable Phenotypic Plasticity

“…SOX2 was shown to contribute to the stable reprogramming of melanoma cells to their undifferentiated counterparts that were resistant to inhibitors of the MAPK signaling pathway independently of which of oncogenic mutations in the MAPK pathway (BRAF or NRAS) was present [70], and SOX2-positive cells were reported in melanoma cell populations exerting a drug-tolerant state to combined dabrafenib and trametinib treatment [71]. Reciprocally excluding SOX2 and MITF expression was reported [72], which was also detected in all vemurafenib-resistant cell lines investigated in our study. Changes in SOX2 expression in 29_TRAR and 17_TRAR cells were less pronounced probably because of an elevated MITF level and high frequency of differentiated cells [27].…”

MITF—the first 25 years

“…Genes implicated in mitosis are also direct MITF targets, including PLK1, encoding a key regulator of M-phase progression, and components of the CENPA and NDC80 complexes that connect mitotic spindle microtubules to kinetochores ). However, the role of MITF as a proproliferative factor has been challenged by the observation that inducible depletion of MITF using shRNA did not block proliferation in all melanoma cell lines tested but did lead to dedifferentiation (Vlcǩová et al 2018). This suggests that other factors may compensate for the absence of MITF or that technical details such as using siRNA, as done in most studies, versus using shRNA, as done by Vlcǩová et al (2018), makes a difference.…”

“…However, the role of MITF as a proproliferative factor has been challenged by the observation that inducible depletion of MITF using shRNA did not block proliferation in all melanoma cell lines tested but did lead to dedifferentiation (Vlcǩová et al 2018). This suggests that other factors may compensate for the absence of MITF or that technical details such as using siRNA, as done in most studies, versus using shRNA, as done by Vlcǩová et al (2018), makes a difference. Evidently, further work is needed to clarify this issue.…”

MITF reprograms the extracellular matrix and focal adhesion in melanoma

“…Two different pathways involved in migration were shown to be regulated by MITF; DIAPH1, a gene implicated in actin polymerization [14], and the guanosine monophosphate reductase (GMPR) gene encoding an enzyme involved in regulating intracellular GTP levels [46]. Surprisingly, however, more recent studies by Falletta et al [47] and Vlckova et al [49] failed to observe any effects on migratory/invasive properties upon MITF knockdown using the same cell lines as were used in the previous studies. Interestingly, knocking down SMAD7 in melanoma cells resulted in a dual invasive-proliferative phenotype without affecting MITF expression [50] Thus, the idea has been proposed that two different modes of invasion operate in melanoma; one with low MITF levels and no proliferation and another with high MITF levels where proliferation and invasion can take place simultaneously [10].…”