Melanoma

Melanoma is a particularly aggressive cancer, well known for its ability to rapidly metastasize to other organs, making early diagnosis crucial. Alarmingly, it is also currently one of the fast growing cancers in the Western world (1), and so further research in this area is particularly important.

Control of protein synthesis in cancer is crucial, needed for their rapidly proliferative growth rates, as well as being implicated in the dedifferentiation processes associated with epithelial-mesenchymal transition and metastasis. In melanoma, one of the most commonly dysregulated pathways is that of MAPK (often through the commonly known BRAF mutations seen in this disease) (2). This pathway is an important regulator of rRNA transcription, and so is intrinsically coupled to the process of translation. Further translation factors, such as eIF3c, have been implicated (3).

Modern leaps in the understanding of melanoma have markedly improved outcomes in recent years, particularly in the guise of immunotherapies. However, the metastatic potential of melanoma remains a grave risk, and late-stage melanoma is particularly difficult to treat. Below, we take a look at some of the papers where translational study has yielded notable insights into the underlying mechanistics of melanoma markers and development.

Translation reprogramming is an evolutionarily conserved driver of phenotypic plasticity and therapeutic resistance in melanoma

Genes & Development, 2017

Falletta, P., Sanchez-del-Campo, L., Chauhan, J., Effern, M., Kenyon, A., Kershaw, C.J., Siddaway, R., Lisle, R., Freter, R., Daniels, M.J. and Lu, X.

The tumour microenvironment is known for its heterologous nature, with there being present an ever-changing flux of environmental cues, thus influencing the cells that inhabit this milieu. This can lead to alterations in the expressed phenotypes, such as drug resistance and/or invasiveness potential. Melanoma is a cancer known for its aggressive and early metastases, and previous analysis of melanoma cell lines revealed they exhibit 2 predominant phenotypes; invasive but poorly proliferative, or proliferative but poorly invasive. This is related to the expression of MITF (microphthalmia-associated transcription factor), with lower expression driving the former phenotype, and high expression the latter. However, the factors that govern its expression across these different melanomas is unknown. While low expression of MIFT is typically associated with senescence, in cancer cells it is associated with invasiveness and drug resistance. Here, the authors look into the mechanisms by which these MIFT-low non-senescent cells are formed, with an aim to investigate the links to invasiveness and resistance.

Key Findings

  • Limitation of glutamine, an essential amino acid for cancer growth and a common occurrence within the tumour microenvironment, was demonstrated to increase the translation of ATF4, a transcriptional repressor and integral member of the integrated stress response (ISR). Concurrent with this was a suppression of MIFT expression driven by ATF4 activation.
  • Knockdown of MITF in isolation led to the appearance of senescence-associated markers. However, when a reduction of MITF expression was brought about via glutamine limitation, such markers were lacking. Indeed, induction of ATF4 expression in nutrient-rich medium was sufficient to avoid senescence, even with artificial MITF knockdown.
  • However, a high ATF4/low MITF expression profile does not lead to an invasive phenotype. This is instead reliant on the upstream factor in the ISR, IF2α, which when phosphorylated, inhibits eIF2B activity. Interestingly, TNFα treatment, representing a different microenvironmental cue, led to similar responses seen with glutamine limitation. Furthermore, both glutamine limitation and ATF4 induction led to a low MITF/AXL ratio, a phenotype strongly associated with drug resistance in melanoma.
  • Interestingly, B16 cell deprived of glutamine, when injected into immunocompromised mice, lead to a massive tumour burden compared to non-deprived cells. Salubrinal, an enzyme that essentially preserves eIF2α phosphorylation, when incubated with B16 cells prior to injection, has a similarly high tumour burden profile compared to glutamine-deprived cells.

Implications

This work brought to light the drivers behind phenotypes that have long been known in the melanoma field, with translation, more specifically a stress-induced translational response, lying behind the classic MITF-separated phenotypes. It also belied the role translation plays in metastasis, sensing stress-based cues and triggers, leading to a switch from a proliferative to invasive phenotypes. Such deeper understanding can’t but only help direct future research decisions.

 

Translational control of tumor immune escape via the eIF4F–STAT1–PD-L1 axis in melanoma

Nature Medicine, 2018

Cerezo, M., Guemiri, R., Druillennec, S., Girault, I., Malka-Mahieu, H., Shen, S., Allard, D., Martineau, S., Welsch, C., Agoussi, S. and Estrada, C.

In many cancer cells, upregulation of PD-L1 on their surface leads to inhibition of T cell’s cytotoxic response. Recent drugs, such as pembrolizumab and nivolumab, that block this inhibition have had some success in improve patient outcomes. However, resistance to these immunotherapies is common, partly due to the variety of mechanisms by which PD-L1 can be upregulated (often through infiltrating lymphocyte-secreted IFN-γ). Separately, the translation initiation-associated complex, eIF4F, has been associated with resistance to a number of anticancer therapeutics in a variety of cancers.  eIF4F is a heterotrimeric complex consisting of eIF4A, eIF4E, and eIF4G, which together function to assist in recruiting ribosomes to the mRNA. eIF4A, in particular, is essential in unwinding complex secondary structure which may inhibit ribosome recruitment and scanning. Expression of the components of this complex have been shown to be altered across a variety of cancers, and inhibitors of eIF4F functionality have been demonstrated to possess strong anti-cancer properties. As such, the authors here aimed to investigate any possibly link between eIF4F and immune escape, with a particular focus on PD-L1 expression.

Key Findings

  • eIF4F inhibition, either through siRNA knockdown of individual components, or through pharmacological treatment with silvestrol, reduced the level of IFN-γ-induced PD-L1 expression. Correspondingly, increased eIF4F expression, obtained via knockout of eIF4F inhibitors eIF4E-BP1/2, results in increases the IFN- γ-induced PD-L1 induction.
  • By analysing genes that were transcriptionally upregulated by IFN-γ treatment, but translational downregulated by silvestrol, STAT1 was identified as gene that may be link between eIF4F and IFN-γ-stimulation PD-L1 expression. Indeed, luciferase reporters containing the STAT1 5’ UTR demonstrated its silvestrol-controlled regulation. This UTR contains predicted G-quadruplexes, hypothesised to be targets of eIF4A helicase activity.
  • Indeed, siRNA mediated knockdown of STAT1is able to elicit a very similar phenotype as is seen with eIF4F knockdown (i.e., decreased IFN-γ-induced PD-L1 expression). Furthermore, overexpression of 5’ UTR-less STAT1 mRNA partially rescues the from impact eIF4F inhibition with regard to PD-L1 expression.
  • Finally, in mice inoculated with carcinogenic BrafV600EPten–/– (BP) melanoma cells, silvestrol treatment resulted in a significantly lower tumour burden as compared to untreated mice. Interestingly, in nude mice, silvestrol treatment had no impact on tumour development, indicating the crucial nature of the immune system acting in synergy with drug-induced melanoma vulnerability.

Implications

This work ably demonstrated yet another role translation plays in the aetiology of cancer. Here, translation found a new-found role within the immunological aspects of melanoma, with potential implications for a further variety of cancers. Immunotherapy is a complex treatment option, with multitudinous regulatory mechanisms. However, this paper highlighted translation, and eIF4F more specifically, as a potential target for future drug development.

Anti-tumour immunity induces aberrant peptide presentation in melanoma

Nature, 2020

Bartok, O., Pataskar, A., Nagel, R., Laos, M., Goldfarb, E., Hayoun, D., Levy, R., Körner, P.R., Kreuger,  I.Z., Champagne, J. and Zaal, E.A., et al

Under normal conditions, the immune system targets cancer cells and prevents them from propagating, using a plethora of mechanisms, including increased T cell infiltration and IFN-γ signalling. To combat these mechanisms, tumours often act to supress the immune system, through a variety of pathways. In response to increase IFN-γ signalling, some tumours are able to deplete levels of tryptophan via IDO1, which suppresses T cell function. However, this strategy also depletes tryptophan, an important amino acid, and reduces translation, for which cancer cells compensate by upregulating other systems. Interestingly however, inhibition of IDO1 does not improve melanoma patient prognosis when combined with PD-1 therapy. As such, it appears there are gaps in the knowledge of the role of IFN-γ signalling on translation in melanoma. In this paper, the authors utilise ribosome profiling to gain a deeper understanding of the impact of IFN-y signalling on translation in this disease.

Key Findings

  • Utilising differential ribosome codon reading, it was determined that IGN-γ treatment induced an accumulation of ribosomes at start codons (corresponding with a global decrease in translation), as well as indicating stalling at tryptophan codons (corresponding with well-characterised tryptophan depletion). More importantly, however, was a drastic increase in occupancy just downstream of tryptophan codons, termed W-bumps.
  • In particular, these W-bumps were found to be associated with the presence of a number of tryptophan codons within a stretch of 8 codons. Later analysis suggested that these W-bumps result from aberrant frameshifting events in an effort to bypass tryptophan codons. This results in disordered, out-of-frame peptides past the frameshifting site. IDO1
  • These post-tryptophan codon, out-of-frame peptides were detected using mass spectroscopy, and we noticeably enriched in IFN-γ-treated cells, as opposed to controls. They are also presented on human leukocyte antigen (HLA-1) molecules, again with a greater enrichment in IFN-γ-treated cells.
  • When dendritic cells derive from healthy donors in were pulsed with a selection of these aberrant, out-of-frame peptides and co-cultured with naive CD8+ T cells from healthy individuals, combinatorial tetramer staining analysis revealed that these T cells developed immunoreactivity to the peptides in question.

Implications

While tryptophan depletion in response to IFN-γ had been a known phenomenon in tumour biology, this paper was the first to reveal the now characterised “W-bumps” downstream of tryptophan codons in these conditions. It demonstrated the role of aberrant ribosomal frameshifting in this event, and the immunogenic consequences of the subsequent novel peptides produced. It has the potential to add to the diagnostic fingerprint of melanoma.

1. Ali Z, Yousaf N, Larkin J. Melanoma epidemiology, biology and prognosis. EJC Suppl. 2013;11(2):81-91.
2. Maldonado JL, Fridlyand J, Patel H, Jain AN, Busam K, Kageshita T, et al. Determinants of BRAF mutations in primary melanomas. J Natl Cancer Inst. 2003;95(24):1878-90.
3. Emmanuel R, Weinstein S, Landesman-Milo D, Peer D. eIF3c: a potential therapeutic target for cancer. Cancer Lett. 2013;336(1):158-66.

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