Uveal Melanoma and Liver Metastasis: Study of Factors Associated with Metastatic Dissemination and Prognostic Implications
The most common genetic alterations in patients with uveal melanoma are the driver mutations in GNAQ, GNA11, BAP1, SF3B1 and EIF1AX. These mutations lead to the sustained activation of various signalling pathways such as MAPK and Hippo-YAP (FIGURE 1), which leads to an increase in cell proliferation and dissemination capacity. The total or partial loss of chromosome 3 and mutations in the BAP1 suppressor gene (located in 3p21.1) is directly associated with liver metastases.
In our line of research, we propose the following working hypothesis:
- Mutations in GNAQ / GNA11 genes and the consequent activation of MAPK signalling pathways may be relevant to the development of uveal melanoma and liver metastases. Likewise, the aberrant expression of growth and migration factors facilitates hematogenous spread and liver colonization.
- Exosomes released by uveal melanoma cells contain certain microRNAs and proteins that are involved in hematogenous spread and metastatic niche preparation for liver colonization. Validation of certain proteins and their targets could provide new prognostic and therapeutic tools.
- To determine the signalling pathways that are essential for the progression of primary uveal melanoma tumours towards liver metastases.
- To study the causes that promote the development of liver metastases.
- To examine the role of exosomes in the preparation of the liver metastatic niche.
- To identify the clinical and prognostic biomarkers associated with metastatic disease.
Clinical data, biological samples, and the genetic information from patients with uveal melanoma are obtained in the hospital setting (oncology, pathology and ophthalmology). In the laboratory, we work with various uveal melanoma cell lines (Mel270, OMM2.3, OMM2.5, UMA and SP6.5) and an animal model in the form of xenografts in immunosuppressed mice (FIGURE 2). We use innovative platforms such as bioluminescence in this research.
We have created a tissue microarray from tissue samples obtained from patients previously treated at our hospital and for whom we have histological results for the tumour, follow-up data, and the genetic/cytogenetic characterization of the cancer. The aim of the present project is to analyze the expression of markers such as BAP1, PRAME and microRNAs, which will allow us to refine tumour stratification thus to determine patient prognosis based on those findings.
We used a panel of uveal melanoma cell lines to screen in vitro for the most relevant tyrosine kinase inhibitors (TKI) belonging to the involved signalling pathways. The approach taken was to screen global cell populations and subpopulations resistant to anoikis that are capable of growing without the need for substrate anchoring. These subpopulations have the characteristics of stem cells, they form spheres (UM-melanospheres) and overexpress markers such as CD133, CD44, P75 and transcription factors such as NANOG, OCT-4 and SOX-2, among others. These cells also have greater activation of certain membrane exclusion transporters; as a result, they are less sensitive to the antiproliferative and proapoptotic effect of TKIs than global populations. Even so, we have identified effective inhibitors of the signalling pathways against SRC, PKC, MEK and JNK, which—used in combination with cytotoxic drugs against the remaining tumour populations—could be an effective treatment for uveal melanoma.
Finally, it is worth emphasizing that cell lines derived from uveal melanoma liver metastases produce the largest number of exosomes in the extracellular medium, as in the case of OMM2.5. We characterized the vesicles secreted by these cells by their size and with protein markers such as ALIX or CD63, which are characteristic of exosomes. The contribution of the exosomes is essential to the process of metastatic dissemination because the hematogenous route reaches the liver and modifies its microenvironment to make it more favourable for the tumour cells and to promote their growth.