Molecular mechanisms of PAX6 survival impact in soft tissue sarcomas and other solid tumors.
This research fund allocation was received in order to acquire knowledge/skills and exploit laboratory facilities abroad, in order to obtain molecular and biological support data for our clinical findings about the prognostic role of PAX6 in non-small cell lung cancer and soft tissue sarcomas. Transcription factor PAX6 has a decisive role in the development of the CNS, eye, pancreas and olfactory epithelium, and the pattern of PAX6 normal expression is restricted to those tissues. PAX6 is detected in various cancer cell lines, but very little is known about its expression in tumors, possible cancer-relevant functions and prognostic value. PAX6 is generally believed to be a tumor suppressor, based mainly on studies in glioblastoma. PAX6 has been detected in tumors of eye and pancreas, but the prognostic significance remains unclear. Soft tissue sarcomas (STS) are a relatively uncommon but deadly group of tumors with poor prognosis and few treatment options. We had previously detected PAX6 in STS - the first tumor group with no developmental links to the normal sites of PAX6 expression. PAX6 appeared to confer a universal negative prognostic impact on DSS, irrespectively of clinical and pathological subgroups analyzed. PAX6 expression correlated with TGF-beta, and reciprocal upregulation may explain their synergistic negative effect on DSS. In non small cell lung cancer we have shown positive prognostic impact of PAX6 in the subgroup of node positive patients. The financing from Helse Nord allowed postdoc fellow Yury Kiselev to gain experience in various methods and their results’ interpretations, including: establishment of stable knockdowns of putative oncogenes in human and mammalian cancer cell lines with the use of lentiviridae, maintenance and RNAi interference in human soft tissue sarcoma cell lines, cell migration analysis with the use of silicon microinserts, proliferation analysis with the use of MTT method, apoptosis analysis with the use of Caspase Glo 3/7 method, work with mRNA expression databases, analysis of gene expression profiles, immunohistochemical staining of rhabdomyosarcoma samples for PAX6. This competence was used to finalise the manuscript on the role of PAX6 in non small cell lung cancer, which was submitted to the Journal of Thoracic Oncology in 2015 (under revision as of February 2015). In regards to soft tissue sarcomas, we received data indicating that PAX6 upregulates proliferation of several sarcoma cell lines, inhibits apoptosis (activation of caspase 3/7) and stimulates cell migration in the wound healing assay. However, for most of the analyses there was no statistical significance, possibly due to high variability of RNAi transfection efficiency. Another challenge was the heterogeneity of the STS tumor group, and it was not possible to cover all the range of subgroups of STS in our TMA data set with corresponding cell lines, as the latter are very rare. Hence, in order to obtain publishable data, a TMA consisting of exclusively rhabdomyosarcoma samples was stained for PAX6, and is going to be analysed in order to see whether PAX6 has a significant prognostic effect in this subgroup. For this subgroup we have 4 cell lines with endogenous expression of PAX6, which can be used to concentrate our efforts in order to avoid heterogeneity as the possible reason for inconclusive results. The projected results of this follow-up cell experiments are expected by summer 2015.
The methods and results obtained during this international research stay financed by Helse Nord scholarship helped to collect further evidence supporting the role of PAX6 as a prognostic marker in soft tissue sarcomas and non small cell lung cancer. Introduction of PAX6 testing into clinical practice may help to stratify patients into high and low risk groups, and potentially aid in selection of the most beneficial targeted therapies for patients depending on their tumor PAX6 status.