Abstract
MicroRNAs (miRs) are increasingly important diagnostic and prognostic markers in cancer but have not been defined in medullary thyroid carcinoma (MTC). MiR microarray profiling was performed on 19 primary MTC tumors, validated with qPCR in 45 cases and correlated with clinical outcomes. MiRs-183 and 375 were overexpressed and miR-9* underexpressed in sporadic vs. hereditary MTC (SMTC; HMTC). MiR-183 and 375 overexpression predicted lateral nodal metastases, residual disease, distant metastases and mortality. MiR-183 knockdown in an MTC cell line (TT cells) reduced cellular proliferation in association with elevated LC3B expression. This is suggestive of increased autophagic flux and potential cell death via autophagy induction. MiRs may subsequently be shown to serve as efficacious therapeutic strategies in MTC with a mechanism based upon autophagy.
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MiRs are small noncoding nucleotides known to exert an influence through post-transcriptional regulation of molecular processes. The role of miRs has been defined in numerous cellular processes both in health and disease, and has recently become a topic of intense investigation in cancer research.
Autophagy as a biological process is complex, and its significance within the context of neoplasia is currently under debate. Our present understanding of the role of autophagy in oncogenesis is somewhat of a double-edged sword. It is well established that autophagy may act as a process ensuring survival in times of cellular stress, possibly assisting in tumor cell preservation and proliferation. Conversely, there is increasing evidence that autophagy may ultimately underpin a mechanism of tumor cell death. Thus, the enticing notion of harnessing the tumoricidal potential of the autophagic process is tempered by the theoretical possibility of facilitating tumor cell proliferation and dissemination. Such contrasting and unique qualities make autophagy a process like no other.
While the molecular basis of autophagy is now coming under increasing scrutiny, there remains a dearth of knowledge regarding the potential influence of miRs. MiR-30a is one miR that has been shown to influence Beclin 1 expression levels and other markers of autophagy in “cancer” cell lines. We sought to examine related phenomena in a cell line model of human MTC.
Following micro-array identification of differential miR expression (showing elevated miR-183 expression in SMTC vs. HMTC tumors), miR-183 was knocked down in a TT cell line. Cellular proliferation is depressed following transfection, without changes in markers of apoptosis. An alternative hypothesis surrounded increased rates of autophagy. Subsequent immunoblotting for LC3B identified elevations in this marker following TT cell line miR-183 knockdown.
As to whether evidence of enhanced autophagic flux sheds light on the mechanism of reduced cellular proliferation following repression of a pathologically overexpressed miR remains to be seen. However, this relatively simple experimentation poses questions within the broad realm of miRs, autophagy and oncogenesis.
What of the relationship between miRs and autophagy? Is there a direct relationship, and at what level is this molecular communication occurring? Have we identified a subclass of regulatory molecules that may be eloquently known as “autophagamiRs”? And if so, how do we best delineate this relationship, and not only identify targets, but define mechanistic pathways? Beyond this, can the translational chemotherapeutic effect of autophagy be harnessed as an efficacious weapon against the burden of malignancy?
Review of the literature on the topic sheds little, if any, light. Target prediction tools geared toward identifying miR targets such as miRbase (www.mirbase.org) and targetscan (www.targetscan.org) have previously identified autophagy-related (ATG) genes in published databases, but require experimental validation.
Investigation within the context of a disease with a well-defined molecular pathogenesis is also of interest. Many cases of MTC are hereditary in origin and possess an identifiable RET gene mutation that has been the focus of much investigation to date; particularly given the poor prognosis that patients with advanced disease suffer in the absence of effective adjuvant therapy beyond surgery. In the course of such work, the TOR kinase pathway has been explored and interestingly would appear to be lying upstream of, and involved in, autophagy induction at the Atg1 level. Could it be that potential “autophagamiRs” such as miR-183 are gatekeepers at this level, in the same way that “rapalogues” have come to be known for their ability to inhibit TOR kinase?
Given the functional influence that miRs have been shown to have within the processes underlying oncogenesis, it would appear that miR-driven therapeutics are not far away. Now that we have evolving evidence of an “autophagamiR”-induced effect, perhaps such advances will rely upon autophagy for their tumoricidal impact. These relationships and the development of potential therapeutic strategies demand further attention.
Acknowledgment
This work was supported by a NSW Cancer Institute Research Innovation Grant. J.S.G. is an Australian Postgraduate Award Scholarship recipient. S.B.S. is a NSW Cancer Institute Fellow.