DDDR-40 - Overcoming Drug Resistance by Targeting Autophagy Regulated by a Long Intergenic Noncoding RNA

Background:  Autophagy is a self-eating process that supports cancer cell survival. Autophagy activation during drug treatment often prolongs cancerous cell survival, causing drug resistance, as seen in glioblastoma multiforme (GBM) to temozolomide (TMZ). Thus, blocking autophagy is an appealing approach to overcoming cancer drug resistance. Long noncoding RNAs (LincRNAs) have emerged as potential targets. LincRNA LINC00467 (renamed ARLINC) was recently identified as a potential autophagy inhibitor in cancer cells. However, ARLINC’s mechanism of inhibition and whether its modulation can suppress autophagy in cancer remains unknown.

Objective: Research Question: Can blocking autophagy restore the sensitivity of cancer cells to chemotherapy?
Hypotheses:
1) ARLINC regulates chemotherapy drug sensitivity by inducing autophagy.
2) Overexpression of ARLINC can increase chemotherapy drug sensitivity, as measured by cell viability and autophagy assays

Methods:
Aim 1) Endogenous ARLINC levels in GBM cell lines will be measured using qRT-PCR.
Aim 2) Cell viability of GBM upregulated with pBabe-ARLINC (experiment) and pBabe (control) then exposed to increasing concentrations of TMZ will be analyzed using MTS assay. Student’s T-test will be used to determine significance.
Aim 3) Autophagy flux will be measured in GBM lines that are transiently transfected with ARLINC compared to the empty vector via Cyto-ID assay.

Results:
Aim 1: Compared to Normal Human Astrocytes (NHA), U87MG had higher levels, LN18 and SF295 and lower, and LN229 had around the same levels of ARLINC.
Aim 2: MTS cell viability assays in SF295 upregulated with ARLINC showed no significant decrease in TMZ sensitivity.
Aim 3: Cyto-ID in U87MG and LN229 upregulated with ARLINC showed no significant decrease in autophagy flux.
Conclusions/Future Work: Results show no effect of ARLINC upregulation on cell viability and autophagy flux. Future work involves knockdown experiments to decrease intracellular levels of ARLINC and determine the effects of upregulation of autophagy on sensitization.