Lysophosphatidic acid reverses Temsirolimus-induced changes in lipid droplets and mitochondrial networks in renal cancer cells.

Ravneet Chhabra,Meera Nanjundan,Vladimir Trajkovic

doi:10.1371/journal.pone.0233887

Abstract

Increased cytoplasmic lipid droplets (LDs) and elevated AKT/mTOR signaling are characteristics of clear cell renal cell carcinoma (ccRCC). Lysophosphatidic acid (LPA), a potent lipid mitogen generated via autotaxin (elevated in ccRCC), can modulate tumor progression but its role in altering chemotherapeutic sensitivity to mTOR inhibitors is unclear and thus is the focus of the studies presented herein. Using malignant (A-498, 769-P and 786-O) and normal immortalized kidney (HK-2) cell lines, we investigated their cellular responsiveness to Temsirolimus (TEMS, mTOR inhibitor) in the absence or presence of LPA by monitoring alterations in AKT/mTOR pathway mediators (via western blotting), LDs (using LipidTOX and real-time PCR to assess transcript changes in modulators of LD biogenesis/turnover), mitochondrial networks (via immunofluorescence staining for TOM20 and TOM70), as well as cellular viability. We identified that TEMS reduced cellular viability in all renal cell lines, with increased sensitivity in the presence of an autophagy inhibitor. TEMS also altered activation of AKT/mTOR pathway mediators, abundance of LDs, and fragmentation of mitochondrial networks. We observed that these effects were antagonized by LPA. In HK-2 cells, LPA markedly increased LD size and abundance, coinciding with phospho-MAPK and phospho-S6 activation, increased diacylglycerol O-acetyltransferase 2 (DGAT2) mRNA (which produces triacylglycerides), and survival. Inhibiting MAPK partially antagonized LPA-induced LD changes. Collectively, we have identified that LPA can reverse the effects of TEMS by increasing LDs in a MAPK-dependent manner; these results suggest that LPA may contribute to the pathogenesis and chemotherapeutic resistance of ccRCC.

Highlights

Renal cell cancer (RCC) is one of the most common urological malignancies
Lysophosphatidic acid (LPA) reverses TEMSR response in RCC cells cells were cultured in Keratinocyte-serum free media (K-SFM, #17-005-042, Fisher Scientific, Pittsburgh, PA) supplemented with 50 μg/ml bovine pituitary extract (BPE, #10450–013) and 5 ng/ml human recombinant epidermal growth factor (EGF, #13028–014). 769-P and 786-O cells were grown in RPMI 1640 media containing 8% fetal bovine serum (FBS) and 1% penicillin/streptomycin whereas A-498 cells were cultured in EMEM media supplemented with 8% FBS and 1% penicillin/streptomycin
To correlate responsiveness of clear cell RCC (ccRCC) cell lines to established forms of treatments with extent of genomic aberrations, we analyzed a subset of malignant clear cell renal cell lines, via The Cancer Genome Atlas (TCGA: [28, 29])

Summary

Introduction

Renal cell cancer (RCC) is one of the most common urological malignancies. Of the five major subtypes of RCC, clear cell RCC (ccRCC) is the most common and lethal subtype; it is a metabolic disease characterized by dysregulated lipid metabolism, altered gene regulation due to multiple genomic aberrations, and increased. There currently exists several first-line targeted therapies which are FDA approved for ccRCC, including mTOR targeting agents [1]. The PI3K/AKT/mTOR pathway is highly dysregulated in ccRCC [4]; targeting mTOR (which modulates cellular survival, blood vessel development, and nutrients) with rapamycin can modulate LD formation [5]. The role of mTOR clinical targeting agents (including Rapalogs such as Temsirolimus (TEMS) [7]) in the regulation of mitochondrial networks and LD biogenesis has not yet been investigated in ccRCC

Methods

Results

Conclusion