Regulation of tumor cell - Microenvironment interaction by the autotaxin-lysophosphatidic acid receptor axis

Abstract

The lipid mediator lysophosphatidic acid (LPA) in biological fluids is primarily produced by cleavage of lysophospholipids by the lysophospholipase D enzyme Autotaxin (ATX). LPA has been identified and abundantly detected in the culture medium of various cancer cell types, tumor effusates, and ascites fluid of cancer patients. Our current understanding of the physiological role of LPA established its role in fundamental biological responses that include cell proliferation, metabolism, neuronal differentiation, angiogenesis, cell migration, hematopoiesis, inflammation, immunity, wound healing, regulation of cell excitability, and the promotion of cell survival by protecting against apoptotic death. These essential biological responses elicited by LPA are seemingly hijacked by cancer cells in many ways; transcriptional upregulation of ATX leading to increased LPA levels, enhanced expression of multiple LPA GPCR subtypes, and the downregulation of its metabolic breakdown. Recent studies have shown that overexpression of ATX and LPA GPCR can lead to malignant transformation, enhanced proliferation of cancer stem cells, increased invasion and metastasis, reprogramming of the tumor microenvironment and the metastatic niche, and development of resistance to chemo-, immuno-, and radiation-therapy of cancer. The fundamental role of LPA in cancer progression and the therapeutic inhibition of the ATX-LPA axis, although highly appealing, remains unexploited as drug development to these targets has not reached into the clinic yet. The purpose of this brief review is to highlight some unique signaling mechanisms engaged by the ATX-LPA axis and emphasize the therapeutic potential that lies in blocking the molecular targets of the LPA system.

Keywords: Cancer stem cell; ENPP2; Invasion; LPA; Metastasis; Therapy resistance.

Fig. 1.

Anti-apoptotic signaling pathways activated by LPA₂.

Fig. 2.

A cross-cancer query of enpp2, the gene encoding ATX using the cBio Cancer Genomics Portal. ATX (encoded by enpp2 gene) is amplified or upregulated predominantly in ovarian cancer. Datasets obtained from The Cancer Genome Atlas.

Fig. 3.

LPA2 inhibition disrupts spheroid formation in 4T1 mouse mammary carcinoma cells. 4T1 cells were plated in triplicate in ultra-low adherence 24-well plates at a density of 7.5 × 10³ cells per well in spheroid formation medium (RPMI with 200 μM L-glutamine, 100 U penicillin, 100 μg/ml streptomycin, 1× N-2 supplement, 20 ng/ml mouse EGF and 20 ng/ml mouse FGF). Cells were incubated at 37 °C with 5% CO₂ for 4 days to allow spheroid formation, then treated with a range of 0–3 μM Amgen 35 (LPA₂ antagonist), BMP-22 (ATX inhibitor) or BESA-3 (ATX/LPA₁ inhibitor) and incubated an additional 3 days. Cells were surveyed under 100× magnification (microscopy panels), and spheroids were manually counted, plotted versus Amgen 35 concentration and GraphPad Prism v. 5.0a was used to perform non-linear regression in a variable slope model to determine the IC₅₀ of Amgen 35 for spheroid formation (dose-response panel). Finally, cell viability was determined using Promega CellTiter Blue reagent as per manufacturer’s instructions. Briefly, 50 μl CellTiter Blue were added to each well, and plates were incubated at 37 °C for 3 h prior to measuring fluorescence at excitation/emission wavelengths of 560/590 nm, respectively. Relative fluorescence was then background-corrected and normalized to percentage vehicle signal for each compound tested (bar graph panel). GraphPad Prism was used to perform one-factor ANOVA with Bonferroni’s post-test to determine whether cell viability differed significantly between vehicle and drug treatment (** = p < 0.01, *** = p < 0.001; n = 3).

Fig. 4.

When DNA damage repair goes awry it can lead to therapy resistance. A new hypothesis.

Fig. 5.

ATX and LPA₂ play key roles in resistance against radiation-induced programmed cell death. (A) LPA protects HPAF II human pancreatic cancer cells against gemcitabine-induced apoptosis. HPAF II cells were cultured in growth media (DMEM F12 supplemented with heat inactivated 10% FBS, 200 μM L-glutamine, 100 U penicillin and 100 μg/ml streptomycin). Next day, cells were serum starved for 24 h followed by exposure to 10 μM Gemcitabine with or without LPA (at 3 μM or 10 μM). Caspase 3/7 activity was measured after 24 h of treatment using the CaspaseGlo^® Kit (Promega). (B) LPA2 and (C) ATX expression are upregulated in 4T1 CSC upon exposure to Paclitaxel or γ-irradiation. 4T1 CSC were generated as described in Fig. 3 and spheroids were allowed to form for 3 days. At day 4, CSCs were either treated with 80 nM of Paclitaxel (PCTXL) or fractionated doses of γ-irradiation (6Gy/day for 4 days). CSCs were harvested for QPCR analysis after 4 days of PCTXL treatment or 24 h after the last dose of fractionated radiation. GraphPad Prism was used to perform one-factor ANOVA with Bonferroni’s post-test to determine the significance in LPA2 or ATX expression in vehicle versus PCTXL or radiation treatment, respectively (*** = p < 0.001; n = 3). (D) The ATX inhibitor BMP-22 enhances 4T1 cell killing by PCTXL. 4T1 cells were plated in quadruplicate in 96-well plates at a density of 5 × 10³ cells per well in RPMI with 1% (v/v) charcoal-stripped FBS and incubated overnight at 37 °C with 5% CO₂. Cells were then treated for 24 h with 3 μM BMP-22 followed by 48 h treatment with a dose range of 0–1 μM PT in RPMI with 1% (v/v) charcoal-stripped FBS. After 48 h incubation, cells were allowed to recover in RPMI with 1% (v/v) charcoal-stripped FBS for an additional 72 h. Viability was then assessed via Promega CellTiter Blue (as per manufacturer’s instructions) and normalized to % vehicle control samples. Data were plotted using GraphPad Prism v. 5.0a, and non-linear regression analysis was performed in a variable slope model in order to determine LD₅₀ concentrations for PCTXL in the presence and absence of BMP-22.