ATM Deficiency Confers Specific Therapeutic Vulnerabilities in Bladder Cancer

Ataxia-telangiectasia mutated (ATM) plays a central role in the cellular response to DNA damage and ATM alterations are common in several tumor types including bladder cancer. However, the specific impact of ATM alterations on therapy response in bladder cancer is uncertain. Here, we combine preclinical modeling and clinical analyses to comprehensively define the impact of ATM alterations on bladder cancer. We show that ATM loss is sufficient to increase sensitivity to DNA-damaging agents including cisplatin and radiation. Furthermore, ATM loss drives sensitivity to DNA repair-targeted agents including poly(ADP-ribose) polymerase (PARP) and Ataxia telangiectasia and Rad3 related (ATR) inhibitors. ATM loss alters the immune microenvironment and improves anti-PD1 response in preclinical bladder models but is not associated with improved anti-PD1/PD-L1 response in clinical cohorts. Last, we show that ATM expression by immunohistochemistry is strongly correlated with response to chemoradiotherapy. Together, these data define a potential role for ATM as a predictive biomarker in bladder cancer

Lysophosphatidate Induces Chemo-Resistance by Releasing Breast Cancer Cells from Taxol-Induced Mitotic Arrest

Taxol is a microtubule stabilizing agent that arrests cells in mitosis leading to cell death. Taxol is widely used to treat breast cancer, but resistance occurs in 25-69% of patients and it is vital to understand how Taxol resistance develops to improve chemotherapy. The effects of chemotherapeutic agents are overcome by survival signals that cancer cells receive. We focused our studies on autotaxin, which is a secreted protein that increases tumor growth, aggressiveness, angiogenesis and metastasis. We discovered that autotaxin strongly antagonizes the Taxol-induced killing of breast cancer and melanoma cells by converting the abundant extra-cellular lipid, lysophosphatidylcholine, into lysophosphatidate. This lipid stimulates specific G-protein coupled receptors that activate survival signals.In this study we determined the basis of these antagonistic actions of lysophosphatidate towards Taxol-induced G2/M arrest and cell death using cultured breast cancer cells. Lysophosphatidate does not antagonize Taxol action in MCF-7 cells by increasing Taxol metabolism or its expulsion through multi-drug resistance transporters. Lysophosphatidate does not lower the percentage of cells accumulating in G2/M by decreasing exit from S-phase or selective stimulation of cell death in G2/M. Instead, LPA had an unexpected and remarkable action in enabling MCF-7 and MDA-MB-468 cells, which had been arrested in G2/M by Taxol, to normalize spindle structure and divide, thus avoiding cell death. This action involves displacement of Taxol from the tubulin polymer fraction, which based on inhibitor studies, depends on activation of LPA receptors and phosphatidylinositol 3-kinase.This work demonstrates a previously unknown consequence of lysophosphatidate action that explains why autotaxin and lysophosphatidate protect against Taxol-induced cell death and promote resistance to the action of this important therapeutic agent

Lysophosphatidate releases the cells from G2/M phase.

<p>Cells were preincubated with 50 nM Taxol for 24 h and cells passing through S-phase were labeled for 2 h with BrdU. Cells were then incubated for 12 h with 10% delipidated serum in the presence or absence of 50 nM Taxol or 5 µM LPA (Protocol B). The progression of cells treated with Taxol alone or Taxol and LPA through S, G2/M and G1 phases of the cell cycle (Panels B and C, respectively) was quantified by FACS analysis (Panel A). DNA histograms were composed with CellQuest software. The histogram for time 0 for all treatments shows the same results, repeated for clarity of the figure. Results are means ± SD for three independent experiments.</p

Lysophosphatidate releases MCF-7 cells from Taxol-induced arrest in G2/M and cell death.

<p>MCF-7 cells were preincubated with 10% delipidated serum and 50 nM Taxol for 26 h. Taxol was then maintained or removed in the presence of 10% delipidated serum and 5 µM LPA was added as indicated (Protocol C). Cells were then stained with DAPI, anti-phospho-histone and with anti-tubulin at the times indicated. The Panels show the percentage of: A) cells in mitosis, B) percentage of cells in mitosis that had abnormal spindles, C) mononucleated cells, D) multinucleated cells and E) dead and dying cells. Total number of cells (100%) includes mononucleated, multinucleated, dead and dying and mitotic cells. Time zero shows 26 h pretreatment with Taxol. Results are means ± SD (where large enough to be shown) for three independent experiments.</p

LPA causes dissociation Taxol from the polymerized tubulin compartment.

<p>Approximately 2×10⁶ cells were seeded in 6 cm dishes. After growing cells to confluence, the medium was replaced with 3 ml RPMI 1640 containing 10% of charcoal-treated FBS containing 50 nM [³H]Taxol (0.5 µCi). Cells were preincubated for 26 h. After this, 5 µM LPA and different concentrations of inhibitors were added as indicated. Polymerized tubulin (Panel A) and soluble fractions (Panel B) were then separated and the distribution of [³H]Taxol was determined. Panels C and D show the dose response curves for LY294002 and Wortmannin, respectively. Results are means ± SD for three independent experiments. Panels A and B, * a significant difference of <i>P<0.05.</i></p

Lysophosphatidate releases MDA-MB-468 cells from Taxol-induced arrest in G2/M and cell death.

<p>MDA-MB-468 cells were preincubated with 10% delipidated serum and 50 nM Taxol for 26 h. Taxol was then maintained or removed in the presence of 10% delipidated serum and 5 µM LPA was added as indicated (Protocol C). Cells were stained with DAPI, anti-phospho-histone and with anti-tubulin at the times indicated. The Panels show the percentage of: A) cells in mitosis, B) percentage of cells in mitosis that had abnormal spindles, C) mononucleated cells, D) multinucleated cells and E) dead and dying cells. Total number of cells (100%) includes mononucleated, multinucleated, dead and dying and mitotic cells. Time zero shows 26 h pretreatment with Taxol. Results are means ± SD for three independent experiments.</p

LPA does not affect Taxol expulsion form MCF-7 cells.

<p>Panel A, approximately 2×10⁵ MCF-7 breast cancer cells were grown to confluence in 3.5 cm cell culture dishes. The medium was replaced with 1.5 ml RPMI 1640 medium containing 10% of charcoal-treated FBS with 50 nM Taxol containing 0.5 µCi [³H]Taxol. After incubation for 26 h, the medium was supplemented in some cases with 5 µM LPA or not as indicated and the incubation was continued for another 12 h. Media and cells were collected at the times indicated and Taxol was extracted. Results are expressed as the percentage of total radioactivity recovered in the cells and medium. Panel B, cells were treated and analyzed as above but after 26 h incubation, Taxol was removed and 5 µM LPA was added or not as indicated. The results are expressed as the percentage of the radioactivity added to the medium presented as mean ± S.D. (where large enough to be shown) for three independent experiments.</p

Morphology of Taxol-treated MCF-7 cells.

<p>MCF-7 cells were preincubated with 10% delipidated serum and 50 nM Taxol for 26 h. Cells were then stained with DAPI, anti-phospho-histone and with anti-tubulin. The appearance of the cell in mitosis (assessed by DAPI and P-histone staining) is shown in Panel A. Panel B shows cells that did not stain for P-histone and which were identified as being mononucleated, multinucleated or dead. Panel C shows examples of the appearance of cells that were identified as having abnormal spindles.</p

Involvement of LPA_1/3 and LPA₂ receptors in LPA-induced displacement of Taxol from polymerized tubulin in MCF-7 and MDA-MB-231 cells.

<p>MCF-7 (Panel A) and MDA-MB-231 cells (Panel B) were preincubated for 26 h with [³H]Taxol as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020608#pone-0020608-g007" target="_blank">Fig. 7</a>. After this, 5 µM LPA or 1 µM VPC51299, a LPA_1/3 receptor antagonist, was added as indicated and the incubations were continued for a further 12 h. Polymerized tubulin was then isolated and the distribution of [³H]Taxol was determined. Results are means ± SD for three independent experiments. *<i>P<0.05</i> comparing cells incubated in the absence of LPA with those incubated with LPA and/or the LPA_1/3 antagonist, VPC51299.</p

Differential expression of autotaxin and LPA_1-3 receptors in MCF-7, MDA-MB-231 and MDA-MB-468 breast cancer cells.

<p>Results show mRNA concentrations expressed relative to that for cyclophilin for (A) ATX (B) LPA₁ (C) LPA₂ and (D) LPA₃ in cells incubated for 26 h in the presence or absence of Taxol. Results are means ± SD from three independent experiments for ATX and LPA₁ and six independent experiments for LPA₂ and LPA₃. ^*<i>P</i><0.05 and ** p<0.01.</p