ATM deficiency promotes progression of CRPC by enhancing Warburg effect

in Endocrine-Related Cancer
Correspondence should be addressed to H Hu or C Liang or J Huang: hailiang.hu@duke.edu or liang_chaozhao@163.com or jiaoti.huang@duke.edu
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ATM is a well-known master regulator of double strand break (DSB) DNA repair and the defective DNA repair has been therapeutically exploited to develop PARP inhibitors based on the synthetic lethality strategy. ATM mutation is found with increased prevalence in advanced metastatic castration-resistant prostate cancer (mCRPC). However, the molecular mechanisms underlying ATM mutation-driving disease progression are still largely unknown. Here, we report that ATM mutation contributes to the CRPC progression through a metabolic rather than DNA repair mechanism. We showed that ATM deficiency generated by CRISPR/Cas9 editing promoted CRPC cell proliferation and xenograft tumor growth. ATM deficiency altered cellular metabolism and enhanced Warburg effect in CRPC cells. We demonstrated that ATM deficiency shunted the glucose flux to aerobic glycolysis by upregulating LDHA expression, which generated more lactate and produced less mitochondrial ROS to promote CRPC cell growth. Inhibition of LDHA by siRNA or inhibitor FX11 generated less lactate and accumulated more ROS in ATM-deficient CRPC cells and therefore potentiated the cell death of ATM-deficient CRPC cells. These findings suggest a new therapeutic strategy for ATM-mutant CRPC patients by targeting LDHA-mediated glycolysis metabolism, which might be effective for the PARP inhibitor resistant mCRPC tumors.

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    ATM deficiency promotes CRPC progression. (A and B) Cell proliferation (left panel) and colony formation (right panel) of C4-2/Ctrl vs C4-2/ATM-KO cells and CWRR1/Ctrl vs CWRR1/ATM-KO cells generated by CRISPR/Cas9 technology. Top: nuclear lysates were extracted and ATM expression was determined by Western blot. p84 was used as a loading control. Bottom: cell proliferation was measured by MTS assay at each indicated time points and normalized to Day 0 (n = 3 replicates). Colony formation assay was performed for C4-2/Ctrl vs C4-2/ATM-KO cells and CWRR1/Ctrl vs CWRR1/ATM-KO cells. Top: representative images for formed colonies. Bottom: graphs showing the quantifications of colonies formed (n = 3 replicates). (C) Xenograft tumor growth of C4-2 stable cell lines generated in (A). Tumor volumes were measured twice a week and normalized to Day 0 (n = 5 mice per group). (D) Representative tumors in each group. (E) The weight of xenograft tumors obtained at the end of animal study (n = 5 mice per group). (F) Representative images of H&E and IHC analysis of CD31, Ki-67 and Snail in xenografts obtained from (E). Magnification: 200×. (G) A plot of Quick-score for IHC staining in (F). All data are depicted as mean ± s.d. *P < 0.05 and **P < 0.01 by two-tailed Student’s t-test.

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    ATM deficiency leads to an enhanced glycolytic utilization in CRPC cells. (A) Heat map to show levels of global metabolites in C4-2/Ctrl and C4-2/ATM-KO cells (n = 3 biological replicates). (B) Principal component analysis (PCA) of metabolic distributions in the indicated cell lines. (C) Enriched glucose-related metabolite pathways in C4-2/ATM-KO cells. Statistically significant differences were assessed by MetaboAnalyst software as indicated in the Materials and Methods. (D) LC-MS analysis of cell extractions to compare the levels of glycolytic intermediates between C4-2/Ctrl and C4-2/ATM-KO cells (n = 3). 1,3BPG, 1,3-bisphosphoglycerate; 3PG, 3-phosphoglycerate; F1,6P, fructose 1,6-bisphosphate; F6P, fructose 6-phosphate; G6P, glucose 6-phosphate; Lac, lactate; pPy, phosphoenolpyruvate; Py, pyruvate. (E and F) Glucose consumption and lactate production in paired C4-2/Ctrl and C4-2/ATM-KO, CWRR1/Ctrl and CWRR1/ATM-KO cells. Glucose consumption was calculated as indicated in the Materials and Methods. Results are normalized to the control group (n = 3). All data are depicted as mean ± s.d. *P < 0.05 and **P < 0.01 by two-tailed Student’s t-test.

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    ATM deficiency detours the glucose flux in CRPC cells. C4-2/Ctrl and C4-2/ATM-KO cells were grown in uniformly 13C-labeled glucose medium. The incorporation of 13C atoms from 13C6-Glc in to citrate/isocitrate, α-ketoglutarate (α-KG), succinate, fumarate, malate, lactate, alanine and aspartate are denoted as m + n, where n is the number of 13C atoms. Blue, red, purple and green circles all indicate that 13C atoms are derived from 13C6-Glc but through different pathways. A scheme describing glucose flows is shown in the middle of the figure. CO2 indicates where carbon dioxide is released; GPT2, glutamate-pyruvate transaminase; LDHA, lactate dehydrogenase A; PC, pyruvate carboxylase. All data are depicted as mean ± s.d. *P < 0.05 and **P < 0.01 by two-tailed Student’s t-test.

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    ATM deficiency upregulates LDHA to promote CRPC progression. (A) 13C-isotopomer tracing analysis of glucose-derived glycolytic metabolites in C4-2/Ctrl and C4-2/ATM-KO cells (n = 3). 1,3BPG, 1,3-bisphosphoglycerate; 3PG, 3-phosphoglycerate; F1,6 P, fructose 1,6-bisphosphate; F6P, fructose 6-phosphate; G6P, glucose 6-phosphate; Lac, lactate; pPy, phosphoenolpyruvate; Py, pyruvate. (B) RT-qPCR to determine the mRNA levels of glycolytic enzymes. Data are normalized to the control group (n = 3 independent experiments). HK, hexokinase; LDHA, lactate dehydrogenase A; PFK, phosphofructokinase; PGK, phosphoglycerate kinase; PK, pyruvate kinase. (C) Western blot to determine LDHA protein expression. GAPDH was used as a loading control. (D) Relative level of intracellular ROS. Results are normalized to control group (n = 3 independent experiments). (E) C4-2/Ctrl and C4-2/ATM-KO cells were transfected with siRNAs targeting either a scramble sequence (siCtrl) or LDHA (siLDHA). Western blot confirmed knockdown of LDHA expression by using siRNAs. GAPDH was used as a loading control. (F) Intracellular ROS production was detected with DCFDA fluorescence and monitored by flow cytometry at 24 h post-transfection with siCtrl or siLDHA. Left: median fluorescent intensity. Right: the representative image of histograms of ROS (n = 3 independent experiments). (G) Cell viability of C4-2/Ctrl and C4-2/ATM-KO cells transfected with siCtrl and siLDHA was determined by MTS assay at the indicated time points (n = 3 replicates). All data are depicted as mean ± s.d. *P < 0.05 and **P < 0.01 by two-tailed Student’s t-test.

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    LDHA inhibitor FX11 suppresses ATM-deficient CRPC cell growth. (A) IC50 values of FX11 were measured on C4-2/Ctrl and C4-2/ATM-KO cells treated with a series concentration of FX11 for 72 h. (B) Lactate production of C4-2/Ctrl and C4-2/ATM-KO cells treated with either DMSO or FX11 for 72 h. Results are normalized to DMSO group and set as ‘1’. (C) Intracellular ROS production was detected with DCFDA fluorescence and monitored by flow cytometry at 72 h post-treatment with either DMSO or 3 μM FX11. Left: median fluorescent intensity. Right: the representative image of histograms of ROS (n = 3 independent experiments). (D) Cell viability of C4-2/Ctrl and C4-2/ATM-KO cells treated with either DMSO or FX11 was determined by MTS assay at the indicated time points (n = 3 replicates). (E and F) Tumor growth of C4-2/Ctrl and C4-2/ATM-KO xenografts treated intraperitoneally with either DMSO or FX11 when tumor reached ~200 mm3. Tumor volumes were measured every 4 days. Top: Representative tumor images in each group. Bottom: The growth curve of xenograft tumors after treatment of vehicle (DMSO) or FX11. All data are depicted as mean ± s.d. *P < 0.05 and **P < .01 by two-tailed Student’s t-test.

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    ATM deficiency upregulates LDHA expression via enhancing c-Myc level. (A) Western blot to determine c-Myc protein expression in C4-2/Ctrl and C4-2/ATM-KO cells. GAPDH was used as a loading control. (B and C) C4-2/Ctrl and C4-2/ATM-KO cells were transfected with siRNAs targeting either a scramble sequence (siCtrl) or c-Myc (sic-Myc). RT-qPCR confirmed that LDHA mRNA was downregulated after c-Myc knockdown in both C4-2/Ctrl and C4-2/ATM-KO cells (B) and Western blot confirmed knockdown of c-Myc expression by siRNAs and the subsequent downregulation of LDHA protein expression (C). GAPDH was used as a loading control. (D) A working model of up-regulated LDHA contributing to the progression of ATM-deficient CRPC.

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