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. 2024 Sep;11(35):e2310126.
doi: 10.1002/advs.202310126. Epub 2024 Jul 23.

Targeting Transient Receptor Potential Melastatin-2 (TRPM2) Enhances Therapeutic Efficacy of Third Generation EGFR Inhibitors against EGFR Mutant Lung Cancer

Zhen Chen  1 , Karin A Vallega  1 , Vijay K Boda  2 , Zihan Quan  3 , Dongsheng Wang  1 , Songqing Fan  3 , Qiming Wang  4 , Suresh S Ramalingam  1 , Wei Li  2 , Shi-Yong Sun  1
Affiliations

Targeting Transient Receptor Potential Melastatin-2 (TRPM2) Enhances Therapeutic Efficacy of Third Generation EGFR Inhibitors against EGFR Mutant Lung Cancer

Zhen Chen et al. Adv Sci (Weinh). 2024 Sep.

Abstract

There is an urgent need to fully understand the biology of third generation EGFR-tyrosine kinase inhibitors (EGFR-TKIs), particularly osimertinib, and to develop mechanism-driven strategies to manage their acquired resistance. Transient receptor potential melastatin-2 (TRPM2) functions as an important regulator of Ca2+ influx, but its role in mediating therapeutic efficacies of EGFR-TKIs and acquired resistance to EGFR-TKIs has been rarely studied. This study has demonstrated a previously undiscovered role of suppression of TRPM2 and subsequent inhibition of Ca2+ influx and induction of ROS and DNA damage in mediating apoptosis induction and the therapeutic efficacy of osimertinib against EGFR mutant NSCLC. The rebound elevation represents a key mechanism accounting for the emergence of acquired resistance to osimertinib and other third generation EGFR-TKIs. Accordingly, targeting TRPM2 is a potentially promising strategy for overcoming and preventing acquired resistance to osimertinib, warranting further study in this direction including the development of cancer therapy-optimized TRPM2 inhibitors.

Keywords: EGFR‐TKIs; TRPM2; apoptosis; calcium; lung cancer; osimertinib.

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Conflict of interest statement

SSR is on consulting/advisory board for AstraZeneca, BMS, Merck, Roche, Tesaro and Amgen.

Figures

Figure 1
Figure 1
Osimertinib suppresses TRPM2 expression at both mRNA (A,B) and protein (C–G) levels accompanied with inhibition of Ca2+ influx (H) and induction of ROS (I) and DNA damage (J) in EGFRm NSCLC cell lines or tumors. A, RNA‐seq data generated from both PC‐9 and HCC827 cells exposed to DMSO or 100 nM osimertinib (Osim) for 14 h. B, RT‐qPCR detection of TRPM2 suppression by osimertinib in the indicated cell lines treated with DMSO or 100 nM osimertinib for 14 h. Each column is the mean ± SE of three independent experiments. Statistical differences were assessed with two‐sided unpaired Student's t‐test. C–E, The given cell lines were exposed to different concentrations of osimertinib as indicated for 24 h (C), 200 nM osimertinib for varied times as indicated (D), 200 nM indicated EGFR‐TKIs for 24 h (E). The proteins of interest were detected with Western blotting. F and G, TRPM2 in the indicated cell lines exposed to DMSO or 100 nM osimertinib for 24 h (F) or PC‐9 tumors treated with 15 mg kg−1 osimertinib for 9 days (G) was detected with IF staining. H, The given cell lines were exposed to DMSO or 200 nM osimertinib for 16 h and then subject to detection of Ca2+ influx using the Invitrogen™Fluo‐4 Direct™ Calcium Assay Kit. The cells were bathed in normal physiological saline HBSS and challenged with 3 mM H2O2 to induce a cytosolic Ca2+ rise. I and J, Both PC‐9 and HCC827 cells were exposed to 200 nM osimertinib for 24 h and then subject to detection of intracellular ROS generation with the H2DCFDA assay (I) and of γ‐H2AX foci using IF staining (J).
Figure 2
Figure 2
VDR is a predicted putative transcriptional factor within the TRPM2 5′‐flanking regulatory region (A), ranks top among genes suppressed by osimertinib in RNA‐seq analysis (B) and mediates TRPM2 downregulation by osimertinib in EGFRm NSCLC cells (C–F) via a VDRE present in the promoter region of TRPM2 (H–J) independent of c‐Myc (G). A, Putative transcriptional factors were predicted with PROMO program. B, Alterations in transcriptional factors in RNA‐seq data generated from both PC‐9 and HCC827 cells exposed to DMSO or 100 nM osimertinib (Osim) for 14 h are presented in the heatmap. C, Both PC‐9 and HCC827 cells were exposed to DMSO or 100 nM for the indicated times. D and G, The tested cell lines were transfected with the indicated siRNAs for 48 h. E, The indicated cell lines were infected with lentiviruses carrying VDR shRNA followed by puromycin selection. F, The indicated cell lines expressing vector (V) and VDR gene, respectively, were exposed to DMSO or 200 nM osimertinib for 16 h. After the aforementioned treatments, the proteins of interest were detected with Western blotting. H, Reporter constructs harboring the TRPM2 promoter region and deleted regions. I, The indicated cell lines were transfected with the given reporter constructs for 24 h followed with 200 nM osimertinib for another 16 h. Cells were then harvested for luciferase assay. The data are the means ± SD of four replicate determinations. Statistical differences were assessed with two‐sided unpaired Student's t‐test. J, The indicated biotin‐oligos were incubated from whole‐cell protein lysates prepared from the indicated cell lines treated with DMSO or 200 nM osimertinib for 16 h. Pulldown assay was conducted with the streptavidin‐agarose beads followed by Western blotting to detect VDR bound to the oligos. Scr, scramble oligo; wt, VDRE‐wt oligo.
Figure 3
Figure 3
Enforced expression of ectopic TRPM2 gene in EGFRm NSCLC cell lines attenuates the ability of osimertinib to induce apoptosis (A,B), decrease cell survival (C), promote ROS generation (D), cause DNA damage (D) and inhibit tumor growth (E–G). A‐D, The indicated cell lines expressing vector (V) or TRPM2 gene were exposed to DMSO or 200 nM osimertinib for 24 h (A), 48 h (B), 72 h (C), or 16 h (D). The proteins of interest were detected with Western blotting (A). Annexin V‐positive cells were determined with flow cytometry (B). Cell numbers were estimated with the SRB assay (C). ROS generation and DNA damage were detected with H2DCFDA and γ‐H2AX foci assays, respectively. The data are means ± SDs of triplicate (B) or four replicate determinations (C). CF, cleaved form. E‐G, Mice inoculated with the indicated cell lines were treated with vehicle or osimertinib (5 mg kg−1, og, daily). Tumor sizes were measured at the indicated times (E) and photographed at the end of the treatments (F). Tumor weights were recorded at the end of the treatment (G). The data are means ± SEs (n = 6). Statistical differences were assessed with two‐sided unpaired Student's t‐test.
Figure 4
Figure 4
TRPM2 expression is elevated at both mRNA (A,B) and protein (C,D) levels in EGFRm NSCLC cell lines with osimertinib acquired resistance, which exhibit increased Ca2+ influx (E), and in the majority of EGFRm NSCLC tissues after relapse from EGFR‐TKI treatment (F,G) A, Heatmap for the expression of TRPM2 and other‐related genes from RNA‐seq data between PC‐9 and PC‐9/AR cells. B, RT‐qPCR detection of TRPM2 mRNA levels in the indicated cell lines. The data are means ± SDs of four replicate determinations. Statistical differences were assessed with two‐sided unpaired Student's t‐test. C and D, Western blotting detection of TRPM2 protein in the indicated cell lines with or without exposure to different concentrations of osimertinib (Osim) as indicated for 16 h. E, Detection of Ca2+ influx in the indicated cell lines using the Invitrogen™Fluo‐4 Direct™ Calcium Assay Kit. The cells were bathed in normal physiological saline HBSS and challenged with 3 mM H2O2 to induce a cytosolic Ca2+ rise. F and G, TRPM2 in human EGFRm NSCLC issues were detected with IHC (F) and the representative IHC pictures are shown (G).
Figure 5
Figure 5
Genetic suppression of TRPM2 expression via gene knockdown in osimertinib‐resistant cells restores their responses to osimertinib in inducing apoptosis (A,B), decreasing cell survival (C) and enhancing suppression of tumor growth (D–F) with augmented effects on decreasing Ki‐67 and inducing PARP cleavage (G) in vivo. A‐C, The indicated cell lines expressing pLKO.1 or shTRPM2 were exposed to DMSO or 200 nM osimertinib (Osim) for 24 h (A), 48 h (B) or 72 h (C). The proteins of interest were detected with Western blotting (A). Annexin V‐positive cells were determined with flow cytometry (B). Cell numbers were estimated with the SRB assay (C). The data are means ± SDs of triplicate (B) or four replicate (C) determinations. CF, cleaved form. D‐F, Mice inoculated with the indicated cell lines were treated with vehicle or osimertinib (5 mg kg−1, og, daily). Tumor sizes were measured at the indicated times (D) and photographed at the end of the treatments (E). Tumor weights were recorded at the end of the treatment as well (F). The data are means ± SEs (n = 6). G, Ki‐67 and cPARP in the indicated tissues were detected with IHC. NS, not significant. Statistical differences were assessed with two‐sided unpaired Student's t‐test.
Figure 6
Figure 6
D9 in combination with osimertinib synergistically decreases cell survival (A), inhibits colony formation and growth (B), induces apoptosis (C,D), reduces Ca2+ influx (E), and induces ROS production and DNA damage (F) in osimertinib‐resistant EGFRm NSCLC cell lines and augments the growth inhibition of osimertinib‐resistant tumors in vivo, as does the combination of ACA and osimertinib (G,H). A, The given cell lines were treated with varied concentrations of the tested agents either alone or in combinations for 3 days. Cell numbers were then measured by the SRB assay (A) and CIs were then calculated and presented inside the graphs. The data are means ± SDs of four replicate determinations. B, The tested cell lines seeded in 12‐well plates were treated with 50 nM osimertinib, 250 nM D9, or their combination, which were repeated with fresh medium every 3 days. After 10 days, the cells were fixed, stained with crystal violet dye, imaged, and counted. Columns are means ± SDs of triplicate determinations. C and D, The tested cell lines were exposed to 200 nM osimertinib, 2.5 µM D9, or their combination for 36 h (C) or 48 h (D). The proteins of interest were detected with Western blotting (C) and apoptotic cells were detected with annexin V staining/flow cytometry (D). Each column represents mean ± SD of triplicate treatments. E, The indicated cell lines exposed to DMSO, 100 nM osimertinib, 2.5 µM D9, or the combination of osimertinib and D9 for 36 h were bathed in normal physiological saline HBSS and challenged with 2.5 mM H2O2 to induce a cytosolic Ca2+ rise followed with the detection of Ca2+ influx using the Invitrogen™Fluo‐4 Direct™ Calcium Assay Kit. F, HCC827/AR cells were exposed to DMSO, 100 nM osimertinib, 2.5 µM D9, or the combination of osimertinib and D9 for 36 h and then assayed for ROS generation with H2DCFDA dye and for p‐H2AX foci formation by p‐H2AX staining. G and H, Mice inoculated with the indicated cell lines were treated with vehicle, osimertinib (5 mg kg−1, og, daily), D9 (25 mg kg−1, ip, daily), or the combination of osimertinib and D9. Tumor sizes were measured at the indicated times (G) and photographed at the end of the treatments. Tumor weights were recorded at the end of the treatment (H). The data are means ± SEs (n = 6). *, P < 0.05; **, P < 0.01; ***, P < 0.001. Statistical differences were evaluated with one‐way ANOVA test.
Figure 7
Figure 7
Osimertinib in combination with D9 synergistically decreases the survival of EGFRm NSCLC cell lines with primary resistance to osimertinib that possess elevated TRPM2 expression (A), eliminates DTCs (B) and regresses different EGFRm PDX tumors in vivo with long‐term remissions (D–F). A, Detection of TRPM2 basal levels in the indicated cell lines with Western blotting. B and C, The given cell lines were exposed to varied concentrations of osimertinib (Osim), D9 alone as indicated and their combination for 3 days. Cell numbers were then determined with the SRB assay and CIs were calculated (C). The data are means ± SDs of four replicate determinations. D, The indicated cell lines seeded in 12‐well plates were treated with 50 nM osimertinib, 250 nM D9, or their combination; these treatments were repeated with fresh medium every 2 days. After 5 or 10 days, the cells were fixed, stained with crystal violet dye and imaged. E‐G, The indicated PDXs in nude mice (6 tumors per group) were treated with vehicle, 5 mg kg−1 osimertinib (daily, og), 25 mg kg−1 D9 (daily, ip) or their combination. Tumor growth curves for each tumor are presented.
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