Mutant PIK3CA as a negative predictive biomarker for treatment with a highly selective PIM1 inhibitor in human colon cancer

Abstract

Significant improvement in targeted therapy for colorectal cancer (CRC) has occurred over the past few decades since the approval of the EGFR inhibitor cetuximab. However, cetuximab is used only for patients possessing the wild-type oncogene KRAS, NRAS, and BRAF, and even most of these eventually acquire therapeutic resistance, via activation of parallel oncogenic pathways such as RAS-MAPK or PI3K/Akt/mTOR. The two aforementioned pathways also contribute to the development of therapeutic resistance in CRC patients, due to compensatory and feedback mechanisms. Therefore, combination drug therapies (versus monotherapy) targeting these multiple pathways may be necessary for further efficacy against CRC. In this study, we identified PIK3CA mutant (PIK3CA MT) as a determinant of resistance to SMI-4a, a highly selective PIM1 kinase inhibitor, in CRC cell lines. In CRC cell lines, SMI-4a showed its effect only in PIK3CA wild type (PIK3CA WT) cell lines, while PIK3CA MT cells did not respond to SMI-4a in cell death assays. In vivo xenograft and PDX experiments confirmed that PIK3CA MT is responsible for the resistance to SMI-4a. Inhibition of PIK3CA MT by PI3K inhibitors restored SMI-4a sensitivity in PIK3CA MT CRC cell lines. Taken together, these results demonstrate that sensitivity to SMI-4a is determined by the PIK3CA genotype and that co-targeting of PI3K and PIM1 in PIK3CA MT CRC patients could be a promising and novel therapeutic approach for refractory CRC patients.

Keywords: PIK3CA; PIM1; colorectal cancer; mutant KRAS; predictive marker.

Figure 1.

Cell death efficacy of SMI-4a against various CRC cell lines. Cell death rates evaluated by trypan blue exclusion after 72 h treatment with 30 μM SMI-4a in nine CRC cell lines (top). Expression of PIM kinases by Western blot and RT-PCR (middle) was conducted to analyze the genotype of major oncogenic mutation in CRC cell lines (bottom). Experiments were conducted three times in triplicate.

Figure 2.

PIK3CA mutation as a negative predictive biomarker of SMI-4a treatment in CRC cell lines. (a) Colony-forming assays of SW620 (PIK3CA WT) and HCT116 (PIK3CA MT) after 30 μM of SMI-4a treatment. Cells were continuously exposed to SMI-4a for 16 (SW620) to 18 (HCT116) days. Colonies were stained with crystal violet and the number of colonies counted. (left) Representative image of colonies. (right) Quantification of colonies represented as relative colony formation ratios (%). ***p < .001 indicates significantly different from control group. (b) Annexin V-FITC/PI staining in SW620 (PIK3CA WT) and HCT116 (PIK3CA MT) after 30 μM SMI-4a treatment for 72 h. (left) Representative flow cytometry plots of annexin V-FITC/PI-stained apoptotic cells. (right) the percentages of apoptotic cells were statistically compared, with significant differences denoted by ***p < .001. (c) Expression of PI3K downstream effectors (left) and downstream signaling molecules related to cell proliferation and apoptosis (right) in SW480 (PIK3CA WT) and DLD-1 (PIK3CA MT) cell lines treated with 30 μM SMI-4a. (d) Cell death rates evaluated by trypan blue exclusion after 72 h SMI-4a (30 μM) treatment in DLD-1 PIK3CA WT (351) vs. MT (353) isogenic cell lines. ***p < .001 indicates significantly different from WT group. Experiments were repeated three times in triplicate.

Figure 3.

Overexpression of mt PIK3CA induces resistance to SMI-4a treatment. PIK3CA-E547K and PIK3CA-H1047R were overexpressed in four (COLO 320HSR, COLO 205, SW620, and SW480) PIK3CA WT CRC cell lines, and cell death rates evaluated after 72 h of 30 μM SMI-4a treatment, by trypan blue exclusion assay. ***p < .001 indicates significantly different from control group. Experiments were repeated three times in triplicate.

Figure 4.

Sensitivity to SMI-4a is determined by PIK3CA genotype in in vivo tumor xenograft and PDX models. (a) COLO 205 and HT-29 ×enografts were subcutaneously infected into nude mice and 50- and 100-mg/kg SMI-4a administered daily by P.O. injection. Representative images of tumors and relative tumor sizes in COLO 205 (top) and HT-29 (bottom) xenograft models. (b) Colon tumors from patients grown in nude mice followed by 50- or 100-mg/kg SMI-4a injected daily by oral gavage. Representative images of tumors and relative tumor sizes in PIK3CA WT (left) and PIK3CA MT (right) mouse models. *p < 0.05 and ***p < 0.001 indicate significantly different from control group, respectively.

Figure 5.

Combinational effect of SMI-4a and PIK3CA inhibitors on CRC cell lines. (a) DLD-1 (PIK3CA MT) cells were treated with the PIK3CA inhibitors BEZ235, BKM120, and GDC-0941 for 72 h, and cell death rates evaluated by trypan blue exclusion. (b) Western blot analysis after BKM120 and/or SMI-4a in DLD-1 cells treated with increasing doses of BKM120 and/or SMI-4a for 48 h and lysed with RIPA buffer. Lysates were subjected to western blot analysis and expression levels of p-Akt, p-STAT3, and STAT3 was anlalyzed. Beta-actin was used as a loading control. (c) HCT116 (PIK3CA MT), DLD-1 (PIK3CA MT), SW480 (PIK3CA WT), and SW620 (PIK3CA WT) CRC cell lines were treated with increasing doses of BKM120 and/or 30 μM SMI-4a for 72 h. Cell death rates were evaluated by trypan blue exclusion assay. (d) DLD-1 (PIK3CA MT) isogenic cell line (353) was treated with 2 μM of BKM120 and/or 30 μM SMI-4a for 72 h. Cell death rates were evaluated by trypan blue exclusion assay. *p < .05, **p < .01, and ***p < .001 indicate significantly different from control group, respectively. Experiments were conducted three times in triplicate.

Figure 6.

Schematic illustration of mutant PIK3CA genotype as a negative predictive biomarker of SMI-4a efficacy against PIK3CA mutated CRC.