Positive feedback between oncogenic KRAS and HIF-1α confers drug resistance in colorectal cancer

Abstract

Approximately 30%-50% of colorectal cancers (CRCs) harbor the somatic mutated KRAS gene. KRAS G12V, one of the most common KRAS mutations in CRCs, is linked to increased tumor aggressiveness, less response to anti-epidermal growth factor receptor (EGFR) therapy, and poor survival rate. In this study, we sought to determine whether resistance to EGFR inhibitors in colorectal cancer cells harboring KRAS G12V mutation is associated with hypoxia. Our data indicated that HIF-1α was induced by KRAS G12V signaling at transcription level. Hypoxia or HIF-1α overexpression could increase KRAS G12V activity. Therefore, a positive feedback between hypoxia and KRAS G12V activation was formed. Cetuximab, an EGFR inhibitor, which has a minor effect on KRAS-mutant CRCs, could effectively inhibit the proliferation of CRC cells harboring KRAS G12V mutation when combined with HIF-1α inhibitor PX-478. Our data indicated that hypoxia was involved in resistance to anti-EGFR therapy, and a combination therapy might be necessary for CRC patients with KRAS mutation.

Keywords: EGFR inhibitor resistance; KRAS G12V; combination therapy.

Figure 1

Induction of HIF-1α by KRAS G12V signaling in CRC cells. Notes: (A) Five CRC cell lines were checked for HIF-1α expression level by Western blot: 1) HT29; 2) CaCo2; 3) Colo205; 4) HTC116; and 5) SW480. (B) Colon cancer cell lines HT29 and CaCo2 transduced with v or G12V retroviruses (pBabe-puro). Cells were then selected with 5 μg/mL puromycin to obtain stably expressing KRAS G12V cells. These cells were confirmed by Western blot using KRAS antibody. HIF-1α expressions were compared between control and KRAS G12V overexpressed cells by Western blot (left panel) or by real-time PCR (right panel). *P<0.05. (C) PI3K/AKT and MAPK signaling pathways were analyzed by detecting pAKT and pERK levels in control or KRAS G12V-expressing cells. (D) HT29 cells were transfected with two siRNAs (si-1 and si-2) targeting 3′-UTR of KRAS; knockdown effect was analyzed by Western blot (top panel). Wild-type KRAS and KRAS G12V were overexpressed in HIF-1α knocked-down HT29 cells and HIF-1α level was analyzed by Western blot (bottom panel). (E) KRAS was knocked down by siRNA in SW480 cells, and HIF-1α expression was examined by real-time PCR (top panel) and Western blot (bottom panel). *P<0.05. Abbreviations: C-KRAS, CaCo2 cells with KRAS G12V overexpression; CRC, colorectal cancer; C-v, CaCo2 control cells; G12V, KRAS G12V; H-KRAS, HT29 cells with KRAS G12V overexpression; H-v, HT29 control cells; NC, non-targeting control; PCR, polymerase chain reaction; si-1, KRAS siRNA 1; si-2, KRAS siRNA 2; v, control.

Figure 2

Hypoxia activates RAS and its downstream signaling. Notes: (A) Both HT29 and CaCo2 cells were cultured in N or H conditions for 24 hours, and then cell lysates were collected to examine GTP-bound RAS, pAKT, and pERK levels using corresponding specific antibodies. (B) HT29 and CaCo2 cells were transfected with v or H1α expression plasmids. Forty-eight hours after transfection, cell lysates were collected to detect HIF-1α, RAS-GTP, pAKT, and pERK. Abbreviations: H, hypoxic; H1α, HIF-1α; N, normoxic; v, control.

Figure 3

HIF-1α inhibitor sensitized KRAS G12V CRC cells to cetuximab treatment. Notes: (A) Control or KRAS G12V-overexpressed CaCo2 cells were treated with or without 200 nM cetuximab for 48 hours. HIF-1α protein level was detected by Western blot. (B) Cells were the same as those in (A) but were treated with or without cetuximab. Cell proliferation was analyzed at the time points indicated by MTT. (C) Control CaCo2 or KRAS G12V overexpressed cells were treated with cetuximab or PX-478 individually or in combination. Cell proliferation was analyzed at the indicated time. Cell lysates were obtained after 48 hours of treatment to examine HIF-1α expression. (D) KRAS was knocked down in SW480 cells and the cells with/without cetuximab treatment were examined for proliferation rate by MTT at the indicated time points. HIF-1α level was checked by Western blot. (E) CaCo2 cells were cultured under normoxic or hypoxic conditions for 24 hours before cetuximab treatment. Then, proliferation rate was examined by MTT. *P<0.05. Images are representative of three independent experiments. Abbreviations: C+P, combination treatment with cetuximab and PX-478; ce, cetuximab treatment; CRC, colorectal cancer; ctrl, control treatment; KRAS, CaCo2 cells with KRAS G12V overexpression; KRAS-ce, cells with KRAS G12V mutation treated with cetuximab; NC, nonspecific siRNA control; px, PX-478 treatment; si, siRNA targeting HIF-1α; v, CaCo2 cells with vector control; v-ce, control cells treated with cetuximab.

Figure 4

Combination of HIF-1α and EGFR inhibitor effectively inhibits the reproductive ability of CRC cells. Notes: (A) Colon cancer patient samples carrying WT KRAS or KRAS G12V were obtained from Beijing Chao-Yang Hospital, Beijing, People’s Republic of China. The samples were fixed with 4% paraformaldehyde and then subjected to immunohistochemistry staining for HIF-1α. Brown color indicated positive staining. (B) SW480 cells treated with ctrl, ce, px, or C+P. After 8 hours of treatment, cells were counted and seeded at 200 cells per well in a six-well plate. Each treatment were triplicated. Fifteen days later when there were visible colonies, these cells were stained with 0.5% crystal violet. The colonies were counted and quantified. *P<0.05. Abbreviations: C+P, ce plus px; ce, cetuximab; CRC, colorectal cancer; ctrl, control; EGFR, epidermal growth factor receptor; px, PX-478; WT, wild-type.