p53-armed oncolytic adenovirus induces autophagy and apoptosis in KRAS and BRAF-mutant colorectal cancer cells

Abstract

Colorectal cancer (CRC) cells harboring KRAS or BRAF mutations show a more-malignant phenotype than cells with wild-type KRAS and BRAF. KRAS/BRAF-wild-type CRCs are sensitive to epidermal growth factor receptor (EGFR)-targeting agents, whereas KRAS/BRAF-mutant CRCs are resistant due to constitutive activation of the EGFR-downstream KRAS/BRAF signaling pathway. Novel therapeutic strategies to treat KRAS/BRAF mutant CRC cells are thus needed. We recently demonstrated that the telomerase-specific replication-competent oncolytic adenoviruses OBP-301 and p53-armed OBP-702 exhibit therapeutic potential against KRAS-mutant human pancreatic cancer cells. In this study, we evaluated the therapeutic potential of OBP-301 and OBP-702 against human CRC cells with differing KRAS/BRAF status. Human CRC cells with wild-type KRAS/BRAF (SW48, Colo320DM, CACO-2), mutant KRAS (DLD-1, SW620, HCT116), and mutant BRAF (RKO, HT29, COLO205) were used in this study. The antitumor effect of OBP-301 and OBP-702 against CRC cells was analyzed using the XTT assay. Virus-mediated modulation of apoptosis, autophagy, and the EGFR-MEK-ERK and AKT-mTOR signaling pathways was analyzed by Western blotting. Wild-type and KRAS-mutant CRC cells were sensitive to OBP-301 and OBP-702, whereas BRAF-mutant CRC cells were sensitive to OBP-702 but resistant to OBP-301. Western blot analysis demonstrated that OBP-301 induced autophagy and that OBP-702 induced autophagy and apoptosis in human CRC cells. In BRAF-mutant CRC cells, OBP-301 and OBP-702 suppressed the expression of EGFR, MEK, ERK, and AKT proteins, whereas mTOR expression was suppressed only by OBP-702. Our results suggest that p53-armed oncolytic virotherapy is a viable therapeutic option for treating KRAS/BRAF-mutant CRC cells via induction of autophagy and apoptosis.

Fig 1. Structures of telomerase-specific replication-competent oncolytic adenoviruses.

A OBP-301 is a telomerase-specific replication-competent oncolytic adenovirus in which the hTERT promoter drives expression of the E1A and E1B genes. B OBP-702 is a p53-armed OBP-301 variant in which the Egr1 promoter drives expression of the human wild-type p53 gene. Ad5, adenovirus serotype 5; hTERT, human telomerase reverse transcriptase; IRES, internal ribosome entry site; ITR, inverted terminal repeat.

Fig 2. OBP-301 reduces the viability of human CRC cells with wild-type and mutant KRAS.

A, B, C Human CRC cells with different KRAS/BRAF mutation status, KRAS/BRAF wild type (A), mutant KRAS (B), and mutant BRAF (C), were treated with OBP-301 at the indicated multiplicity of infection (MOI) for 72 h. Cell viability was quantified using the XTT assay. Uninfected (mock-treated) cells were shown as virus-infected cells at an MOI of 0. Cell viability was calculated relative to that of the mock-treated group, which was set at 100%. Cell viability data are expressed as mean ± SD (n = 5). The Student’s t-test was used to evaluate the significance of differences. *P<0.05; **P<0.01 (versus an MOI of 0).

Fig 3. OBP-301 induces autophagy in human CRC cells.

A, B, C Human CRC cells with different KRAS/BRAF mutation status, KRAS/BRAF wild type (A), mutant KRAS (B), and mutant BRAF (C), were treated with OBP-301 at the indicated MOI for 72 h. Cell lysates were prepared and subjected to Western blot analysis of PARP, cleaved PARP (C-PARP), p62, E1A, and p53 expression. β-Actin was assayed as a loading control. Uninfected (mock-infected) cells were shown as virus-infected cells at an MOI of 0.

Fig 4. OBP-702 reduces the viability of human CRC cells independent of KRAS/BRAF mutation status.

A, B, C Human CRC cells with different KRAS/BRAF mutation status, KRAS/BRAF wild type (A), mutant KRAS (B), and mutant BRAF (C), were treated with OBP-702 at the indicated MOI for 72 h. Cell viability was quantified using the XTT assay. Uninfected (mock-treated) cells were shown as virus-infected cells at an MOI of 0. Cell viability was calculated relative to that of the mock-treated group, which was set at 100%. Cell viability data are expressed as mean ± SD (n = 5). The Student’s t-test was used to evaluate the significance of differences. *P<0.05; **P<0.01 (versus an MOI of 0).

Fig 5. OBP-702 induces apoptosis and autophagy in human CRC cells.

A, B, C Human CRC cells with different KRAS/BRAF mutation status, KRAS/BRAF wild type (A), mutant KRAS (B), and mutant BRAF (C), were treated with OBP-702 at the indicated MOI for 72 h. Cell lysates were prepared and subjected to Western blot analysis of PARP, cleaved PARP (C-PARP), p62, E1A, and p53 expression. β-Actin was assayed as a loading control. Uninfected (mock-infected) cells were shown as virus-infected cells at an MOI of 0.

Fig 6. OBP-702 suppresses the EGFR-MEK-ERK and AKT-mTOR signaling pathways in BRAF-mutant CRC cells.

A BRAF-mutant HT29 cells were treated with OBP-301 or OBP-702 at the indicated MOI for 72 h. Cell lysates were prepared and subjected to Western blot analysis of EGFR, MEK, ERK, AKT, and mTOR expression. B BRAF-mutant HT29 cells were treated with OBP-301, DL312, or Ad-p53 at the indicated MOI for 72 h. Cell lysates were prepared and subjected to Western blot analysis of PARP, C-PARP, p62, E1A, p53, EGFR, MEK, ERK, AKT, and mTOR expression. β-Actin was assayed as a loading control. Uninfected (mock-infected) cells were shown as virus-infected cells at an MOI of 0. C Outline of the EGFR-MEK-ERK and AKT-mTOR signaling pathways in BRAF-mutant CRC cells infected with OBP-301 or OBP-702.