FOXA1, induced by RC48, regulates HER2 transcription to enhance the tumorigenic capacity of lung cancer through PI3K/AKT pathway

Abstract

Lung cancer remains the tumor with the highest global incidence and mortality rates. Current primary treatment modalities encompass targeted therapy, immunotherapy, and chemotherapy; however, a subset of patients derives no benefit from these interventions. Recently, the HER2-targeting antibody drug Disitamab vedotin (RC48) was approved and introduced primarily for gastric and bladder cancers, with minimal investigation in the field of lung cancer. This study demonstrates that FOXA1 directly binds to the promoter region of HER2, influencing the HER2/PI3K/AKT signaling pathway, which consequently modulates factors that foster lung cancer proliferation and impede apoptosis. Unlike FOXA1, HER2 does not influence the expression of FOXA1. Intriguingly, in lung cancer cells, RC48 not only impacts the HER2/PI3K/AKT pathway but also affects the FOXA1/HER2/PI3K/AKT pathway, thereby exerting a robust antitumor effect. In clinical specimens, heightened expressions of FOXA1 and HER2 correlate positively with clinical progression and poorer prognosis. These findings suggest that FOXA1 may serve as a potential biomarker or therapeutic target in future non-small cell lung cancer (NSCLC) treatments, and ongoing research may position RC48 as a transformative agent in lung cancer therapy.

Keywords: FOXA1; HER2; RC48; apoptosis.; proliferation.

Figure 1

Expression of FOXA1 gene in para-cancerous tissues (PT) and NSCLC. (A) mRNA expression of FOXA1 is decreased in PT compared with NSCLC tissues by real-time PCR assay. Data are presented were presented as mean ± SD for three independent experiments (***P<0.001). (B) Western blot analysis of FOXA1 expression in 11 cases of NSCLC tissue samples compared with 4 PT tissues. (C) FOXA1 protein was expressed in NSCLC and PT samples (original magnification: 400X). A. a). Negative staining of FOXA1 in PT. b). Negative staining of FOXA1 in NSCLC. c) Weak staining of FOXA1 in NSCLC. d). Strong cytoplasmic FOXA1 staining in NSCLC. (D) Expression of FOXA1 protein predicts overall survival of NSCLC patients. Patients with positive FOXA1 had worse survival than those with negative FOXA1 (P = 0.006).

Figure 2

FOXA1 promotes tumor cell proliferation and apoptosis. (A) FOXA1 expression levels in A549 cell with knockdown of FOXA1 detected by qRT-PCR. (B) CCK-8 assay analyzed the effect of silencing FOXA1 on cell viability. (C) Cell cycle of A549 cell transfected with FOXA1 inhibitors (n = 3). (D) Colony formation assay analyzed the effect of silencing FOXA1 on cell proliferation. (E) A549 cell apoptosis after si-FOXA1 treatment detected by fow cytometry. (F) Enrichment of cell cycle-related and apoptosis-associated proteins in the cotransfected cells was determined using western blotting. Student's t-test. mean± SD (*P <0.05).

Figure 3

FOXA1 binds to a specific sequence within the HER2 gene promoter. (A) The ENCODE project (https://www.encodeproject.org/,accessed on 1 March 2024), TRANSFAC and the TFFFIND search tool from the Piptools package were used to identify segments of the HER2 proximal gene promoter in NSCLC cells that contained binding sequences for FOXA1. Five such sequences were identified (sequence logo for the FOXA1 consensus motif shown, accessed on 1 March 2024), of which one was synthesized as oligonucleotides for binding assays. (B) CHIP experiment shows 5 binding sites of FOXA1 in HER2 promoter region. (C) Amplification of HER2-binding sites after Ch-IP using an antibody against FOXA1. An IgG antibody was used as the negative control. (D) Relative luciferase activity of the indicated promoter vectors A549 cell transfected with FOXA1 plasmids. (E) HER2 mRNA expression with the treatment of silencing FOXA1 as analyzed by qRT-PCR. (F) HER2 protein expression with the treatment of silencing FOXA1 as analyzed by western blot; Student's t-test. mean± SD (*P <0.05, *** P <0.001).

Figure 4

Effects of RC48 on cell cycle arrest, apoptosis induction and suppression of HER2-mediated cell signaling. (A) HER2 expression levels in A549 cell with knockdown of HER2 detected by qRT-PCR. (B) FOXA1 expression levels in A549 cell with knockdown of HER2 detected by qRT-PCR. (C) HER2 and FOXA1 protein expressions with the treatment of silencing HER2 as analyzed by western blot. (D)HER2 and FOXA1 protein expressions with the treatment of RC48 as analyzed by western blot. (E) CCK-8 assay analyzed the effect of RC48 and knockdown of HER2 on cell viability. (F) A549 cell cycle arrest results analyzed by FCM. A549 cells were treated with siHER2 and RC48. (G) A549 cell apoptosis after si-HER2 and RC48 treatment detected by fow cytometry. (H) Representative Western blots results of CCNB1, P21, P53, Caspase3, Cleaved-Caspase3, Bcl2 and Bax in the A549 cells. Interference of HER2 signal pathway related proteins, P-HER2 and P-AKT, detected by Western blot. Student's t-test, mean ± SD (*P <0.05, ** P <0.001).

Figure 5

(A) Immunohistochemical staining of HER2 and FOXA1 in adenocarcinoma (×40): a: FOXA1 negative b: FOXA1 positive c:HER2 negative d: HER2 positive. (B) Overall survival in groups with HER2 expression. (C) Overall survival in different groups. (D) Correlations between FOXA1and HER2 expression levels were calculated. Two tailed Spearman's correlation analysis ( r= 0.5398, P < 0.0001).

Figure 6

Proposed model linking FOXA1 and HER2 in NSCLC. FOXA1 is capable of translocating to the nucleus and turning on the transcription of HER2, whereupon a subsequent upregulation of HER2 protein expression would be predicted to drive tumorigenic signaling via pathways including the activation of PI3K and AKT. HER2 cannot regulate the expression of FOXA1, but the targeted drug RC48 can affect the HER2/PI3K/AKT signaling pathway by regulating the expression of FOXA1.