Fig 1: PIK3CA mutations enriched in advanced HER2+ breast tumours and associated with worse efficacy of anti-HER2 therapy for locally advanced or advanced HER2-positive patients. (A) The variant allele frequency (VAF) of PIK3CA mutations increases among advanced HER2+ subgroup (left, HER2+ all cases; middle, HR+HER2+ cases; right, HR-HER2+ cases). The efficacy of trastuzumab-based therapy in advanced HER2+ patients is shown in the waterfall plot (B) and the spider plot (C). The y-axis represents the percentage of maximum tumour reduction compared to the baseline data. (D) Statistical analysis of the progressive response (PD+SD) for patients carrying PIK3CA-WT or PIK3CA mutations in the advanced HER2+ cohort using a fisher's exact test. (E) Objective response rate according to PIK3CA mutation status in locally advanced HER2+ patients receiving neoadjuvant therapy, using a ?2 test of association. (F) Comparison of VAF distribution between the PR+CR and PD+SD groups in HR-HER2+ breast cancer patients harbouring PIK3CA mutation (VAF threshold > 12.23%). OR, overall response; CR, complete response; PR, partial response; PD, progressive disease
Fig 2: BYL719 synergises with lapatinib to suppress the proliferation of HER2+ cells harbouring PIK3CA mutation. (A) SK-BR-3 cells expressing PIK3CA WT and PIK3CA H1047R were treated with lapatinib, BYL719 or both as indicated. The percentage inhibition (left) and CI (right) at each concentration of the drugs are presented. Each score represents data from three independent experiments. (B) Activation status of the RTK-PI3K-MAPK pathway in PIK3CA WT, PIK3CA H1047R, PIK3CA E545K cells treated with lapatinib (500 nM), BYL719 (500 nM) and both. (C) Immunoblot analysis of key molecules among the RTK-PI3K-MAPK pathway in HER2+ breast cancer cell line HCC1954 with an endogenous PIK3CA H1047R mutation treated with lapatinib (500 nM), BYL719 (500 nM) and both. (D) Drug administration model for NOD/SCID mice bearing MCF-10CA1a cells over-expressing HER2 and PIK3CA H1047R mutant. Mice were randomly divided into the following four treatment groups (n = 6 to 8 per group): vehicle control, 100 mg/kg lapatinib, 25 mg/kg BYL719, or combination of 100 mg/kg lapatinib and 25 mg/kg BYL719. Tumour volumes (E) and tumour weights (F) of mice bearing PIK3CA H1047R cells treated with vehicle, lapatinib (100 mg/kg body weight), BYL719 (25 mg/kg body weight), or both drugs in combination for the indicated times. Error bars, mean ± SEM (n = 5 per treatment group). (G) Tumour tissues of mice bearing PIK3CA WT and PIK3CA H1047R cells treated with vehicle, lapatinib (100 mg/kg body weight), BYL719 (25 mg/kg body weight), or both drugs in combination for 22 days was evaluated by IHC for HER2, phospho-HER2, phospho-AKT (Ser473), phospho-ERK, Ki67 and H&E staining. (H) Clinical validation from patient-derived organoids with wild-type PIK3CA (PDO A) and mutated PIK3CA H1047R (PDO B) in the presence of the HER2 inhibitor lapatinib (0.4 µM) and the PI3K inhibitor BYL719 (1 µM) treated for 5 days. The same organoid's image were captured at Day 0, 1, 3, 5
Fig 3: The PIK3CA functional mutations mediate the negative feedback inhibition of RTKs which contributes to suppression of HER2+ cells proliferation. (A) Proliferation viability of SK-BR-3 cells expressing PIK3CA wild-type and mutants using a CCK-8 assay. Xenograft tumour volumes (B) and tumour weights (C) of mice bearing PIK3CA WT and PIK3CA H1047R cells. Error bars, mean ± SEM (n = 5 per treatment group). (D) Immunoblot detecting activation of the key molecules among RTK-PI3K-MAPK pathway in HER2+ cells harbouring pCDH-NC, PIK3CA-WT and mutants. Control referred to SK-BR-3 with empty plasmid pCDH (pCDH-NC). (E) Tumour tissues of mice bearing PIK3CA WT and PIK3CA H1047R cells were harvested and subjected to IHC analysis for HER2, phospho-HER2, phospho-AKT (Ser473), phospho-ERK, Ki67 and H&E staining. (F) Expression levels of RTK transcripts were examined by quantitative PCR (qPCR) in SK-BR-3 cells carrying PIK3CA mutant and control. The results are shown as the mean ± SD of three independent experiments
Fig 4: Genomic characteristics of the RTK-PI3K-MAPK pathway in the FUSCC cohort. (A) The genomic landscape of alterations among RTK-PI3K-MAPK pathway in FUSCC breast cancer sequencing cohort classified by molecular subtypes. (B) A comparison of somatic alteration frequencies for the RTK-PI3K-MAPK-related genes between the FUSCC and MSKCC cohorts. (C) Overall alteration frequency of the RTK, PI3K and MAPK pathways per subtype. (D) Mutual co-occurrence (blue) and mutual exclusivity (red) of gene mutations among the RTK-PI3K-MAPK pathway in the FUSCC HR±HER2+ breast cancer cohort. Scale bar: value of log10 ratio of odds ratio (OR); value scaled with colour intensity. * p < .05, ** p < .01, *** p < .001
Fig 5: A diagram showing the adaptive strategies targeting mutated-PI3Ka and enriched-HER2 in breast cancer progression. PIK3CA functional mutations suppress the expression and activation of HER2 and other RTKs through a negative feedback loop, thus acting as a protective factor for treatment-naïve HER2+ breast cancer. Meanwhile, mutated-PI3Ka confers anti-HER2 resistance for patients receiving continuous trastuzumab-based neoadjuvant/adjuvant treatment. The tumours harbouring PIK3CA functional mutations are sensitive to the PI3K-specific inhibitor BYL719, but the therapeutic effects will be interfered with rescued RTKs activation by abolishing the negative feedback. The combination of BYL719 and lapatinib shows a promising synergetic efficacy to target mutated-PI3Ka and enriched-HER2 simultaneously, offering a therapeutic potential to address multiple pathogens and improve outcomes for advanced HER2+ patients
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