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Gene expression profile of RAS/RAF–wild-type colorectal cancer patient-derived spheroids

ABSTRACT: We would like to propose a novel parameter that helps predict the chemosensitivity of cancer patients to FGFR inhibitors (FGFRi). Although FGFRi were introduced into the clinical trials on a variety of cancer types, they were found to be particularly efficacious on urothelial cancer and on cholangiocarcinoma for which the FDA had approved several compounds based on clinical studies. The enrollment criteria in such clinical trials were genomic changes in the FGFR genes including amplifications, translocations that caused gene fusions, and point mutations. By scrutinizing the published data on these reports, we have noticed an unresolved issue. Namely, most of these reports showed waterfall plots where tumors of some good fractions of enrolled patients responded well, demonstrating efficacy of the FGFRi. Importantly, however, there were sizable subsets in which tumors did not respond to the FGFRi despite that the patients’ tumors carried the genomic changes that met the enrollment criteria. As already established, both FGFR and EGFR share the downstream signaling pathway of MAPK activation as well as other pathways. Accordingly, it is conceivable that chemotherapeutic inhibition of FGFR alone may leave the MAPK signaling unaffected when the signaling through EGFR is relatively strong. To test this hypothesis, we calculated the FGFR to EGFR mRNA ratio (F/E for short) based on the RNA sequencing data from the Broad Institute Cancer Cell Line Encyclopedia (https://portals.broadinstitute.org/ccle) of cholangiocarcinoma and urothelial cancer cell lines for which FGFRi chemosensitivity had been published in preclinical studies. Because human FGFR has four paralogs FGFR1–4, we first summed up the expression levels of FGFR1–4 mRNAs by adding the read numbers per MB of FGFR paralogs (sumFGFR). To take EGFR expression into consideration, we then divided this number with that of EGFR; F/E. In six biliary tract cancer cell lines tested, two responsive cell lines had higher F/E ratios of 9.5 and 9.0, whereas four non-responsive lines had substantially lower ratios of 0.1–1.8. In 22 urothelial cancer cell lines, four of the five responsive lines showed F/E of 2.8–4.9, whereas 17 non-responsive lines had 0.01–2.7. To obtain further insights into the F/E ratio, we looked into our patient-derived colorectal cancer (CRC)-stem cell lines. Seven (28%) of 25 RAS/RAF-wild type CRC-stem cell lines responded to the pan-FGFRi erdafitinib singly in culture whereas 21 (84%) did respond to erdafitinib in combination with an EGFR inhibitor (erlotinib). The seven singly responsive cell lines showed relatively high F/E ranging 11–207 with the mean of 46, whereas the 18 non-responsive lines had the ratio of 3.5–37 with the mean at 12. These results suggest that F/E can be a useful biomarker also for colorectal cancer chemosensitivity where EGFR expression level can vary widely. Taken together, F/E is another strong predictor of responses to FGFRi that is as useful as the current genomic criteria that are based on the FGFR genomic changes.

ORGANISM(S): Homo sapiens

PROVIDER: GSE189212 | GEO | 2021/11/22

REPOSITORIES: GEO

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Project description:Despite initial and often dramatic responses of epidermal growth factor receptor (EGFR)-addicted lung tumors to the EGFR-specific tyrosine kinase inhibitors (TKIs), gefitinib and erlotinib, nearly all develop resistance and relapse. To explore novel mechanisms mediating acquired resistance, we employed non-small-cell lung cancer (NSCLC) cell lines bearing activating mutations in EGFR and rendered them resistant to EGFR-specific TKIs through chronic adaptation in tissue culture. In addition to previously observed resistance mechanisms including EGFR-T790M 'gate-keeper' mutations and MET amplification, a subset of the seven chronically adapted NSCLC cell lines including HCC4006, HCC2279 and H1650 cells exhibited marked induction of fibroblast growth factor (FGF) 2 and FGF receptor 1 (FGFR1) mRNA and protein. Also, adaptation to EGFR-specific TKIs was accompanied by an epithelial to mesenchymal transition (EMT) as assessed by changes in CDH1, VIM, ZEB1 and ZEB2 expression and altered growth properties in Matrigel. In adapted cell lines exhibiting increased FGF2 and FGFR1 expression, measures of growth and signaling, but not EMT, were blocked by FGFR-specific TKIs, an FGF-ligand trap and FGFR1 silencing with RNAi. In parental HCC4006 cells, cell growth was strongly inhibited by gefitinib, although drug-resistant clones progress within 10 days. Combined treatment with gefitinib and AZD4547, an FGFR-specific TKI, prevented the outgrowth of drug-resistant clones. Thus, induction of FGF2 and FGFR1 following chronic adaptation to EGFR-specific TKIs provides a novel autocrine receptor tyrosine kinase-driven bypass pathway in a subset of lung cancer cell lines that are initially sensitive to EGFR-specific TKIs. The findings support FGFR-specific TKIs as potentially valuable additions to existing targeted therapeutic strategies with EGFR-specific TKIs to prevent or delay acquired resistance in EGFR-driven NSCLC. Examination of mRNA levels in DMSO and gefitinib-resistant cultures of HCC4006 and HCC827. Each group has two replicates.

Project description:Introduction: The clinical benefit of EGFR tyrosine kinase inhibitor (TKI) treatment in non-small cell lung cancer (NSCLC) patients with activating EGFR mutations is temporary, as virtually all patients develop acquired EGFR TKI resistance that occurs via diverse mechanisms. Here, we identified increased FGFR1 expression as such a resistance mechanism and using pathways analysis and drug combination testing we identified a novel combination treatment to control growth of these resistant tumors. Methods: Novel erlotinib-resistant NSCLC cell lines were generated and analyzed by mass spectrometry-based proteomics to identify altered pathways associated with erlotinib resistance. The altered pathways were further analyzed in gefitinib and osibenib resistant cell lines. Small molecule inhibitor combinations were used to block the altered pathways and investigate growth reduction in vitro and in two xenograft mouse models. FGFR1 mRNA levels were examined in pre- and (post?)- EGFR TKI treatment clinical tumor samples. Results: Proteomic analysis revealed increased expression of FGFR1 and AXL as well as increased Akt and ERK1/2 activation in a panel of novel erlotinib-resistant HCC827 cell lines. Combined treatment with erlotinib or osimertinib and a panel of small molecule inhibitors targeting AXL/MET, FGFRs, Akt, PI3K/mTOR, MEK or ERK1/2 showed that the most prominent re-sensitization to EGFR TKI occurred with the pan-FGFR inhibitor, PD173074. Interestingly, simultaneous blockade of components of the Akt pathway using specific Akt or dual PI3K-mTOR inhibitors combined with inhibitors targeting the FGFR family exhibited the most efficient growth inhibition of FGFR1 overexpressing EGFR TKI-resistant cell lines. Phosphorylation of proteins downstream of Akt, including PRAS40, FOXO and S6 ribosomal protein, were completely abrogated by PD173074 combined with the Akt inhibitor GSK2141795 . Combination treatment with PD173074 and an Akt inhibitor exhibited synergistic growth inhibition in vivo in two FGFR1 overexpressing NSCLC EGFR TKI-resistant animal models. Conclusion: The significant growth inhibition in vitro and in vivo observed with PD173074 combined with Akt compared to either drug alone imply that inhibition of several key targets may be beneficial in controlling erlotinib-resistant NSCLC. The complete abrogation of PRAS40, FOXO and S6 phosphorylations by PD173074 combined with an Akt inhibitor indicates that the Akt pathway is no longer active.

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Gene expression profile of RAS/RAF–wild-type colorectal cancer patient-derived spheroids

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