HER2-Overexpressing Breast Cancers Amplify FGFR Signaling upon Acquisition of Resistance to Dual Therapeutic Blockade of HER2.

Purpose: Dual blockade of HER2 with trastuzumab and lapatinib or pertuzumab has been shown to be superior to single-agent trastuzumab. However, a significant fraction of HER2-overexpressing (HER2+) breast cancers escape from these drug combinations. In this study, we sought to discover the mechanisms of acquired resistance to the combination of lapatinib + trastuzumab.Experimental Design: HER2+ BT474 xenografts were treated with lapatinib + trastuzumab long-term until resistance developed. Potential mechanisms of acquired resistance were evaluated in lapatinib + trastuzumab-resistant (LTR) tumors by targeted capture next-generation sequencing. In vitro experiments were performed to corroborate these findings, and a novel drug combination was tested against LTR xenografts. Gene expression and copy-number analyses were performed to corroborate our findings in clinical samples.Results: LTR tumors exhibited an increase in FGF3/4/19 copy number, together with an increase in FGFR phosphorylation, marked stromal changes in the tumor microenvironment, and reduced tumor uptake of lapatinib. Stimulation of BT474 cells with FGF4 promoted resistance to lapatinib + trastuzumab in vitro Treatment with FGFR tyrosine kinase inhibitors reversed these changes and overcame resistance to lapatinib + trastuzumab. High expression of FGFR1 correlated with a statistically shorter progression-free survival in patients with HER2+ early breast cancer treated with adjuvant trastuzumab. Finally, FGFR1 and/or FGF3 gene amplification correlated with a lower pathologic complete response in patients with HER2+ early breast cancer treated with neoadjuvant anti-HER2 therapy.Conclusions: Amplification of FGFR signaling promotes resistance to HER2 inhibition, which can be diminished by the combination of HER2 and FGFR inhibitors. Clin Cancer Res; 23(15); 4323-34. ©2017 AACR.

Screening a novel FGF3 antagonist peptide with anti-tumor effects on breast cancer from a phage display library.

Accumulating evidence has suggested that fibroblast growth factor 3 (FGF3) is expressed in breast cancer and correlates with the stage and grade of the disease. In the present study, a specific FGF3­binding peptide (VLWLKNR, termed FP16) was isolated from a phage display heptapeptide library with FGF3. The peptide FP16 contained four identical (WLKN) amino acids and demonstrated high homology to the peptides of the 188­194 (TMRWLKN) site of the high­affinity FGF3 receptor fibroblast growth factor receptor 2. Functional analyses indicated that FP16 mediated significant inhibition of FGF3­induced cell proliferation, arrested the cell cycle at the G0/G1 phase by increasing proliferation­associated protein 2G4, suppressing cyclin D1 and proliferating cell nuclear antigen, and inhibited the FGF3­induced activation of extracellular signal­regulated kinase 1/2 and Akt kinase. Taken together, these results demonstrated that the peptide FP16, acting as an FGF3 antagonist, is a promising therapeutic agent for the treatment of breast cancer.

A mutation in cnot8, component of the Ccr4-not complex regulating transcript stability, affects expression levels of developmental regulators and reveals a role of Fgf3 in development of caudal hypothalamic dopaminergic neurons.

While regulation of the activity of developmental control genes at the transcriptional level as well as by specific miRNA-based degradation are intensively studied, little is known whether general cellular mechanisms controlling mRNA decay may contribute to differential stability of mRNAs of developmental control genes. Here, we investigate whether a mutation in the deadenylation dependent mRNA decay pathway may reveal differential effects on developmental mechanisms, using dopaminergic differentiation in the zebrafish brain as model system. In a zebrafish genetic screen aimed at identifying genes controlling dopaminergic neuron development we isolated the m1061 mutation that selectively caused increased dopaminergic differentiation in the caudal hypothalamus, while other dopaminergic groups were not affected. Positional cloning revealed that m1061 causes a premature stop codon in the cnot8 open reading frame. Cnot8 is a component of the Ccr4-Not complex and displays deadenylase activity, which is required for removal of the poly (A) tail in bulk mRNA turnover. Analyses of expression of developmental regulators indicate that loss of Cnot8 activity results in increased mRNA in situ hybridization signal levels for a subset of developmental control genes. We show that in the area of caudal hypothalamic dopaminergic differentiation, mRNA levels for several components of the FGF signaling pathway, including Fgf3, FGF receptors, and FGF target genes, are increased. Pharmacological inhibition of FGF signaling or a mutation in the fgf3 gene can compensate the gain of caudal hypothalamic dopaminergic neurons in cnot8m1061 mutants, indicating a role for Fgf3 in control of development of this dopaminergic population. The cnot8m1061 mutant phenotype provides an in vivo system to study roles of the Cnot8 deadenylase component of the mRNA decay pathway in vertebrate development. Our data indicate that attenuation of Cnot8 activity differentially affects mRNA levels of developmental control genes.

A screen for hoxb1-regulated genes identifies ppp1r14al as a regulator of the rhombomere 4 Fgf-signaling center.

Segmentation of the vertebrate hindbrain into multiple rhombomeres is essential for proper formation of the cerebellum, cranial nerves and cranial neural crest. Paralog group 1 (PG1) hox genes are expressed early in the caudal hindbrain and are required for rhombomere formation. Accordingly, loss of PG1 hox function disrupts development of caudal rhombomeres in model organisms and causes brainstem defects, associated with cognitive impairment, in humans. In spite of this important role for PG1 hox genes, transcriptional targets of PG1 proteins are not well characterized. Here we use ectopic expression together with embryonic dissection to identify novel targets of the zebrafish PG1 gene hoxb1b. Of 100 genes up-regulated by hoxb1b, 54 were examined and 25 were found to represent novel hoxb1b regulated hindbrain genes. The ppp1r14al gene was analyzed in greater detail and our results indicate that Hoxb1b is likely to directly regulate ppp1r14al expression in rhombomere 4. Furthermore, ppp1r14al is essential for establishment of the earliest hindbrain signaling-center in rhombomere 4 by regulating expression of fgf3.

Bortezomib, dexamethasone, and fibroblast growth factor receptor 3-specific tyrosine kinase inhibitor in t(4;14) myeloma.

PURPOSE: Novel drugs including targeted approaches have changed treatment paradigms for multiple myeloma (MM) and may also have therapeutic potential in the poor-prognosis t(4;14) subset; t(4;14) results in overexpressed and activated fibroblast growth factor receptor 3 (FGFR3). Blocking this receptor tyrosine kinase (RTK) induces apoptosis in t(4;14)+ MM cells and decreases adhesion to bone marrow stromal cells (BMSC). Using combinations of novel drugs, we investigated potential enhancement of single-agent activities within the tumor cells, targeting of the marrow micromilieu, or circumvention of drug resistance in t(4;14)+ MM. EXPERIMENTAL DESIGN: We tested effects on apoptosis and related signaling pathways in the t(4;14)+ MM subset, applying drug combinations including a FGFR3 tyrosine kinase inhibitor (RTKI), the proteasome inhibitor bortezomib, and dexamethasone. RESULTS: RTKI, bortezomib, and dexamethasone were active as single agents in t(4;14)+ MM. RTK inhibition triggered complementary proapoptotic pathways (e.g., decrease of Mcl-1, down-regulation of p44/42 mitogen-activated protein kinase, and activation of proapoptotic stress-activated protein/c-Jun NH(2)-terminal kinases). Synergistic or additive effects were found by combinations of RTKI with dexamethasone or bortezomib. In selected cases of t(4;14)+ MM, triple combinations were superior to dual combinations tested. Prevention from MM cell apoptosis by BMSC or exogenous interleukin-6 was circumvented by drug combinations. In t(4;14)+, N-ras-mutated NCI-H929 cells, resistance to RTKI was overcome by addition of dexamethasone. Notably, the combination of RTKI and dexamethasone showed additive proapoptotic effects in bortezomib-insensitive t(4;14)+ MM. CONCLUSIONS: Combining novel drugs in poor-prognosis t(4;14)+ MM should take into account at least bortezomib sensitivity and probably Ras mutational status.

FGF signaling is required for {beta}-catenin-mediated induction of the zebrafish organizer.

We have used the maternal effect mutant ichabod, which is deficient in maternal beta-catenin signaling, to test for the epistatic relationship between beta-catenin activation, FGF signaling and bozozok, squint and chordin expression. Injection of beta-catenin RNA into ichabod embryos can completely rescue normal development. By contrast, when FGF signaling is inhibited, beta-catenin did not induce goosecoid and chordin, repress bmp4 expression or induce a dorsal axis. These results demonstrate that FGF signaling is necessary for beta-catenin induction of the zebrafish organizer. We show that FGFs function downstream of squint and bozozok to turn on chordin expression. Full rescue of ichabod by Squint is dependent on FGF signaling, and partial rescue by FGFs is completely dependent on chordin. By contrast, Bozozok can rescue the complete anteroposterior axis, but not notochord, in embryos blocked in FGF signaling. Surprisingly, accumulation of bozozok transcript in beta-catenin RNA-injected ichabod embryos is also dependent on FGF signaling, indicating a role of FGFs in maintenance of bozozok RNA. These experiments show that FGF-dependent organizer function operates through both bozozok RNA accumulation and a pathway consisting of beta-catenin-->Squint-->FGF-->Chordin, in which each component is sufficient for expression of the downstream factors of the pathway, and in which Nodal signaling is required for FGF gene expression and FGF signaling is required for Squint induction of chordin.