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Supplementary Methods, radiologist's reading of CT images, supplementary figure legends, supplementary references

microCT scans

SFigure 1: Histological examination of mouse lungs; SFigure 2: Tissue sections from human lung adenocarcinoma; SFigure 3: Cabozantinib (CBT) and vandetanib (VDT) resistant cells; SFigure 4: Comparison of in vitro inhibition of RET, RETV804L and RETV804M by ponatinib, cabozantinib, vandetanib, and lenvatinib; SFigure 5: Ponatinib treatment schedule and body weight measurements; SFigure 6: Lung section histology from Dox-induced C/KR mice and treated with vehicle or ponatinib for 1 months; SFigure 7: μCT examination of dox-induced C/KR mice with lung tumors before and after one month treatment with vehicle or ponatinib.

The expression and role of methyltransferase SET and MYND domain-containing protein 5 (SMYD5) in inflammatory bowel disease (IBD) is completely unknown. Here, we investigated the role and underlying mechanism of epithelial SMYD5 in IBD pathogenesis and progression.The expression levels of SMYD5 and the mitochondrial transcriptional coactivator peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) were examined by Western blot, immunofluorescence staining, and immunohistochemistry in intestinal epithelial cells (IECs) and in colon tissues from human IBD patients and colitic mice. Mice with Smyd5 conditional knockout in IECs and littermate controls were subjected to dextran sulfate sodium-induced colitis and the disease severity was assessed. SMYD5-regulated mitochondrial biogenesis was examined by quantitative reverse-transcription polymerase chain reaction and transmission electron microscopy, and the mitochondrial oxygen consumption rate was measured in a Seahorse Analyzer system (Agilent, Santa Clara, CA). SMYD5 and PGC-1α interaction was determined by co-immunoprecipitation assay. PGC-1α degradation and turnover (half-life) were analyzed by cycloheximide chase assay. SMYD5-mediated PGC-1α methylation was assessed via in vitro methylation assay followed by mass spectrometry for identification of methylated lysine residues.Up-regulated SMYD5 and down-regulated PGC-1α were observed in intestinal epithelia from IBD patients and colitic mice. Smyd5 depletion in IECs protected mice from dextran sulfate sodium-induced colitis. SMYD5 was critically involved in regulating mitochondrial biology such as mitochondrial biogenesis, respiration, and apoptosis. Mechanistically, SMYD5 regulates mitochondrial functions in a PGC-1α-dependent manner. Furthermore, SMYD5 mediates lysine methylation of PGC-1α and subsequently facilitates its ubiquitination and degradation.SMYD5 attenuates mitochondrial functions in IECs and promotes IBD progression by enhancing PGC-1α degradation in a methylation-dependent manner. Strategies to decrease SMYD5 expression and/or increase PGC-1α expression in IECs might be a promising therapeutic approach to treat IBD patients.

Background and Aims The expression and role of methyltransferase SET and MYND domain-containing protein 5 (SMYD5) in inflammatory bowel diseases (IBD) is completely unknown. Here, we investigated the role and the underlying mechanism of epithelial SMYD5 in IBD pathogenesis and progression. Methods The expression and subcellular localization of SMYD5 and peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α) were examined by Western blot analysis, immunofluorescence staining, and immunohistochemistry in intestinal epithelial cells (IECs) and in colon tissues from human IBD patients and mice with experimental colitis. Mice with Smyd5 conditional knockout in IECs and littermate controls were subjected to DSS-induced experimental colitis and the disease severity and inflammation were assessed. SMYD5-regulated mitochondrial biogenesis was examined by RT-qPCR and transmission electron microscopy and mitochondrial oxygen consumption rate was measured in a Seahorse Analyzer system. The interaction between SMYD5 and PGC-1α was determined by co-immunoprecipitation assay. PGC-1α degradation and turnover (half-life) were analyzed by cycloheximide chase assay. SMYD5-mediated PGC-1α methylation was measured via in vitro methylation followed by mass spectrometry to identify the specific lysine residues that were methylated. Results Up-regulated SMYD5 and down-regulated PGC-1α were observed in IECs from IBD patients and mice with experimental colitis. However, Smyd5 depletion in IECs protected mice from DSS-induced colitis. SMYD5 was critically involved in regulating mitochondrial biology such as mitochondrial biogenesis, respiration, and apoptosis. Mechanistically, SMYD5 regulated mitochondrial functions in a PGC-1α dependent manner. Further, SMYD5 mediated lysine methylation of PGC-1α and facilitated its ubiquitination and proteasomal degradation. Conclusion SMYD5 attenuates mitochondrial functions in IECs and promotes IBD progression by enhancing the proteasome-mediated degradation of PGC-1α protein in a methylation-dependent manner. Strategies to decrease SMYD5 expression and/or increase PGC-1α expression in IECs might be a promising therapeutic approach to treat patients with IBD.

RET fusions have been found in lung adenocarcinoma, of which KIF5B–RET is the most prevalent. We established inducible KIF5B-RET transgenic mice and KIF5B–RET-dependent cell lines for preclinical modeling of KIF5B–RET-associated lung adenocarcinoma. Doxycycline-induced CCSP-rtTA/tetO-KIF5B-RET transgenic mice developed invasive lung adenocarcinoma with desmoplastic reaction. Tumors regressed upon suppression of KIF5B–RET expression. By culturing KIF5B–RET-dependent BaF3 (B/KR) cells with increasing concentrations of cabozantinib or vandetanib, we identified cabozantinib-resistant RETV804L mutation and vandetanib-resistant-RETG810A mutation. Among cabozantinib, lenvatinib, ponatinib, and vandetanib, ponatinib was identified as the most potent inhibitor against KIF5B–RET and its drug-resistant mutants. Interestingly, the vandetanib-resistant KIF5B-RETG810A mutant displayed gain-of-sensitivity (GOS) to ponatinib and lenvatinib. Treatment of doxycycline-induced CCSP-rtTA/tetO-KIF5B-RET bitransgenic mice with ponatinib effectively induced tumor regression. These results indicate that KIF5B-RET–associated lung tumors are addicted to the fusion oncogene and ponatinib is the most effective inhibitor for targeting KIF5B–RET in lung adenocarcinoma. Moreover, this study finds a novel vandetanib-resistant RETG810A mutation and identifies lenvatinib and ponatinib as the secondary drugs to overcome this vandetanib resistance mechanism. Mol Cancer Ther; 15(10); 2521–9. ©2016 AACR.

In addition to being positively regulated by prandial activity, bile acid production is also negatively controlled by the endocrine fibroblast growth factor 19 (FGF19) or the mouse ortholog FGF15 from the ileum that represses hepatic cholesterol 7 α-hydroxylase (Cyp7a1) expression through activating FGF receptor four (FGFR4). However, how these two regulatory mechanisms interplay to control bile acid homeostasis in the body and the downstream pathways by which FGFR4 regulates Cyp7a1 expression are not fully understood. Here we report that hepatocyte FGFR substrate 2α (FRS2α), a scaffold protein essential for canonical FGFRs to activate the ERK and AKT pathways, was required for the regulation of bile acid production by the FGF15/19-FGFR4 signaling axis. This occurred through limiting the extent of increases in Cyp7a1 expression induced by prandial activity. Excess FGFR4 kinase activity reduced the amplitude of the increase whereas a lack of FGFR4 augmented the increase of Cyp7a1 expression in the liver. Ablation of Frs2α alleles in hepatocytes abrogated the regulation of Cyp7a1 expression by FGFR4. Together, the results demonstrate that FRS2α-mediated pathways are essential for the FGF15/FGF19-FGFR4 signaling axis to control bile acid homeostasis.

Understanding the cellular and molecular mechanisms underlying the self-renewal and differentiation of dental epithelial stem cells (DESCs) that support the unlimited growth potential of mouse incisors is critical for developing novel tooth regenerative therapies and unraveling the pathogenesis of odontogenic tumors. However, analysis of DESC properties and regulation has been limited by the lack of an in vitro assay system and well-documented DESC markers. Here, we describe an in vitro sphere culture system to isolate the DESCs from postnatal mouse incisor cervical loops (CLs) where the DESCs are thought to reside. The dissociated cells from CLs were able to expand and form spheres for multiple generations in the culture system. Lineage tracing indicated that DESC within the spheres were epithelial in origin as evident by lineage tracing. Upon stimulation, the sphere cells differentiated into cytokeratin 14- and amelogenin-expressing and mineral material-producing cells. Compared to the CL tissue, sphere cells expressed high levels of expression of Sca-1, CD49f (also designated as integrin α6), and CD44. Fluorescence-activated cell sorting (FACS) analyses of mouse incisor CL cells further showed that the CD49f(Bright) population was enriched in sphere-forming cells. In addition, the CD49f(Bright) population includes both slow-cycling and Lgr5(+) DESCs. The in vitro sphere culture system and identification of CD49f(Bright) as a DESC marker provide a novel platform for enriching DESCs, interrogating how maintenance, cell fate determination, and differentiation of DESCs are regulated, and developing tooth regenerative therapies.

Background: Understanding of the self-renewal and differentiation of dental epithelial stem cells (DESCs) is important for tooth regeneration therapies. Results: Depletion of FGF signaling suppressed self-renewal and led to differentiation of DESCs. Conclusion: FGF signaling is essential for maintenance of DESCs. Significance: The finding sheds new light on the mechanism by which the homeostasis, expansion, and differentiation of DESCs are regulated. A constant supply of epithelial cells from dental epithelial stem cell (DESC) niches in the cervical loop (CL) enables mouse incisors to grow continuously throughout life. Elucidation of the cellular and molecular mechanisms underlying this unlimited growth potential is of broad interest for tooth regenerative therapies. Fibroblast growth factor (FGF) signaling is essential for the development of mouse incisors and for maintenance of the CL during prenatal development. However, how FGF signaling in DESCs controls the self-renewal and differentiation of the cells is not well understood. Herein, we report that FGF signaling is essential for self-renewal and the prevention of cell differentiation of DESCs in the CL as well as in DESC spheres. Inhibiting the FGF signaling pathway decreased proliferation and increased apoptosis of the cells in DESC spheres. Suppressing FGFR or its downstream signal transduction pathways diminished Lgr5-expressing cells in the CL and promoted cell differentiation both in DESC spheres and the CL. Furthermore, disruption of the FGF pathway abrogated Wnt signaling to promote Lgr5 expression in DESCs both in vitro and in vivo. This study sheds new light on understanding the mechanism by which the homeostasis, expansion, and differentiation of DESCs are regulated.

BACKGROUND Despite dramatic positive effects, there is evidence that the androgen receptor (AR) may negatively influence prostate tumor progression. Understanding the AR repressor function and how it is subverted is of particular importance in anti-androgen and AR intervention strategies. METHODS AR, resident FGFR2IIIb, and ectopic FGFR1 were expressed by transfection in the AR-negative epithelial cell line DTE that predominates in cell culture of AR-positive androgen-responsive model Dunning R3327 rat prostate tumors. Androgen-responsiveness at transcription was measured by a luciferase reporter. Cell population growth rates were assessed by cell counts, DNA synthesis, and expression of cell cycle genes. AR variants (ARVs) were assessed by immunochemistry and nuclease protection of mRNA. RESULTS Expression of AR inhibited cell population growth of AR-negative DTE cells at the G1–S phase of the cell cycle. Ectopic FGFR1, but not resident FGFR2IIIb abrogated the growth inhibitory effects of AR. Appearance of ARVs was coincident with co-expression of FGFR1 and AR and abrogation of the AR-dependent inhibition of cell growth. CONCLUSIONS DTE cells may represent non-malignant AR-negative progenitors whose population is restricted by activation of AR in vivo. Ectopic expression of epithelial FGFR1, a common observation in tumors, overrides the inhibition of AR and thus may contribute to evolution of androgen and AR independent tumors. These results are consistent with the notion that some tumor cells are negatively restricted by AR and are unleased by androgen-deprivation or ectopic expression of FGFR1. ARV's may play a role in the bypass of the negative restrictions of AR. Prostate 71:1691–1700, 2011. © 2011 Wiley-Liss, Inc.

Defects in lymphangiogenesis are added to the broad clinical manifestations of DiGeorge syndrome, caused by deletion of the T box transcription factor Tbx1., Lymphatic dysfunction causes several human diseases, and tumor lymphangiogenesis is implicated in cancer spreading. TBX1 is the major gene for DiGeorge syndrome, which is associated with multiple congenital anomalies. Mutation of Tbx1 in mice recapitulates the human disease phenotype. In this study, we use molecular, cellular, and genetic approaches to show, unexpectedly, that Tbx1 plays a critical role in lymphatic vessel development and regulates the expression of Vegfr3, a gene that is essential for lymphangiogenesis. Tbx1 activates Vegfr3 transcription in endothelial cells (ECs) by binding to an enhancer element in the Vegfr3 gene. Conditional deletion of Tbx1 in ECs causes widespread lymphangiogenesis defects in mouse embryos and perinatal death. Using the mesentery as a model tissue, we show that Tbx1 is not required for lymphatic EC differentiation; rather, it is required for the growth and maintenance of lymphatic vessels. Our findings reveal a novel pathway for the development of the lymphatic vessel network.

Fibroblast growth factor (FGF) family signaling mediates cell-to-cell communication in development and organ homeostasis in adults. Of the FGF receptor (FGFR) isotypes, FGFR4 is the sole resident isotype present in mature parenchymal hepatocytes. FGFR1 that is normally associated with activated nonparenchymal cells appears ectopically in hepatoma cells. Ectopic expression and chronic activity of FGFR1 in hepatocytes accelerates diethylnitrosamine (DEN)-initiated hepatocarcinogenesis by driving unrestrained cell proliferation and tumor angiogenesis. Hepatocyte FGFR4 mediates liver's role in systemic cholesterol/bile acid and lipid metabolism and affects proper hepatolobular restoration after damage without effect on cell proliferation. Here we ask whether FGFR4 plays a role in progression of hepatocellular carcinoma (HCC). We report that although spontaneous HCC was not detected in livers of FGFR4-deficient mice, the ablation of FGFR4 accelerated DEN-induced hepatocarcinogenesis. In contrast to FGFR1 that induced a strong mitogenic response and depressed rate of cell death in hepatoma cells, FGFR4 failed to induce a mitogenic response and increased the rate of cell death. FGFR1 but not FGFR4 induced cyclin D1 and repressed p27 expression. Analysis of activation of Erk, JNK, and PI3K-related AKT signaling pathways indicated that in contrast to FGFR1, FGFR4 failed to sustain Erk activation and did not activate AKT. These differences may underlie the opposing effects of FGFR1 and FGFR4. These results suggest that in contrast to ectopic FGFR1 that is a strong promoter of hepatoma, resident FGFR4 that mediates differentiated hepatocyte metabolic functions also serves to suppress hepatoma progression.

Prostate stem cells (P-SCs) are capable of giving rise to all three lineages of prostate epithelial cells, which include basal, luminal, and neuroendocrine cells. Two types of P-SCs have been identified in both human and mouse adult prostates based on prostasphere or organoid cultures, cell lineage tracing, renal capsule implantation, and expression of luminal- and basal-specific proteins. The sphere-forming P-SCs are from the basal cell compartment that express P63, and are therefore designated as basal P-SCs (P-bSCs). Luminal P-SCs (P-lSCs) express luminal cytokeratins and Nkx3.1. Herein, we report that the type 2 FGF receptor (FGFR2) signaling axis is crucial for preserving stemness and preventing differentiation of P-bSCs. FGFR2 signaling mediated by FGFR substrate 2α (FRS2α) is indispensable for formation and maintenance of prostaspheres derived from P63(+) P-bSCs. Ablation of Fgfr2 in P63(+) cells in vitro causes the disintegration of prostaspheres. Ablation of Fgfr2 in vivo reduces the number of P63-expressing basal cells and enriches luminal cells. This suggests a basal stem cell-to-luminal cell differentiation. In addition, ablation of Fgfr2 in P63(+) cells causes defective postnatal development of the prostate. Therefore, the data indicate that FGFR2 signaling is critical for preserving stemness and preventing differentiation of P-bSCs.

Bone metastasis is the major cause of morbidity and mortality of prostate cancer (PCa). Fibroblast growth factor 9 (FGF9) has been reported to promote PCa bone metastasis. However, the mechanism by which overexpression of FGF9 promotes PCa progression and metastasis is still unknown. Herein, we report that transgenic mice forced to express FGF9 in prostate epithelial cells (F9TG) developed high grade prostatic intraepithelial neoplasia (PIN) in an expression level- and time-dependent manner. Moreover, FGF9/TRAMP bigenic mice (F9TRAMP) grew advanced PCa earlier and had higher frequencies of metastasis than TRAMP littermates. We observed tumor microenvironmental changes including hypercellularity and hyperproliferation in the stromal compartment of F9TG and F9TRAMP mice. Expression of TGFβ1, a key signaling molecule overexpressed in reactive stroma, was increased in F9TG and F9TRAMP prostates. Both in vivo and in vitro data indicated that FGF9 promoted TGFβ1 expression via increasing cJun-mediated signaling. Moreover, in silico analyses showed that the expression level of FGF9 was positively associated with expression of TGFβ1 and its downstream signaling molecules in human prostate cancers. Collectively, our data demonstrated that overexpressing FGF9 in PCa cells augmented the formation of reactive stroma and promoted PCa initiation and progression.

Fibroblast growth factor (FGF) signaling mediates cell-to-cell communication in development and organ homeostasis in adults. Of the four FGF receptor (FGFR) tyrosine kinases, only FGFR4 is expressed in mature hepatocytes. Although FGFR1 is expressed by hepatic cell progenitors and adult nonparenchymal cells, ectopic expression is commonly observed in hepatoma cells. Here, we determined whether ectopic FGFR1 is a cause or consequence of hepatocellular carcinoma by targeting a constitutively active human FGFR1 to mouse hepatocytes. Livers of transgenic mice exhibited accelerated regeneration after partial hepatectomy but no signs of neoplastic or preneoplastic abnormalities for up to 18 months. However, in diethylnitrosamine-treated mice, the chronic FGFR1 activity promoted an incidence of 44% adenomas at 4 months and 38% hepatocellular carcinoma at 8 months. No adenoma or hepatocellular carcinoma was observed in diethylnitrosamine-treated wild-type (WT) livers at 4 or 8 months, respectively. At 10 and 12 months, tumor-bearing livers in transgenic mice were twice the size of those in WT animals. Isolated hepatoma cells from the transgenic tumors exhibited a growth advantage in culture. Advanced hepatocellular carcinoma in the transgenic livers exhibited a reduced rate of necrosis. This was accompanied by a mean microvessel density of 2.7 times that of WT tumors and a markedly higher level of vascular endothelial growth factor. In cooperation with an initiator, the persistent activity of ectopic FGFR1 in hepatocytes is a strong promoter of hepatocellular carcinoma by driving cell proliferation at early stages and promoting neoangiogenesis at late stages of progression. (Cancer Res 2006; 66(3): 1481-90)

Tissue homeostasis in normal prostate and two-compartment nonmalignant prostate tumors depends on harmonious two-way communications between epithelial and stromal compartments. Within the fibroblast growth factor (FGF) family, signaling to an epithelial cell-specific FGF receptor (FGFR) 2IIIb-heparan sulfate complex from stromal-specific FGF7 and FGF10 delivers directionally specific instruction from stroma to epithelium without autocrine interference. Using a two-compartment transplantable prostate tumor model in which survival of stromal cells in vivo depends on epithelial cells, we show that signaling from epithelial FGF9 to stromal FGFR3 potentially mediates epithelial-to-stromal communication that also is directionally specific. FGF9 mRNA was expressed exclusively in the epithelial cells derived from well-differentiated, two-compartment Dunning R3327 rat prostate tumors. In contrast, FGFR3 was expressed at functionally significant levels only in the derived stromal cells. Competition binding and immunoprecipitation assays revealed that FGF9 only bound to an FGFR on the stromal cells. FGF9 also failed to covalently cross-link to clonal lines of stromal cells devoid of FGFR3 that expressed FGFR1 and FGFR2IIIc. Furthermore, FGF9 specifically stimulated DNA synthesis in stromal cells expressing FGFR3. These results demonstrate a directionally specific paracrine signaling from epithelial FGF9 and stromal FGFR3. Similar to the FGF7/FGF10 to FGFR2IIIb signaling from the stroma to the epithelium, the directional specificity from epithelium to stroma appears set by a combination of cell-specific expression of isoforms and cell-context specificity of FGFR isotypes for FGF.

Genomic ablation of hepatocyte-specific fibroblast growth factor receptor (FGFR)4 in mice revealed a role of FGF signaling in cholesterol and bile acid metabolism and hepatolobular restoration in response to injury without effect on liver development or hepatocyte proliferation. Although the potential role of all 23 FGF polypeptides in the liver is still unclear, the most widely studied prototypes, FGF1 and FGF2, are present and have been implicated in liver cell growth and function in vitro. To determine whether FGF1 and FGF2 play a role in response to injury and fibrosis, we examined the impact of both acute and chronic exposure to carbon tetrachloride (CCl(4)) in the livers of FGF1- and FGF2-deficient mice. After acute CCl(4) exposure, FGF1(-/-)FGF2(-/-) mice exhibited an accelerated release of serum alanine aminotransferase similar to FGFR4 deficiency, but no effect on overall hepatolobular restoration or bile acid metabolism. FGF1(-/-)FGF2(-/-) mice exhibited a normal increase in alpha-smooth muscle actin and desmin associated with activation and migration of hepatic stellate cells to damage, but a reduced level of hepatic stellate cell-derived matrix collagen alpha1(I) synthesis. Liver fibrosis resulting from chronic CCl(4) exposure was markedly decreased in the livers of FGF1/FGF2-deficient mice. These results suggest an agonist role for FGF1 and FGF2 in specifically insult-induced liver matrix deposition and hepatic fibrogenesis and a potential target for the prevention of hepatic fibrosis.

Exons IIIb and IIIc of the FGFR2 gene are alternatively spliced in a mutually exclusive manner in different cell types. A switch from expression of FGFR2IIIb to FGFR2IIIc accompanies the transition of nonmalignant rat prostate tumor epithelial cells (DTE) to cells comprising malignant AT3 tumors. Here we used transfection of minigenes with and without alterations in reading frame and with and without introns to examine how translation affects observed FGFR2 splice products. We observed that nonsense mutations in other than the last exon led to a dramatic reduction in mRNA that is abrogated by removal of downstream introns in both DTE and AT3 cells. The mRNA, devoid of both IIIb and IIIc exons (C1-C2), is a major splice product from minigenes lacking an intron downstream of the second common exon C2. From these observations, we suggest that repression of exon IIIc and activation of exon IIIb inclusion in DTE cells lead to the generation of both C1-IIIb-C2 and C1-C2 products. However, the C1-C2 product from the native gene is degraded due to a frameshift and a premature termination codon caused by splicing C1 and C2 together. Derepression of exon IIIc and repression of exon IIIb lead to the generation of both C1-IIIc-C2 and C1-C2 products in AT3 cells, but the C1-C2 product is degraded. The C1-IIIb-IIIc-C2 mRNA containing a premature termination codon in exon IIIc was present, but at apparently trace levels in both cell types. The nonsense-mediated mRNA decay pathway and cell type-dependent rates of inclusion of exons IIIb and IIIc result in the mutually exclusive expression of FGFR2IIIb and IIIc.

Heparan sulfate-regulated transmembrane tyrosine kinase receptor FGFR4 is the major FGFR isotype in mature hepatocytes. Fibroblast growth factor has been implicated in the definition of liver from foregut endoderm where FGFR4 is expressed and stimulation of hepatocyte DNA synthesis in vitro. Here we show that livers of mice lacking FGFR4 exhibited normal morphology and regenerated normally in response to partial hepatectomy. However, the FGFR4 (-/-) mice exhibited depleted gallbladders, an elevated bile acid pool and elevated excretion of bile acids. Cholesterol- and bile acid-controlled liver cholesterol 7alpha-hydroxylase, the limiting enzyme for bile acid synthesis, was elevated, unresponsive to dietary cholesterol, but repressed normally by dietary cholate. Expression pattern and cholate-dependent, cholesterol-induced hepatomegaly in the FGFR4 (-/-) mice suggested that activation of receptor interacting protein 140, a co-repressor of feed-forward activator liver X receptor alpha, may mediate the negative regulation of cholesterol- and bile acid-controlled liver cholesterol 7alpha-hydroxylase transcription by FGFR4 and cholate. The results demonstrate that transmembrane sensors interface with metabolite-controlled transcription networks and suggest that pericellular matrix-controlled liver FGFR4 in particular may ensure adequate cholesterol for cell structures and signal transduction.

Background Endocrine FGF21 and FGF19 target adipocytes and hepatocytes through betaKlotho (KLB) and FGFR tyrosine kinases effecting glucose, lipid and energy metabolism. Both factors alleviate obesity and metabolic abnormalities which are contributing factors to breast tumor progression. Genomic manipulation of hepatic FGFR4 has uncovered roles of endocrine FGF signaling in both metabolic and cellular homeostasis. Here we determined whether systemic and microenvironmental metabolic alterations caused by the FGFR4 deficiency affect tumorigenesis in breast where FGFR4 is negligible. Breast tumors were induced in the bigenic mice with ablation of FGFR4 and overexpression of TGFα that activates Her2 in the ductal and lobular epithelium surrounded by adipocytes. Mammary tumorigenesis and alterations in systemic and breast microenvironmental metabolic parameters and regulatory pathways were analyzed. Results Ablation of FGFR4 had no effect on cellular homeostasis and Her2 activity of normal breast tissue. However, the absence of FGFR4 reduced TGFα–driven breast tumor incidence and progression and improved host survival. Notable increases in hepatic and serum FGF21, ileal FGF15/19, adiponectin and adipsin, and decreases in systemic Fetuin A, IGF-1, IGFBP-1, RBP4 and TIMP1 were observed. The ablation affected adipogenesis and secretory function of adipocytes as well as lipogenesis, glycolysis and energy homeostasis associated with the functions of mitochondria, ER and peroxisomes in the breast and tumor foci. Treatment with a chemical inhibitor of NAMPT involved in the pathways inhibited the growth and survival of breast tumor cells and tumor-initiating cell-containing spheres. The FGFR4 ablation also caused elevation of inflammatory factors in the breast. Conclusions Although the primary role of FGFR4 in metabolism occurs in hepatocytes, its ablation results in a net inhibitory effect on mammary tumor progression. We suggest that the tumor-delaying effect of FGFR4 deficiency may be in large part due to elevated anti-obesogenic FGF21 that triggers tumor-suppressing signals from both peripheral and breast adipocytes. The predominant changes in metabolic pathways suggested roles of metabolic effects from both peripheral and breast adipocytes on metabolic reprogramming in breast epithelial cells that contribute to the suppression of tumor progression. These results provide new insights into the contribution of systemic and microenvironmental metabolic effects controlled by endocrine FGF signaling to breast carcinogenesis.

Evolution of unresponsiveness to homeostasis-promoting signals from the microenvironment is a hallmark of malignant tumor cells. In Dunning R3327 model rat prostate tumors that are comprised of distinct stromal and epithelial compartments, progression from non-malignant, androgen-responsive tumors to malignancy is characterized by loss of compartmentation coincident with a loss of resident epithelial cell FGFR2IIIb that receives instructive signals from stromal FGF7 and FGF10. Restoration of FGFR2IIIb to malignant tumor cells restores responsiveness to stromal cells, restores distinct stromal and epithelial compartments, and retards malignant progression. Cultured stromal cells from two-compartment tumors are comprised of smooth muscle α-actin-positive cells that express predominantly FGFR3 and fibroblast-like cells devoid of α-actin and FGFR3. Here, we show that it is primarily the smooth muscle cell-like α-actin-expressing stromal cells that survive, morphologically differentiate, and delay tumor incidence and size in the presence of malignant cells in which FGFR2IIIb has been restored. Expression of FGFR3 by transfection in the fibroblast-like stromal cells conferred ability to respond similar to the smooth muscle cell-like stromal cells in which FGFR3 is normally resident. These results highlight the importance of the two-way communication back and forth between stroma and epithelium that is mediated by signaling within the FGFR family during progression to malignancy.

In organs involved in metabolic homeostasis, transmembrane α and βklothos direct FGFR signaling to control of metabolic pathways. Coordinate expression of βklotho and FGFR4 is a property of mature hepatocytes. Genetic deletion of FGFR4 or βklotho in mice disrupts hepatic cholesterol/bile acid and lipid metabolism. The deletion of FGFR4 has no effect on the proliferative response of hepatocytes after liver injury. However, its absence results in accelerated progression of dimethynitrosamine-initiated hepatocellular carcinomas, indicating that FGFR4 suppresses hepatoma proliferation. The mechanism underlying the FGFR4-mediated hepatoma suppression has not been addressed. Here we show that βklotho expression is more consistently down-regulated in human and mouse hepatomas than FGFR4. Co-expression and activation by either endocrine FGF19 or cellular FGF1 of the FGFR4 kinase in a complex with βklotho restricts cell population growth through induction of apoptotic cell death in both hepatic and nonhepatic cells. The βklotho-FGFR4 partnership caused a depression of activated AKT and mammalian target of rapamycin while activating ERK1/2 that may underlie the pro-apoptotic effect. Our results show that βklotho not only interacts with heparan sulfate-FGFR4 to form a complex with high affinity for endocrine FGF19 but also impacts the quality of downstream signaling and biological end points activated by either FGF19 or canonical FGF1. Thus the same βklotho-heparan sulfate-FGFR4 partnership that mediates endocrine control of hepatic metabolism plays a role in cellular homeostasis and hepatoma suppression through negative control of cell population growth mediated by pro-apoptotic signaling.

Fibroblast growth factor (FGF) family signaling mediates cell-to-cell communication in development and organ homeostasis in adults. Of the FGF receptor (FGFR) isotypes, FGFR4 is the sole resident isotype present in mature parenchymal hepatocytes. FGFR1 that is normally associated with activated nonparenchymal cells appears ectopically in hepatoma cells. Ectopic expression and chronic activity of FGFR1 in hepatocytes accelerates diethylnitrosamine (DEN)-initiated hepatocarcinogenesis by driving unrestrained cell proliferation and tumor angiogenesis. Hepatocyte FGFR4 mediates liver's role in systemic cholesterol/bile acid and lipid metabolism and affects proper hepatolobular restoration after damage without effect on cell proliferation. Here we ask whether FGFR4 plays a role in progression of hepatocellular carcinoma (HCC). We report that although spontaneous HCC was not detected in livers of FGFR4-deficient mice, the ablation of FGFR4 accelerated DEN-induced hepatocarcinogenesis. In contrast to FGFR1 that induced a strong mitogenic response and depressed rate of cell death in hepatoma cells, FGFR4 failed to induce a mitogenic response and increased the rate of cell death. FGFR1 but not FGFR4 induced cyclin D1 and repressed p27 expression. Analysis of activation of Erk, JNK, and PI3K-related AKT signaling pathways indicated that in contrast to FGFR1, FGFR4 failed to sustain Erk activation and did not activate AKT. These differences may underlie the opposing effects of FGFR1 and FGFR4. These results suggest that in contrast to ectopic FGFR1 that is a strong promoter of hepatoma, resident FGFR4 that mediates differentiated hepatocyte metabolic functions also serves to suppress hepatoma progression.

OBJECTIVE—Fibroblast growth factor (FGF) family signaling largely controls cellular homeostasis through short-range intercell paracrine communication. Recently FGF15/19, 21, and 23 have been implicated in endocrine control of metabolic homeostasis. The identity and location of the FGF receptor isotypes that mediate these effects are unclear. The objective was to determine the role of FGFR4, an isotype that has been proposed to mediate an ileal FGF15/19 to hepatocyte FGFR4 axis in cholesterol homeostasis, in metabolic homeostasis in vivo. RESEARCH DESIGN AND METHODS—FGFR4−/− mice—mice overexpressing constitutively active hepatic FGFR4—and FGFR4−/− with constitutively active hepatic FGFR4 restored in the liver were subjected to a normal and a chronic high-fat diet sufficient to result in obesity. Systemic and liver-specific metabolic phenotypes were then characterized. RESULTS—FGFR4-deficient mice on a normal diet exhibited features of metabolic syndrome that include increased mass of white adipose tissue, hyperlipidemia, glucose intolerance, and insulin resistance, in addition to hypercholesterolemia. Surprisingly, the FGFR4 deficiency alleviated high-fat diet–induced fatty liver in obese mice, which is also a correlate of metabolic syndrome. Restoration of FGFR4, specifically in hepatocytes of FGFR4-deficient mice, decreased plasma lipid levels and restored the high-fat diet–induced fatty liver but failed to restore glucose tolerance and sensitivity to insulin. CONCLUSIONS—FGFR4 plays essential roles in systemic lipid and glucose homeostasis. FGFR4 activity in hepatocytes that normally serves to prevent systemic hyperlipidemia paradoxically underlies the fatty liver disease associated with chronic high-fat intake and obesity.

The new precision oncology requires identification of driver oncogenes, validation of oncogene addiction, and development of oncogene-targeted drugs. Lung cancer is molecularly heterogeneous characterized by various genetic alterations, many of them occur at low frequencies. Recurrent RET fusion genes have been found in 1-2% of non-small cell lung cancer (NSCLC). Among them KIF5B-RET fusion is the most prevalent and occurs specifically in NSCLC. To develop an in vivo animal model for evaluation of KIF5B-RET fusion in lung adenocarcinoma, we generated transgenic mice containing a doxycycline (Dox)-inducible tetO-KIF5B-RET transgene. To express KIF5B-RET in lung type II epithelial cells, tetO-KIF5B-RET mice were crossed with CCSP-rtTA mice. CCSP-rtT/tetO-KIF5B-RET bitransgenic mice were induced with Dox and lung tissues were analyzed. KIF5B-RET mRNA and protein were detected in the lungs of Dox-induced CCSP-rtTA/tetO-KIF5B-RET bitransgenic mice. Histological examinations showed lung hyperproliferative lesions and tumors in Dox-induced CCSP-rtTA/tetO-KIF5B-RET bitransgenic mice. Thus, KIF5B-RET fusion is a driver oncogene sufficient to induce lung tumors in transgenic mice. Citation Format: Qingling Huang, Valentina E. Schneeberger, Noreen Luetteke, Chengliu Jin, Domenico Coppola, Jie Wu. Generation and characterization of inducible KIF5B-RET mouse model of non-small cell lung cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2299. doi:10.1158/1538-7445.AM2015-2299

Inappropriate fibroblast growth factor (FGF) signaling is involved in most tissue-specific pathologies including cancer. Previously we showed that inappropriate expression and chronic activity of FGF receptor (FGFR) 1 in hepatocytes accelerated diethylnitrosamine (DEN)-initiated hepatocarcinogenesis. Here we showed that although widely expressed FGF1 and FGF2 are frequently upregulated in hepatocellular carcinoma (HCC), germline deletion of both FGF1 and FGF2 had no effect on DEN-initiated hepatocarcinogenesis. Thus overexpression of FGF1 or FGF2 may be a consequence rather than contributor to hepatoma progression. FGF21 is the first of 22 homologues whose expression has been reported to be preferentially in the liver. We showed that similar to FGF1 and FGF2, FGF21 mRNA was upregulated in neoplastic and regenerating liver after partial hepatectomy (PH) and CCl4 administration. In situ hybridization analysis confirmed that in contrast to FGF1 and FGF2, expression of FGF21 mRNA was limited to hepatocytes. Forced overexpression of FGF21 in hepatocytes by gene targeting had no apparent impact on normal liver development and compensatory response to injury. Surprisingly, overexpression of FGF21 delayed the appearance of DEN-induced liver tumors. At 8 and 10 mo, only 10% and 30% of transgenic mice, respectively, developed adenomas compared to 50% (all adenomas) and 80% (60% adenoma/20% HCC) in the wild-type (WT) mice. However, the incidence and burden of HCC at 10 mo and later was equal in the FGF21 transgenic and WT mice. We propose that FGF21 may delay development of adenomas through activation of resident hepatocyte FGFR4 at early times, but counteracts the delay by acceleration of progression to HCC through interaction with ectopic FGFR1 once it appears in hepatoma cells. This indicates a dual function of FGF21 that may reflect changes in FGFR isotype during progression of differentiated hepatoma cells.

The fibroblast growth factor (FGF) receptor complex is a regulator of adult organ homeostasis in addition to its central role in embryonic development and wound healing. FGF receptor 4 (FGFR4) is the sole FGFR receptor kinase that is significantly expressed in mature hepatocytes. Previously, we showed that mice lacking mouse FGFR4 (mR4(-/-)) exhibited elevated fecal bile acids, bile acid pool size, and expression of liver cholesterol 7alpha-hydroxylase (CYP7A1), the rate-limiting enzyme for canonical neutral bile acid synthesis. To prove that hepatocyte FGFR4 was a negative regulator of cholesterol metabolism and bile acid synthesis independent of background, we generated transgenic mice overexpressing a constitutively active human FGFR4 (CahR4) in hepatocytes and crossed them with the FGFR4-deficient mice to generate CahR4/mR4(-/-) mice. In mice expressing active FGFR4 in liver, fecal bile acid excretion was 64%, bile acid pool size was 47%, and Cyp7a1 expression was 10-30% of wild-type mice. The repressed level of Cyp7a1 expression was resistant to induction by a high cholesterol diet relative to wild-type mice. Expression of CahR4 in mR4(-/-) mouse livers depressed bile acid synthesis below wild-type levels from the elevated levels observed in mR4(-/-). Levels of phosphorylated c-Jun N-terminal kinase (JNK), which is part of a pathway implicated in bile acid-mediated repression of synthesis, was 30% of wild-type levels in mR4(-/-) livers, whereas CahR4 livers exhibited an average 2-fold increase. However, cholate still strongly induced phospho-JNK in mR4(-/-) livers. These results confirm that hepatocyte FGFR4 regulates bile acid synthesis by repression of Cyp7a1 expression. Hepatocyte FGFR4 may contribute to the repression of bile acid synthesis through JNK signaling but is not required for activation of JNK signaling by bile acids.

Because of specificity for both heparin/heparan sulfate and the receptor complex on epithelial cells relative to other fibroblast growth factor (FGF) homologues, there is considerable interest in clinical and commercial applications of FGF7 (also called keratinocyte growth factor or KGF) that require large quantities at reasonable cost. Production of recombinant FGF7 from bacteria suffers from lower yields and recovery relative to FGF1 and FGF2. Fusion of FGF7 at the N-terminus with glutathione-S-transferase (GST) followed by removal of GST by proteolysis while bound to natural ligand heparin improved the intrinsically low yields from Escherichia coli hosts to 3.2 mg per liter per OD(600), which was still only 10% of that for FGF1. Yield of the GST-FGF7 fusion product was improved to about 17 mg per liter per OD(600) in strain BL21(DE3)pLysS by inclusion of 10-100mM magnesium chloride (MgCl(2)) in the culture medium. This improved by about five times the yields of fully active 54ser-FGF7 after proteolytic excision of the GST portion from GST-FGF7 immobilized on heparin-Sepharose. This simple enhancement improves the cost-effectiveness of production of recombinant FGF7 in bacteria for clinical and commercial applications.

BACKGROUND Prostate-specific gene ablation provides a powerful tool for functional characterization of genes that have impact on embryonic development or on other organs, specifically in the prostate. Uniform expression of Cre with high recombinase activity in the prostate is needed for prostate-specific gene ablation based on Cre-loxP recombinations. Currently, available strains of Cre transgenic mice only express Cre recombinase adequately in certain lobes of the prostate. In other lobes, the expression is low and mosaic. Additional strains of transgenic mice expressing high levels of prostate-specific Cre in all prostate lobes would be useful to study the impact of genome manipulation in all prostate lobes. METHODS The ARR2PB composite promoter with improved capacity to drive androgen-responsive gene expression was used to initiate expression of a transgene bearing the cDNA encoding a recently modified Cre recombinase with improved recombination activity. In addition, an insulator element from the chicken globin locus that minimized negative effect on transcription of the transgene imposed by chromosome structure was employed. The derived transgenic founders were crossed with the Z/AP reporter mouse and Fgfr2f/f mice bearing loxP flanking the FGFR2 locus. Immunochemical and mRNA analyses were employed to test expression and efficacy of the Cre recombinase in the prostate and other tissues. RESULTS The ARR2PBi-Cre transgenic mouse specifically and uniformly expressed Cre recombinase in the dorsal, lateral, ventral, and anterior lobes of the prostate, seminal vesicles, and ductus deferens. The Cre recombinase in these tissues effectively excised loxP flanked DNA fragments in the Z/AP reporter that triggered expression of β-galactosidase, and the loxP–flanked FGFR2f/f locus resulting in specific ablation of FGFR2 in the prostate. CONCLUSIONS Compared with the currently available prostate-specific Cre strains, the new ARR2PBi-Cre strain exhibited higher and more uniform expression of Cre recombinase in the prostate as well as in seminal vesicles and ductus deferens. This provides an additional tool for efficient hormone-dependent gene targeting in epithelial cells of all lobes of the adult prostate, seminal vesicle, and ductus deferens. Prostate 57: 160–164, 2003. © 2003 Wiley-Liss, Inc.

Homeostasis of normal prostate and two-compartment nonmalignant prostate tumors is dependent on two-way communication between epithelial and stromal compartments. Independence of epithelial cells on controlling instructions from stroma is a hallmark of extremely malignant epithelial cell tumors. To better understand the evolution of stromal independence during malignant progression, we performed a clonal analysis of stromal cells derived from a well-defined model of two-way stromal-epithelial cell communication that loses response to stroma during prostate tumor progression. Directionally specific signaling from stroma to epithelium contributes to homeostasis between the two compartments. Stromal cells were characterized in respect to expression and activity of isotypes of the fibroblast growth factor (FGF) family of ligands and receptors in addition to morphology and cytoskeletal markers. One stromal subtype (DTS1) exhibited a fibroblast-like morphology and did not display smooth muscle cell (SMC) alpha-actin. The other (DTS2) exhibited SMC alpha-actin and an SMC-like morphology in vitro. Both subtypes expressed FGF7 and equally low levels of FGFR2IIIc mRNA, whereas fibroblast growth factor receptor (FGFR) 1 predominated in DTS1 cells. DTS1 cells also expressed FGF10 and no detectable FGFR3, whereas the absence of FGF10 and presence of FGFR3 distinguished DTS2 cells. Epithelial cell-derived FGF9 bound to FGFR and stimulated growth of specifically FGFR3-positive DTS2 cells, not the FGFR3-negative DTS1 cells. These results demonstrate stromal cell heterogeneity in signal reception of FGF from epithelium. This correlated with potential heterogeneity in the response back to epithelial cells. Epithelium-dependent control of a stromal cell phenotype within a tumor may be a determinant of whether tumors remain in nonmalignant homeostasis or progress to malignancy.

Carbon tetrachloride (CCl(4)) intoxification in rodents is a commonly used model of both acute and chronic liver injury. Recently, we showed that mice in which FGFR4 was ablated from the germline exhibited elevated cholesterol metabolism and bile acid synthesis coincident with unrepressed levels of cytochrome P450 7A (CYP7A), the rate-limiting enzyme in cholesterol disposal. Of the four fibroblast growth factor (FGF) receptor genes expressed in adult liver, FGFR4 is expressed specifically in mature hepatocytes. To determine whether FGFR4 plays a broader role in liver-specific metabolic functions, we examined the impact of both acute and chronic exposure to CCl(4) in FGFR4-deficient mice. Following acute CCl(4) exposure, the FGFR4-deficient mice exhibited accelerated liver injury, a significant increase in liver mass and delayed hepatolobular repair. Chronic CCl(4) exposure resulted in severe fibrosis in livers of FGFR4-deficient mice compared to normal mice. Analysis at both mRNA and protein levels indicated an 8-hour delay in FGFR4-deficient mice in the down-regulation of cytochrome P450 2E1 (CYP2E1) protein, the major enzyme whose products underlie CCl(4)-induced injury. These results show that hepatocyte FGFR4 protects against acute and chronic insult to the liver and prevents accompanying fibrosis. The results show that FGFR4 acts by promotion of processes that restore hepatolobular architecture rather than cellularity while limiting damage due to prolonged CYP2E1 activity.

Background The prairie vole (Microtus ochrogaster) is an emerging animal model for biomedical research because of its rich sociobehavioral repertoire. Recently, lentiviral transgenic technology has been used to introduce the gene encoding the green fluorescent protein (GFP) into the prairie vole germline. However, the efficiency of transgenesis in this species is limited by the inability to reliably produce large numbers of fertilized embryos. Here we examined several factors that may contribute to variability in superovulation success including, age and parentage of the female, and latency to mating after being placed with the male. Methods Females produced from 5 genetically distinct breeder lines were treated with 100 IU of pregnant mare serum gonadotrophin (PMSG) and immediately housed with a male separated by a perforated Plexiglas divider. Ovulation was induced 72 hr later with 30 IU of human chorionic gonadotropin (hCG) and 2 hrs later mating was allowed. Results Superovulation was most efficient in young females. For example, females aged 6-11 weeks produced more embryos (14 +/- 1.4 embryos) as compared to females aged 12-20 weeks (4 +/- 1.6 embryos). Females aged 4-5 weeks did not produce embryos. Further, females that mated within 15 min of male exposure produced significantly more embryos than those that did not. Interestingly, there was a significant effect of parentage. For example, 12 out of 12 females from one breeder pair superovulated (defined as producing 5 or more embryos), while only 2 out of 10 females for other lines superovulated. Conclusions The results of this work suggest that age and genetic background of the female are the most important factors contributing to superovulation success and that latency to mating is a good predictor of the number of embryos to be recovered. Surprisingly we found that cohabitation with the male prior to mating is not necessary for the recovery of embryos but is necessary to recover oocytes. This information will dramatically reduce the number of females required to generate embryos for transgenesis in this species.

Background Endocrine FGF19 and FGF21 exert their effects on metabolic homeostasis through fibroblast growth factor receptor (FGFR) and co-factor betaKlotho (KLB). Ileal FGF19 regulates bile acid metabolism through specifically FGFR4-KLB in hepatocytes where FGFR1 is not significant. Both FGF19 and FGF21 activate FGFR1-KLB whose function predominates in adipocytes. Recent studies using administration of FGF19 and FGF21 and genetic ablation of KLB or adipocyte FGFR1 indicate that FGFR1-KLB mediates the response of adipocytes to both FGF21 and FGF19. Here we show that adipose FGFR1 regulates lipid metabolism through direct effect on adipose tissue and indirect effects on liver under starvation conditions that cause hepatic stress. Methods We employed adipocyte-specific ablations of FGFR1 and FGFR2 genes in mice, and analyzed metabolic consequences in adipose tissue, liver and systemic parameters under normal, fasting and starvation conditions. Results Under normal conditions, the ablation of adipose FGFR1 had little effect on adipocytes, but caused shifts in expression of hepatic genes involved in lipid metabolism. Starvation conditions precipitated a concurrent elevation of serum triglycerides and non-esterified fatty acids, and increased hepatic steatosis and adipose lipolysis in the FGFR1-deficient mice. Little effect on glucose or ketone bodies due to the FGFR1 deficiency was observed. Conclusions Our results suggest an adipocyte-hepatocyte communication network mediated by adipocyte FGFR1 that concurrently dampens hepatic lipogenesis and adipocyte lipolysis. We propose that this serves overall to mete out and extend lipid reserves for neural fuels (glucose and ketone bodies), while at the same time governing extent of hepatosteatosis during metabolic extremes and other conditions causing hepatic stress.