Cancer esearch cular and Cellular Pathobiology oblast Growth Factor Receptor 4 Regulates Tumor sion by Coupling Fibroblast Growth Factor Signaling to R acellular Matrix Degradation

nloaded rrant expression and polymorphism of fibroblast growth factor receptor 4 (FGFR4) has been linked to progression and anticancer drug resistance. We describe here a novel mechanism of tumor progression trix degradation involving epithelial-to-mesenchymal transition in response to membrane-type 1 matrix oproteinase (MT1-MMP, MMP-14) induction at the edge of tumors expressing the FGFR4-R388 risk varoth FGFR4 and MT1-MMP were upregulated in tissue biopsies from several human cancer types includeast adenocarcinomas, where they were partially coexpressed at the tumor/stroma border and tumor on front. The strongest overall coexpression was found in prostate carcinoma. Studies with cultured te carcinoma cell lines showed that the FGFR4-R388 variant, which has previously been associated with ancer prognosis, increased MT1-MMP–dependent collagen invasion. In this experimental model, knockof FGFR4-R388 or MT1-MMP by RNA interference blocked tumor cell invasion and growth in collagen. as coupled with impaired phosphorylation of FGFR substrate 2 and Src, upregulation of E-cadherin, and ssion of cadherin-11 and N-cadherin. These in vitro results were substantiated by reduced MT1-MMP t and in vivo growth of prostate carcinoma cells after the FGFR4-R388 gene silencing. In contrast, down of the alternative FGFR4-G388 allele enhanced MT1-MMP and invasive tumor cell growth in vivo ithin three-dimensional collagen. These results will help to explain the reported association of the and w FGFR4-R388 variant with the progression and poor prognosis of certain types of tumors. Cancer Res; 70(20); 7851–61. ©2010 AACR.


Introduction
Tumor progression is frequently associated with aberrant activation of the fibroblast growth factor (FGF) pathway (1,2).Cancer-associated genetic alterations such as singlenucleotide polymorphisms (SNP), translocations, missense mutations, and gene amplifications or altered expression have been reported for each of the four FGF receptors (FGFR; refs.[1][2][3][4].FGFR4 upregulation occurs in advanced prostate, breast, pancreatic, and gynecologic cancers as well as in rhabdomyosarcomas (3)(4)(5)(6)(7), and in breast cancer, it has been associated with resistance to chemotherapy (8).Furthermore, a SNP in codon 388 of the human FGFR4 gene has been linked to poor prognosis of patients with several types of tumors such as adenocarcinomas of the breast, prostate, and colon as well as head and neck squamous cell carcinomas and melanomas (9)(10)(11)(12)(13)(14).In the FGFR4-R388 risk variant, Gly 388 in the transmembrane domain is changed to arginine, resulting in enhanced stability and prolonged activation of the receptor (15).
FGFRs are activated by the binding of two FGF molecules in a complex with heparin sulfate proteoglycan to their extracellular part.This triggers FGFR dimerization, leading to autophosphorylation of multiple tyrosine residues in the intracellular tyrosine kinase domain (1,2).Subsequent phosphorylation of docking proteins, FGFR substrate 2α and β (FRS2α/β), and downstream signaling through Ras/mitogen-activated protein kinase, phosphatidylinositol 3-kinase (PI3K)/Akt, phospholipase Cγ (PLCγ), and Src pathways then serve to control cell proliferation, differentiation, survival, and migration.In vivo, FGFR signaling has been associated with tumor initiation and growth (1,2).Recently, the FGFR4 G385R mutation in the mouse genome (analogous to the human G388R SNP) has been shown to accelerate WAP-TGFα transgene-induced mammary carcinoma development and progression (16).This was associated with increased transformation and migration/invasiveness of FGFR4-R388 mouse embryonic fibroblasts, whereas the cellular and molecular mechanisms of the increased carcinoma progression remained unclear.
On carcinoma progression, the neoplastic epithelium can become invasive through the process of epithelial-tomesenchymal transition (EMT; refs.17,18).This results in loss of apical-basal polarization, dissociation of epithelial cell-cell junctions, and gain of mesenchymal morphology plus an invasive protein expression pattern (17,18).Alternatively, tumor cells can invade collectively without loosing their cell-cell contacts (18,19).In this process, leading cells protrude pseudopods and provide microtracks by extracellular matrix (ECM) degradation, which allows the following carcinoma cells to migrate as a cohort (20).In human and mouse carcinomas, signs of EMT are frequently observed in the leading cells of collectively invasive tumor fronts (18,19).Membrane-type matrix metalloproteinases (MT-MMP) expressed in the tumor cells or stroma mediate both EMT-induced single-cell invasion and collective tumor invasion (17,(20)(21)(22)(23). MT-MMPs can promote invasiveness by cleaving ECM, cell surface receptors, and pericellular proteins or by activating secreted MMPs (17,24,25).We have recently identified an MT1-MMP/FGFR4 complex by a kinome screen (26).In this complex, the FGFR4 G388R polymorphism stabilizes both FGFR4 and MT1-MMP, thus enhancing receptor tyrosine kinase and proteolytic activities.To understand the biological relevance of the FGFR4/ MT1-MMP interaction in tumor progression, we have here examined the coexpression of FGFR4 and MT1-MMP in human tumors.The functions of the FGFR4 allelic variants in MT1-MMP-dependent tumor cell invasion and growth were defined using stable gene silencing in three-dimensional (3D) cell cultures and tumor xenografts.

3D growth and invasion
Collagen invasion was assessed essentially as described (24) and as further explained in Supplementary Data.For 3D cell culture, single-cell suspension (4 × 10 4 cells/mL) was prepared in 2.2 mg/mL collagen I solution followed by casting 50 μL gels onto 24-well plates and cultured for 6 days in complete medium.Sphere-shaped cell colonies that grew in the gels were stained after paraformaldehyde fixation with TRITC-conjugated phalloidin (Sigma) and 4′,6-diamidino-2-phenylindole (DAPI) for confocal imaging or epifluorescence using Zeiss Axiovert 200 microscope.For relative sphere size, spheres in 30 random phase-contrast images of six separate gels were measured (58-106 spheres/shRNA-transduced cell line, n = 3) using ImageJ software.The number of invasive spheres with surrounding single cells, multicellular sprouts, or nodular extensions of at least three single cells in collagen was counted.The results are expressed as relative ratio of invasive spheres to total random spheres.

Tumor growth in mice
Experiments were approved by the State Provincial Office of Southern Finland.PC3 and DU145 cells were lentivirally transduced with a Renilla luciferase-green fluorescent protein fusion reporter protein.Stable cell pools expressing scrambled, MT1-MMP, and FGFR4-targeting shRNAs were selected by puromycin (Sigma).Cell pools with >95% initial knockdown efficiencies (2 × 10 6 cells/mouse) were implanted into the abdominal subcutis of ICR-severe combined immunodeficient (SCID) male mice (5-7 weeks of age; Taconic) and allowed to grow for 6 to 8 weeks.Tumor size was measured with a caliper, and noninvasive bioluminescence was visualized after i.p. injection of coelenterazine (35 μg in 100 μL PBS; Synchem) using a Xenogen IVIS System.
The experiments with five mice per group were repeated twice with similar results.

Histologic analyses and immunohistochemistry
Tumor and normal frozen tissue arrays (FMC401 and BRF401; US Biomax, Inc.) were stained with pretitrated dilutions of polyclonal anti-FGFR4 antibodies (Santa Cruz Biotechnology) and anti-MT1-MMP mAbs produced by immunization of the MT1-MMP −/− mice (28) as described in Supplementary Data.Mouse tumors and lymph nodes were fixed with 4% paraformaldehyde, dehydrated, and embedded in paraffin followed by H&E and Herovici collagen staining.Vessels with intravasated tumor cells were counted from H&E-stained sections.Frozen sections (20 μm) were subjected to immunofluorescence using indicated primary antibodies and Alexa Fluor 488 and 594 secondary antibodies.
qPCR and FGFR4 alleles mRNA was extracted and quantified in the cell and tissue extracts as well as in Tissue Scan Breast Cancer qPCR Array III (OriGene Technologies) essentially as described (32) and as explained in Supplementary Materials and Methods.The fragments of FGFR4 cDNA containing the G388R site (1,329-1,331 bp) were amplified by PCR (primers: TAC-CAGTCTGCCTGGCTC and AGTACGTGCAGAGGCCTT) and digested with BstN1 (New England Biolabs).R388 allele was identified by a specific 126-bp fragment, and G388 allele by two fragments (97 and 29 bp).Unclear results were confirmed by sequencing.

GeneSapiens
GeneSapiens expression data are derived from the database version 3 predating the published version 4. The normalized expression data in version 3 were constructed from 9,783 human tissue samples as described (33), with the exception of using original CDF files from Affymetrix and somewhat different collection of raw source data files.

Statistical analysis
All numerical values represent mean ± SE or SD.Statistical significance was determined using two-tailed Student's t tests.
To define the cell types that express FGFR4 and MT1-MMP in the tumors, tissue section arrays containing 42 normal or malignant breast tissue samples were subjected to immunohistochemistry. Faint FGFR4 staining was detected in luminal epithelial cells in all four biopsies of normal breast, whereas MT1-MMP was undetectable in the normal epithelium and negligible in stroma (Fig. 1C; Supplementary Fig. S1; modestly positive stroma in two of four biopsies).FGFR4 was increased in carcinoma cells (20 of 38 cases; Fig. 1C and D; Supplementary Fig. S1), and MT1-MMP was increased in the reactive stroma (carcinoma in situ, 3 of 8; invasive ductal carcinoma, 17 of 28; invasive lobular carcinoma, 2 of 2; Fig. 1D), as previously reported (4,(21)(22)(23)34).MT1-MMP was also induced in the myoepithelium that was abutting the stroma of in situ breast carcinomas (7 of 8; Fig. 1C; Supplementary Fig. S1) and in poorly differentiated cells of invasive ductal carcinomas (13 of 28; Fig. 1D; Supplementary Fig. S1) relative to normal tissue.These two cell types also expressed FGFR4 and thus represented the unexpectedly restricted MT1-MMP and FGFR4 codistribution in breast carcinomas (Fig. 1C and D).

MT1-MMP and FGFR4 are coexpressed in tumor edges and prostate carcinoma
To assess FGFR4 and MT1-MMP coexpression in different human tumor types, we first used frozen tissue arrays composed of single sections from 14 different malignant and normal tissue types.MT1-MMP upregulation was observed in the tumor cells (9 of 14; Supplementary Table S1) and/or reactive stroma (9 of 14), whereas FGFR4 expression was mainly found in the tumor cells (Supplementary Table S1).In ovarian, stomach, pancreas, lung, and colon tumor sections containing stromal MT1-MMP, further MT1-MMP expression in the FGFR4-expressing carcinoma cells was observed (Supplementary Table S1).This localized particularly to the tumor cells adjacent to the stroma and into the tumor cell foci as shown for a lung squamous cell carcinoma and colon adenocarcinoma (Fig. 2A; Supplementary Fig. S2).
Further analysis using GeneSapiens database (33) revealed that FGFR4 and MT1-MMP were frequently upregulated in same tumor types including carcinomas of the breast, colon, testis, and uterus when compared with normal tissues (Supplementary Fig. S3A and B).However, the correlation plots of FGFR4 and MT1-MMP expression in individual tissue biopsies from different human tumor types revealed weak or no correlation (Fig. 2B).Two interesting exceptions were a positive correlation in prostate cancers, which did not display markedly enhanced mean MT1-MMP or FGFR4 expression (r = 0.633, n = 357, P < 1 × 10 −8 ) and a negative correlation in sarcomas (r = −0.433,n = 161, P = 1 × 10 −8 ).FGFR4 was exceptionally induced in rhabdomyosarcomas (Fig. 2B), whereas only MT1-MMP was strongly expressed in osteosarcomas, synovial sarcomas, and fibrosarcomas.These expression profiles raise the possibility of FGFR4/MT1-MMP interplay particularly at the tumor edges and in the individual tumors or tumor types such as prostate carcinomas that show limited MT1-MMP and/or FGFR4 induction.

FGFR4-R388 enhances MT1-MMP-mediated prostate carcinoma cell invasion
To determine the effects of FGFR4 and MT1-MMP in cell invasion, we assessed MT1-MMP and  S2).MT1-MMP was expressed in cell lines, which displayed signs of EMT and invaded collagen rapidly as single cells (HT-1080 fibrosarcoma, MDA-MB-231 breast carcinoma, and SCC-25 squamous cell carcinoma).In contrast, MDA-MB-453, ZR-75-1, and BT-474 breast carcinoma cells and WiDr colon carcinoma cells expressed FGFR4 but had negligible MT1-MMP and invasiveness in collagen (Supplementary Table S2).This suggests that the restricted coexpression observed at the tumor edges and poorly differentiated cells in vivo was dependent on the local tumor tissue environment and/or the activated myoepithelium.Importantly, PC3 cells coexpressing the FGFR4-R388 risk allele and MT1-MMP invaded collagen gels efficiently, whereas the DU145 cells homozygous for the FGFR4-G388 allele did not, unless they were transfected with FGFR4-R388 cDNA (Fig. 3; Supplementary Fig. S4B).Collagen invasion was blocked by the depletion of MT1-MMP by siRNA (Supplementary Fig. S4B-D).FGFR1 knockdown seemed to instead increase collagen invasion (Supplementary Fig. S4D-F).Notably, FGFR4 siRNAs inhibited 87% of the PC3 cell invasion (Fig. 3), indicating that the FGFR4-R388 risk variant and MT1-MMP cooperatively promote prostate carcinoma cell invasion in 3D collagen.

FGFR4-R388 and FGFR4-G388 have opposite effects on tumor cell invasion
Lentiviral shRNAs were used next for stable FGFR4 and MT1-MMP gene silencing in PC3 and DU145 cells.To investigate tumor cell growth and invasion in 3D environment (19,35), single cells were implanted inside cross-linked collagen matrix.During a 6-day culture, control shRNA-transduced PC3 and DU145 cells both formed sphere-shaped colonies within collagen (Fig. 4A).Consistent with the rapid PC3 cell (FGFR4; R/R) invasion from atop collagen (see Fig. 3), more single cells and collective cell foci had invaded into collagen around the PC3 cell spheres compared with DU145 cells (FGFR4; G/G; Fig. 4A; Supplementary Fig. S5A).MT1-MMP knockdown decreased the growth and invasion of both PC3 and DU145 cell colonies, as expected (Fig. 4A and B; Supplementary Fig. S5A).Importantly, 95% reduction of mRNAs for the FGFR4-R388 risk allele also inhibited the growth and invasion of PC3 cell spheres, simultaneously increasing the epithelial organization in the cells observed by cortical actin staining (Fig. 4A and B).MT1-MMP protein, but not mRNA expression, decreased in parallel (Fig. 4B and C).In contrast, FGFR4-G388 silencing increased MT1-MMP protein (but not mRNA) expression (Fig. 4B and C) as well as the collective invasion of DU145 cells, as observed by multicellular extensions and irregular shape of the spheres in the fixed 3D collagen gels (Fig. 4A).Unexpectedly, MT1-MMP knockdown resulted in increased expression of mRNA for the MT1-MMP-suppressive FGFR4-G388 in the DU145 cells (Fig. 4B).
Considering the observed changes in epithelial organization within the spheres (Fig. 4A), the expression of cell-cell junctional and proinvasive cadherins was investigated.Notably, E-cadherin, important for stable cell-cell contacts and epithelial quiescence, was increased, whereas N-cadherin and particularly cadherin-11, linked to prostate cancer  S1 for more information).Arrowheads indicate MT1-MMP that is coexpressed with FGFR4 in the tumor-stromal borders and invasive carcinoma edges.B, overall coexpression of MT1-MMP and FGFR4 in human cancers was analyzed using GeneSapiens database.Pearson correlation coefficients (r) between the genes, P values for coefficient, and amounts of samples (n) are also shown.(36), were suppressed by either FGFR4-R388 or MT1-MMP knockdown (Fig. 4D).Coincidentally, the FGF2induced phosphorylation of FRS2 and Src was inhibited in PC3 cells, whereas FGFR4-G388 silencing altered neither FRS2 or Src phosphorylation nor cadherins in DU145 cells (Fig. 4D).These shRNA-induced changes did not correlate with ERK, Akt, or PLCγ activation (Fig. 4D; Supplementary Fig. S5B).Furthermore, stable silencing of MT1-MMP or either variant of FGFR4 had minor effects on cell growth in the monolayer cultures (Supplementary Fig. S5C and D).These results indicate that both FGFR4-R388 and MT1-MMP are required for EMT and collagen invasion of PC3 cells.They also indicate the FGFR4-G388 allele has an opposite effect on MT1-MMP and tumor cell invasion in 3D collagen.

FGFR4 controls MT1-MMP-dependent ECM degradation and tumor progression involving EMT in vivo
To investigate the effects of FGFR4 and MT1-MMP in tumorigenesis in vivo, tumor growth, invasion, and collagen content were analyzed after s.c.injection of PC3 and DU145 cells into SCID mice.The growth rates of PC3 tumors were dramatically decreased after stable knockdown of either MT1-MMP or the FGFR4-R388 risk variant (Fig. 5A; Supplementary Fig. S6A; >70% reduction of respective mRNAs in the tumors).Vascular and lymph node invasion decreased accordingly (Fig. 5B; Supplementary Fig. S6B; Supplementary Table S3).Notably, only control PC3 cells were able to fully colonize the lymph nodes, although tumor cells were occasionally detected in a few lymph vessels after MT1-MMP or FGFR4-R388 knockdown (Supplementary Fig. S6B).Furthermore, human tumor cell mRNA was detected by qPCR only in the lungs of mice carrying control PC3 tumors (Supple-mentary Table S3).Importantly, the severely impaired growth, mitotic index, as well as vascular and lymph node invasion of MT1-MMP and FGFR4-R388 knockdown tumors correlated inversely with the increased tumor collagen content (Fig. 5A and B; Supplementary Figs.S7 and S8A and B).Both the fibrous capsule around the tumor and intratumoral collagen increased markedly, although collagen (collagen IA, IIA, and IV) mRNA levels were not altered in the same tumors (Fig. 5C; Supplementary Fig. S8B and C).MT1-MMP knockdown increased the collagen content of both PC3 and DU145 tumors simultaneously with their decreased growth (Fig. 5A and C; Supplementary Figs.S7 and S8B  and D).In contrast, FGFR4-G388 silencing decreased collagen accumulation and increased the growth and vascular invasion of the less aggressive DU145 tumors (Fig. 5A-C; Supplementary Figs.S7 and S8A, B, and D).
To assess if the opposite effects of FGFR4-R388 and FGFR4-G388 silencing on collagen accumulation and tumor spread involved differential MT1-MMP regulation in vivo, frozen PC3 and DU145 tumor sections were subjected to immunohistochemistry.As expected on the basis of mRNA expression and the MT1-MMP protein regulation by the FGFR4 variants in the corresponding cultured cells (see Fig. 4C and Supplementary Fig. S4A), more MT1-MMP was detected in PC3 tumors than in DU145 tumors (Fig. 6A).The staining was specific because only rare MT1-MMP-expressing cells were observed after MT1-MMP knockdown (Fig. 6A).Importantly, the knockdown of the FGFR4-R388 risk variant also decreased MT1-MMP dramatically in the PC3 tumors, whereas FGFR4-G388 knockdown increased MT1-MMP that became abundant particularly at the edges of the DU145 tumors (Fig. 6A).Strikingly, the impaired growth and invasion of the MT1-MMP and FGFR4-R388 knockdown PC3 tumors were coupled with increased epithelial polarization and some acinar lumen formation (Fig. 6B).Basement membrane collagen IV accumulated around the tumor cells, and cadherins were translocated to cell-cell junctions in these knockdown tumors, which thus displayed increased epithelial differentiation and reduced signs of EMT (Fig. 6B).In contrast, FGFR4-G388 silencing did not result in collagen IV accumulation in the DU145 tumors (Supplementary Fig. S8D).These results indicate that when coexpressed with MT1-MMP, the FGFR4-R388 risk variant stimulates EMT and MT1-MMP-dependent ECM degradation, thus promoting tumor progression.

Discussion
Collective cell invasion and EMT have emerged as important mechanisms for tissue invasion and metastasis of tumor cells, which are the rate-limiting steps in cancer progression (19,(37)(38)(39).MT1-MMP-mediated ECM degradation by tumor cells or stromal cells is essential for both types of invasion through basement membranes and stromal interstitial matrix (19,40,41).We observed an unexpectedly selective coexpression of MT1-MMP and FGFR4 in the human tumor-stromal borders, tumor invasion fronts, and some poorly differentiated invasive breast carcinoma cells.This raised the possibility of a yet undefined regulatory mechanism of tumor invasion involving the MT1-MMP/FGFR4 membrane complexes that are activated by the FGFR4 G388R SNP associated with poor cancer prognosis (26).Indeed, we found that stable silencing of either FGFR4-R388 or MT1-MMP by lentiviral shRNAs inhibited MT1-MMP-mediated human PC3 prostate adenocarcinoma cell invasion into 3D collagen.Simultaneously, FGF-induced activation of the FRS2-Src pathway and tumor progression were blocked along with signs of reversed EMT, or mesenchymalto-epithelial transition.
We found that either FGFR4-R388 or MT1-MMP silencing decreased MT1-MMP in cultured PC3 cells and the corresponding tumor xenografts, which supports our recent results of MT1-MMP protein stabilization by the FGFR4-R388 risk variant.Consistent with MT1-MMP being a major collagenase in vivo (35,42), the reduced MT1-MMP correlated with increased accumulation of collagenous matrix within and around the tumors.These results are important because collagen-containing stroma and cross-linked 3D collagen serve as a physical barrier, which degradation by MT1-MMP facilitates cell growth and invasion (35,40,41).Accordingly, mitotic index as well as vascular and lymph node invasion in vivo decreased after FGFR-R388 or MT1-MMP silencing.MT1-MMP and collagen mRNAs were not affected, suggesting that FGFR4-R388 regulates collagen degradation and thus invasive tumor growth by increasing MT1-MMP protein in the tumor cells.This FGFR4-R388 function was distinct from mitogenic signaling because FGFR4-R388 silencing did not impair FGF signaling through ERK1/2 and PI3K/Akt pathways or cell growth in adherent monolayer cultures.Furthermore, the most potent mitogenic FGFR, FGFR1, was not required for collagen invasion (1)(2)(3).Silencing either one of the FGFR4 alleles or FGFR1 has been reported to inhibit the proliferation and migration of prostate carcinoma cells through a lamininrich matrix (4).However, unlike invasion across basement   membranes or interstitial collagen, these assays do not require matrix proteolysis (17,24,42).MT1-MMP regulation by FGFR4-R388 thus provides a new mechanism of collagen degradation and cell invasion.This helps to explain the contribution of the G388R SNP to the progression of certain tumors and poor prognosis of cancer patients (9)(10)(11)(12)(13)(14)16).
In contrast, the silencing of the alternative FGFR4-G388 alleles resulted in increased DU145 tumor growth and vascular invasion, as well as MT1-MMP protein content particularly at the tumor edges.This was coupled with partial loss of intratumoral collagen and fibrous capsule around the tumors.Because the MT1-MMP mRNA was not affected, also this negative MT1-MMP regulation occurred at the protein level.Interestingly, whereas the inverse regulation of MT1-MMP by the FGFR4 variants was posttranscriptional, stable MT1-MMP silencing instead increased FGFR4-G388 mRNA.Thus, our results suggest that a transcriptional FGFR4-G388 suppression on MT1-MMP induction at the tumor edges could provide the tumor cells that carry this FGFR4 allele with a feedback mechanism to promote invasion.However, the FGFR4 G388R SNP-dependent MT1-MMP/FGFR4 interplay discovered here in cells coexpressing both proteins may not apply in tumors expressing MT1-MMP exclusively in stromal cells and FGFR4 in tumor cells.Indeed, only stromal MT1-MMP has been reported to be induced in MMTV-PymT transgenic mouse mammary carcinoma (21,22), thus explaining why the G385R mutation had no effect on the tumor progression in this breast carcinoma model (16).Importantly, we also observed increased epithelial differentiation of PC3 cells after FGFR4-R388 or MT1-MMP silencing.In vivo, this was observed as acinar morphology and increased cell-cell junctional cadherins and basement membrane components.Many of these components, which support epithelial cell differentiation and quiescence (42), are MT1-MMP substrates (25).Because there were no consistent changes in laminin or type IV collagen mRNAs, we suppose that the MT1-MMP/FGFR4-R388 interaction enhanced the degradation of both basement membranes and fibrillar collagen in the rapidly growing and invasive PC3 tumors.MT1-MMP can also induce shedding of cadherins and other receptors, which could directly contribute to the EMT (43).However, increased MT1-MMP, invasive growth, and vascular invasion of cell cohorts did not involve loss of E-cadherin or upregulation of the proinvasive cadherins in the DU145 cells.In these cells, the FRS2-Src pathway was not altered by FGFR4-G388 silencing, and in the corresponding tumors, basement membrane collagen was mainly restricted to the blood vessels.Therefore, the functional MT1-MMP/FGFR4-R388 interaction seems to contribute to EMT both by preventing basement membrane accumulation and by altering cadherins through FGF signaling.
Tumor cells have recently been found to develop resistance to the therapy through increased invasion (38,45).Better understanding of different invasion mechanisms is thus essential for the development of novel interventions to inhibit tumor progression.Our results of increased MT1-MMP and invasive tumor growth after FGFR4-G388 silencing will help to explain the reported tumor-suppressive functions of this allele (3,46,47).As such, FGFR4 targeting could even be harmful by increasing invasiveness in patients with homozygous G388 alleles.Most importantly, the present results identify the FGFR4-R388 risk variant as a critical enhancer of MT1-MMP, ECM degradation, and cadherin switch independently of mitogenic FGF signaling, providing a mechanistic basis for the proinvasive FGFR-R388 function in tumor progression.FGFR4-R388 could serve as an ideal target to inhibit tumor invasion because it is selectively upregulated in aggressive tumors (2-4), yet unlike MT1-MMP (42,48), it can be deleted from mice without causing obvious developmental or functional defects (3).

Disclosure of Potential Conflicts of Interest
No potential conflicts of were disclosed.

Research
FGFR4 expression in relation to collagen invasion of cultured tumor cells.Consistent with the coexpression in human prostate cancers in vivo, PC3 and DU145 prostate adenocarcinoma cells expressed both MT1-MMP and homozygous FGFR4-R388 or FGFR4-G388 variant as well as FGFR1 (Supplementary Fig. S4A; ref. 4).Unexpectedly, most other tumor cell lines analyzed expressed either MT1-MMP or FGFR4 mRNA (Sup-plementary Table

Figure 1 .
Figure 1.FGFR4 is coexpressed with MT1-MMP in vivo.A, MT1-MMP and FGFR4 mRNA expression and the FGFR4 amino acid 388 sequence in human breast carcinomas.The biopsies from patients with grade 2 or 3 tumors and homozygous G/G, heterozygous R/G, or homozygous R/R FGFR4 allele are indicated.B, chart shows relative values of average mRNA expression in tumors with different FGFR4 alleles (G/G, n = 21; R/G, n = 23; R/R, n = 4).Columns, mean; bars, SE.C and D, FGFR4 and MT1-MMP immunohistochemistry of frozen tissue sections from human normal breast and carcinomas in situ (C) or invasive breast carcinomas (D).Arrowheads indicate MT1-MMP and FGFR4 codistribution in the myoepithelium (carcinoma in situ; C) and in the invasive poorly differentiated breast carcinomas (D).

Figure 2 .
Figure 2. Coexpression of MT1-MMP and FGFR4 in tumor edges and prostate carcinoma.A, FGFR4 and MT1-MMP immunohistochemistry of frozen tissue sections from human lung squamous cell carcinomas and colorectal adenocarcinomas (see Supplementary TableS1for more information).Arrowheads indicate MT1-MMP that is coexpressed with FGFR4 in the tumor-stromal borders and invasive carcinoma edges.B, overall coexpression of MT1-MMP and FGFR4 in human cancers was analyzed using GeneSapiens database.Pearson correlation coefficients (r) between the genes, P values for coefficient, and amounts of samples (n) are also shown.

Figure 3 .
Figure 3. FGFR4-R388 promotes collagen invasion of prostate carcinoma cells.Light micrographs of collagen cross-sections visualize the invasion of PC3 (FGFR4; R/R) and DU145 (FGFR4; G/G) cells after transfection of pCR3.1 empty vector (Mock), FGFR4 siRNAs (FGFR4si), or FGFR4-G and FGFR4-R expression vectors.Quantitative assessment of the invasion is indicated in each micrograph.Results are expressed as the average number of invasive foci per microscopic field (mean ± 1 SE, n = 3).

Figure 4 .
Figure 4. Cooperative FGFR4-R388 and MT1-MMP-dependent EMT or knockdown of the MT1-MMP-suppressive FGFR4-G388 promotes invasive tumor cell growth.A, PC3 (FGFR4; R/R) and DU145 (FGFR4; G/G) cells were embedded in 3D collagen as single-cell suspension after stable MT1-MMP or FGFR4 knockdown by lentiviral shRNAs.Confocal laser scanning micrographs show the cells stained for filamentous actin (red) after the 6-d assay.DAPI counterstaining (blue) visualizes nuclei.The average size of cell spheres and number of the invasive spheres are indicated below each micrograph (58-106 spheres/cell line, mean ± 1 SE, n = 3).B, MT1-MMP and FGFR4 mRNAs were detected by qPCR in the stable knockdown cells.Columns, mean (n = 3); bars, SE.C, endogenous FGFR4 variants regulate MT1-MMP protein expression.The cells were incubated with FGF2 (10 ng/mL) for 15 min and subjected to immunoblotting.Mean values of relative MT1-MMP levels are expressed below each lane (n = 3).D, the cells were subjected to immunoblotting for cadherins and activated signaling intermediates as indicated (n = 3).Ponceau red staining served as a loading control.

Figure 5 .
Figure 5. Endogenous FGFR4 controls MT1-MMP-dependent ECM turnover, tumor growth, and invasion in vivo.A, luminescence images of representative tumors grown in the abdominal subcutis of SCID mice are shown 32 d after the injection of Renilla luciferase expressing scrambled, FGFR4, or MT1-MMP shRNA-transduced PC3 (FGFR4; R/R) and DU145 (FGFR4; G/G) cells.Luminescence scales are shown on the right.Chart illustrates tumor growth curves.Points, mean (n = 5); bars, SE.B, light micrographs of H&E-stained PC3 and DU145 tumor sections.Dashed lines mark tumor-stromal borders and asterisks indicate tumor cells in the stromal vessels of scrambled PC3 tumors and FGFR4 knockdown DU145 tumors.For mitotic indices, mitotic figures relative to total nuclei were quantified (20 microscopic fields/cross-section).Columns, mean (n = 5); bars, SE.Stromal vessels containing tumor cells were quantified for tumor vascular invasion indices (six cross-sections/tumor).Columns, mean (n = 5); bars, SE.C, light micrographs of Herovici collagen staining (red) are shown for PC3 and DU145 tumors.Collagen contents were quantified as red collagen staining.Arrowheads indicate increased collagen.