Transforming properties of TC-1 in human breast cancer: interaction with FGFR2 and beta-catenin signaling pathways

Breast cancer development is associated with gene amplification and over expression that is believed to have a causative role in oncogenesis. Previous studies have demonstrated that over expression of TC-1(C8orf4) mRNA occurs in approximately 50% of breast cancer cell lines and primary tumor specimens. Here, we show that TC-1 has transforming properties in human mammary epithelial (HME) cells and its expression is mechanistically linked to FGFR signaling cascades. In vitro experiments demonstrate that TC-1 over expression mediates both anchorage-independent and growth factor-independent proliferation of HME cells. TC-1 was down regulated by the FGFR inhibitor PD173074 in the breast cancer cell line SUM-52 that also has an FGFR2 gene amplification and over expression. Furthermore, forced expression of FGFR2 in HME cells increased the level of expression of endogenous TC-1 mRNA. TC-1 has been implicated as a modulator of Wnt/beta-catenin signaling in 293 cells and in gastric cancer cells. However, while we did find increased expression of a subset of beta-catenin target genes in TC-1 over expressing cells, we did not find an association of TC-1 with global expression of beta-catenin target genes in our cells. Taken together, our data suggest that TC-1 over expression is transforming and may link with the FGFR pathway in a subset of breast cancer.     

Finite proliferative lifespanin vitro of a human breast cancer cell strain isolated from a metastatic lymph node

Summary We recently described culture conditions that allow proliferation of metastatic human breast cancer cells from biopsy specimens of certain patient samples. These conditions resulted in the development of an immortalized cell strain designated SUM-44PE. These same culture conditions were used to isolate a human breast cancer cell strain from a metastatic lymph node of a separate breast cancer patient. The SUM-16LN human breast cancer cells isolated from this specimen were cultured either in serum-free medium or serum-containing medium supplemented with insulin and hydrocortisone. Unlike the SUM-44PE cells that have proliferated in culture continuously for over two years, SUM-16LN cells proliferated in culture for approximately 200 days and underwent 15 to 20 population doublings before undergoing cell senescence. No cells of this strain proliferated beyond passage 8. SUM-16LN cells were keratin-19 positive and had an aneuploid karyotype. These cells overexpressed p53 protein and had an amplified epidermal growth factor (EGF) receptor gene that resulted in high level expression of tyrosine phosphorylated EGF receptor protein. Despite the presence of high levels of tyrosine phosphorylated EGF receptor in these cells, they proliferated in serum-free, EGF-free medium and did not secrete detectable levels of EGF-like mitogenic growth factor. In addition, these cells were potently growth inhibited by all concentrations of exogenous EGF tested and by the neutralizing EGF receptor antibody Mab 425. These results suggest that the high level of tyrosine phosphorylated EGF receptor present in these cells is the direct result of receptor overexpression and not the result of the presence of a simulatory ligand. Thus, SUM-16LN represents a human breast cancer cell strain that exhibited genetic and cellular characteristics of advanced human breast cancer cells. Nevertheless, these cells exhibited a finite proliferative lifespan in culture, suggesting that cellular immortalization is not a phenotype expressed by all human breast cancer cells.     

KAT6A,a Chromatin Modifier from the 8p11-p12 Amplicon is a Candidate Oncogene in Luminal Breast Cancer

The chromosome 8p11-p12 amplicon is present in 12% to 15% of breast cancers, resulting in an increase in copy number and expression of several chromatin modifiers in these tumors, including KAT6A. Previous analyses in SUM-52 breast cancer cells showed amplification and overexpression of KAT6A, and subsequent RNAi screening identified KAT6A as a potential driving oncogene. KAT6A is a histone acetyltransferase previously identified as a fusion partner with CREB binding protein in acute myeloid leukemia. Knockdown of KAT6A in SUM-52 cells, a luminal breast cancer cell line harboring the amplicon, resulted in reduced growth rate compared to non-silencing controls and profound loss of clonogenic capacity both in mono-layer and in soft agar. The normal cell line MCF10A, however, did not exhibit slower growth with knockdown of KAT6A. SUM-52 cells with KAT6A knockdown formed fewer mammospheres in culture compared to controls, suggesting a possible role for KAT6A in self-renewal. Previous data from our laboratory identified FGFR2 as a driving oncogene in SUM-52 cells. The colony forming efficiency of SUM-52 KAT6A knockdown cells in the presence of FGFR inhibition was significantly reduced compared to cells with KAT6A knockdown only. These data suggest that KAT6A may be a novel oncogene in breast cancers bearing the 8p11-p12 amplicon. While there are other putative oncogenes in the amplicon, the identification of KAT6A as a driving oncogene suggests that chromatin-modifying enzymes are a key class of oncogenes in these cancers, and play an important role in the selection of this amplicon in luminal B breast cancers.     

Blocking of FGFR signaling inhibits breast cancer cell proliferation through downregulation of D-type cyclins

Overexpression of fibroblast growth factor receptor (FGFR) tyrosine kinases has been found in many human breast cancers and has been associated with poor patient prognosis. In order to understand the mechanism by which FGFR mediates breast cancer cell proliferation, we used a low molecular weight compound, PD173074, that selectively inhibits FGFR tyrosine kinase activity and autophosphorylation. This potential anticancer agent caused a G1 growth arrest of MDA-MB-415, MDA-MB-453 and SUM 52 breast cancer cells. Our analyses revealed that FGFR signaling links to the cell cycle machinery via D-type cyclins. PD173074-mediated inhibition of FGFR activity caused downregulation of cyclin D1 and cyclin D2 expression, inhibition of cyclin D/cdk4 activity and, as a consequence, reduction of pRB phosphorylation. Retroviral-mediated ectopic expression of cyclin D1 prevented pRB hypophosphorylation and the cell cycle G1 block in PD173074-treated cells, suggesting a central role for D cyclins in proliferation of FGFR-driven breast cancer cells. The repression of FGFR activity caused downregulation of MAPK in MDA-MB-415 and MDA-MB-453 cells. In SUM 52 cells, both MAPK and PI3K signaling pathways were suppressed. In conclusion, results shown here describe a mechanism by which FGFR promotes proliferation of breast cancer cells.     

FGFR2 isoforms support epithelial-stromal interactions in thyroid cancer progression

Alternate splicing yields two distinct isoforms of the fibroblast growth factor (FGF) receptor FGFR2-IIIb and FGFR2-IIIc varying their extracellular structure in human thyroid cancer, in which FGFR expression is commonly dysregulated. In this study, we characterized the function of these variants in modulating thyroid cancer behavior. Enforced expression of either FGFR2-IIIb or FGFR2-IIIc in thyroid epithelial cancer cells reduced expression of fibronectin, MAGE-A3 and MMP9, while increasing p21 and enhancing Rb dephosphorylation. Consistent with these tumor-suppressive properties, FGFR2-IIIb and FGFR2-IIIc each diminished invasive behavior in vitro and reduced tumor growth and metastasis in vivo. Notably, these effects contrasted with those produced by expression of these FGFR isoforms in fibroblasts, in which they both stimulated cell growth. Moreover, in xenograft tumors generated by coimplantation of epithelial and fibroblast cells expressing that same isoform, there was no significant effect on tumor progression. Conversely, FGFR2-IIIb expression in epithelial cells yielded higher FGF4/FGF7 expression that, in the presence of FGFR2-IIIc-expressing fibroblasts, enhanced tumor progression. Together, our findings highlight the importance of cellular context in assigning growth properties to growth factor receptor isoforms. More specifically, they show how alternative splicing of FGFR2 yields heteroisoforms critical to the growth-promoting actions of FGFs that exert distinct epithelial-stromal effects in thyroid cancer.     

Inhibition of fibroblast growth factor receptor 2 attenuates proliferation and invasion of pancreatic cancer

The alternative splicing of the extracellular domain of fibroblast growth factor receptor (FGFR)‐2 generates the IIIb and IIIc isoforms. Expression of FGFR‐2 IIIb correlates with vascular endothelial growth factor‐A (VEGF‐A) expression and venous invasion of pancreatic ductal adenocarcinoma (PDAC). By contrast, FGFR‐2 IIIc expression correlates with faster development of liver metastasis after surgery, and increased proliferation rates and invasion of the cancer. In this study, we analyzed the expression and roles of total FGFR‐2 (both isoforms) to determine the effectiveness of FGFR‐2‐targeting therapy for PDAC. Immunohistochemically, FGFR‐2 was highly expressed in 25/48 (52.1%) PDAC cases, and correlated with advanced stage cancer. In FISH analysis, FGFR2 was amplified in 3/7 PDAC cell lines. We stably transfected an FGFR‐2 shRNA targeting the IIIb and IIIc isoforms into FGFR2‐amplified PDAC cells. The proliferation rates, migration, and invasion of FGFR‐2‐shRNA‐transfected cells were lower than those of control cells in vitro. In response to FGF‐2, FGFR‐2‐shRNA‐transfected cells showed decreased phosphorylation of ERK compared with control cells. The FGFR‐2‐shRNA‐transfected cells also expressed lower levels of vascular endothelial growth factor‐A than control cells, and formed smaller s.c. tumors in nude mice. These findings suggest that FGFR‐2 is a therapeutic target for inhibition in PDAC.     

Targeting FGFR2 with alofanib (RPT835) shows potent activity in tumour models

Alofanib (RPT835) is a novel selective allosteric inhibitor of fibroblast growth factor receptor 2 (FGFR2). We showed previously that alofanib could bind to the extracellular domain of FGFR2 and has an inhibitory effect on FGF2-induced phoshphorylation of FRS2α. In the present study, we further showed that alofanib inhibited phosphorylation of FRS2α with the half maximal inhibitory concentration (IC50) values of 7 and 9 nmol/l in cancer cells expressing different FGFR2 isoforms. In a panel of four cell lines representing several tumour types (triple-negative breast cancer, melanoma, and ovarian cancer), alofanib inhibited FGF-mediated proliferation with 50% growth inhibition (GI50) values of 16–370 nmol/l. Alofanib dose dependently inhibited the proliferation and migration of human and mouse endothelial cells (GI50 11–58 nmol/l) compared with brivanib and bevacizumab. Treatment with alofanib ablated experimental FGF-induced angiogenesis in vivo. In a FGFR-driven human tumour xenograft model, oral administration of alofanib was well tolerated and resulted in potent antitumour activity. Importantly, alofanib was effective in FGFR2-expressing models. These results show that alofanib is a potent FGFR2 inhibitor and provide strong rationale for its evaluation in patients with FGFR2-driven cancers.     

Increased expression of fibroblast growth factor 8 in human breast cancer

Fibroblast growth factor 8 (FGF8) is an important developmental protein which is oncogenic and able to cooperate with wnt-1 to produce mouse mammary carcinoma. The level of expression of FGF8 mRNA was measured in 68 breast cancers and 24 non-malignant breast tissues. Elevated levels of FGF8 mRNA were found in malignant compared to non-malignant breast tissues with significantly more malignant tissues expressing FGF8 (P=0.019) at significantly higher levels (P=0.031). In situ hybridization of breast cancer tissues and analysis of purified populations of normal epithelial cells and breast cancer cell lines showed that malignant epithelial cells expressed FGF8 mRNA at high levels compared to non-malignant epithelial and myoepithelial cells and fibroblasts. Although two of the receptors which FGF8 binds to (FGFR2-IIIc, FGFR3-IIIc) are not expressed in breast cancer cells, an autocrine activation loop is possible since expression of fibroblast growth factor receptor (FGFR) 4 and FGFR1 are retained in malignant epithelial cells. This is the first member of the FGF family to have increased expression in breast cancer and a potential autocrine role in its progression.     

Fibroblast growth factor receptor expression reflects cellular differentiation in human oral squamous carcinoma cell lines

This study examined the expression of fibroblast growth factor receptor 2 (FGFR 2) splice variants, IIIb and IIIc, in normal and malignant human oral keratinocytes and in normal oral fibroblasts by RT-PCR using both exon-specific primers and primers common to both FGFR 2 isoforms. Fibroblasts expressed exclusively FGFR 2/IIIc whilst the normal and malignant keratinocytes co-expressed FGFR 2/IIIb and FGFR 2/IIIc. Well- differentiated keratinocytes expressed proportionally more FGFR 2/IIIb than IIIc whereas the poorly-differentiated cells expressed more FGFR 2/IIIc than IIIb. The normal and malignant keratinocytes, but not fibroblasts, expressed an additional amplification product, which consisted of both IIIb and IIIc of FGFR 2 joined by an extra base pair and with the intronic sequence removed. The results indicate that the expression of FGFR 2 isoforms reflects the degree of cellular differentiation in normal and malignant human oral keratinocytes and that receptor complexes of FGFR 2/IIIb and IIIc may regulate ligand- receptor interactions.      

Expression of fibroblast growth factor (FGF)-8 isoforms and FGF receptors in human ovarian tumors

FGF-8 is a mitogenic growth factor, which is widely expressed during embryonic development but only at a very low level in adult tissues. Alternative splicing of the human FGF-8 gene potentially allows coding for 4 protein isoforms (a, b, e, f), which differ in their transforming capacity. The FGF-8 isoforms preferentially activate the receptors FGFR1IIIc, FGFR2IIIc, FGFR3IIIc and FGFR4. FGF-8 is over-expressed in human breast and prostate cancers. Expression has also been found in RT-PCR studies of human ovarian and testicular cancers. The present study was undertaken to examine which FGF-8 isoforms are expressed in ovarian cancer and whether FGF-8 receptors are also expressed. Specimens from 5 normal human ovaries and 51 ovarian tumors (1 benign tumor, 8 borderline malignancies, 42 malignant tumors of different histopathological types) were studied by RT-PCR and immunohistochemistry. FGF-8 isoform b was expressed in all ovarian tumors and in all 7 ovarian-cancer cell lines studied. Isoform a was co-expressed in 9 malignant ovarian tumors. FGF-8 mRNA was not detected by RT-PCR of 3 normal ovary samples. Immunohistochemical staining localized FGF-8 protein to cancer cells. In general, the increased intensity of FGF-8 staining was associated with loss of differentiation within the tumors (Bowker's test, p = 0.37). FGF-8 staining of surface epithelium observed on 2 normal ovaries was very faint. RT-PCR showed that FGFR1IIIc, FGFR2IIIc and FGFR4 were the FGF-8 receptors expressed in normal ovaries and in ovarian tumors. FGF-8 receptor immunoreactivity was preferentially found in normal ovary surface epithelium and tumor cells but also in some stromal cells. Collectively, our results show that ovarian cancers of a wide variety of histological types expressing receptors for FGF-8 have acquired the capacity of expressing FGF-8. This suggests that FGF-8 has an important role in ovarian tumorigenesis.     

Cytotoxicity of a novel fibroblast growth factor receptor targeted immunotoxin on a human ovarian teratocarcinoma cell line

Basic fibroblast growth factor (bFGF) is an important tumor-associated growth factor that contributes to proliferation and angiogenesis of tumor. The high-affinity receptor for bFGF, fibroblast growth factor receptor (FGFR), is found to be overexpressed in a number of tumor cells. For the purpose of exploring the significance of bFGF/FGFR in tumor-targeted therapy, a recombinant immunotoxin contained the N-terminal 389 residues of diphtheria toxin (DT), and the full length of human bFGF was designed, expressed, and purified. The bioactivity of the product was evaluated by testing the cytotoxicity on PA-1 cells (a human ovarian teratocarcinoma cell line with high-level expression of FGFR) in vitro. The immunotoxin showed a significant cytotoxicity on PA-1 cells (IC(50) 8 - 9 ng/mL), and this effect could be antagonized by equine diphtheria antitoxin (DAT), bFGF, anti-bFGF monoclonal antibody (MAb), and anti- FGFR polyclonal antibody (PAb), respectively. Additionally, Chinese hamster ovary (CHO) cells and Hep-2 cells (a human epidermoid laryngocarcinoma cell line) with low expression of FGFR were tested to be resistant to the immunotoxin. The results indicated that FGFR might be an effective target for tumor therapy, and bFGF-mediated immunotoxin could be a potential candidate in the treatment of cancer.     

Mechanisms of FGFR3 actions in endocrine resistant breast cancer

Although endocrine therapy has dramatically improved the treatment of breast cancer therapeutic resistance and tumour recurrence occurs, even in estrogen receptor (ER) positive cases. Identifying and understanding the molecular mechanisms which underpin endocrine resistance is therefore important if future therapeutic strategies are to be developed. Members of the fibroblast growth factor (FGF) and fibroblast growth factor receptor (FGFR) families have been implicated in breast cancer development and progression. Our results demonstrate that culture of michigan cancer foundation - 1 (MCF)7 cells with FGF1 results in reduced sensitivity to tamoxifen in vitro. Furthermore, our tissue microarray expression data demonstrates that FGFR3 expression is increased in tamoxifen resistant breast tumours. To confirm that activation of FGFR3 reduced sensitivity to tamoxifen we used an inducible activation system and a constitutively active mutant of FGFR3 expressed in MCF7 cells. Activation of FGFR3 reduced sensitivity to tamoxifen and Fulvestrant but did not lead to phosphorylation of ER demonstrating that FGFR3 does not feedback to modulate ER activity. FGFR3 activation in MCF7 cells stimulated activation of the mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase (PI3K) signalling pathways, both of which have been implicated in tamoxifen resistance in breast cancer. Furthermore, our data indicates that activation of phospholipase C gamma is a key-signalling event regulating MAPK and PI3K activation and that its activation reduces sensitivity to tamoxifen. Therefore, we hypothesise that FGFRs could play an integral part, not only in breast cancer development but also in resistance to endocrine-therapy.     

Enhanced invasion and tumor growth of fibroblast growth factor 8b-overexpressing MCF-7 human breast cancer cells

Fibroblast growth factor 8 (FGF-8) is a secreted heparin-binding protein, which has mitogenic and transforming activity. Increased expression of FGF-8 has been found in human breast cancer, and it has a potential autocrine role in its progression. Human FGF-8 is alternatively spliced to generate four protein isoforms (a, b, e, and f). Isoform b has been shown to be the most transforming. In this work, we studied the role of FGF-8b in the growth (in vitro and in vivo) of MCF-7 human breast cancer cells, which proliferate in an estrogen-dependent manner. Constitutive overexpression of FGF-8b in MCF-7 cells down-regulated FGF-8b-binding receptors FGF receptor (FGFR) 1IIIc, FGFR2IIIc, and FGFR4 found to be expressed in these cells. FGF-8b overexpression led to an increase in the anchorage-independent proliferation rate in suspension culture and colony formation in soft agar, when MCF-7 cells were cultured with or without estradiol. FGF-8b also provided an additional growth advantage for cells stimulated with estradiol. In addition, FGF-8b-transfected cells invaded more actively through Matrigel than did control cells. This was possibly due to the increased secretion of matrix metalloproteinase 9. In vivo, FGF-8b-transfected MCF-7 cells formed faster growing tumors than vector-only-transfected cells when xenografted into nude mice. The tumors formed by FGF-8b-transfected cells were more vascular than the tumors formed by vector-only-transfected cells. In conclusion, FGF-8b expression confers a growth advantage to MCF-7 breast carcinoma cells, both in vitro and in vivo. In addition to stimulation of proliferation, this growth advantage probably arises from increased invasion and tumor vascularization induced by FGF-8b. The results suggest that FGF-8b signaling may be an important factor in the regulation of tumorigenesis and progression of human breast cancer.     

Fibroblast growth factor 8 is expressed at higher levels in lactating human breast and in breast cancer

Fibroblast growth factor 8 can transform NIH3T3 cells and its expression has been found to be associated with breast and prostate cancer. Following our finding that fibroblast growth factor 8 mRNA expression is increased in breast cancer, we have undertaken an immunohistochemistry study of fibroblast growth factor 8 expression in a series of human breast tissues and other normal tissues. Our findings confirm increased expression of fibroblast growth factor 8 in malignant breast tissue but also show significant fibroblast growth factor 8 expression in non-malignant breast epithelial cells. No significant difference in fibroblast growth factor 8 expression was found between different grades of ductal carcinoma, lobular carcinoma and ductal carcinoma in-situ or cancer of different oestrogen receptor, progesterone receptor or nodal status. The highest levels of fibroblast growth factor 8 expression were found in lactating breast tissues and fibroblast growth factor 8 was also detected in human milk. A survey of other normal tissues showed that fibroblast growth factor 8 is expressed in the proliferative cells of the dermis and epithelial cells in colon, ovary fallopian tube and uterus. Fibroblast growth factor 8 appears to be expressed in several organs in man and appears to have an importance in lactation.     

Differential expression of antiapoptotic gene BAG-1 in human breast normal and cancer cell lines and tissues.

BAG-1 is an antiapoptotic protein that binds to and enhances the antiapoptotic activity of Bcl-2. It binds several growth factor and hormone receptors and modulates their function. BAG-1 was also shown recently to be expressed as four protein isoforms, p50, p46, p33, and p29, through alternative translation initiation. Although many apoptosis-associated genes have been linked to oncogenesis of human breast cancer, the role of BAG-1 has not been fully elucidated. In this study, we examined the expression of BAG-1 RNA or protein isoforms and its interacting antiapoptotic proteins, Bcl-2 and BcI-X(L), in breast normal and tumor cell lines and tissues by Northern or Western blot analysis. We provide convincing evidence that both BAG-1 RNA and protein are overexpressed in human breast cancer cell lines. More importantly, we found that the expression of two isoforms of BAG-1, p46 and p33, was also much higher in breast primary tumors. The expression of Bcl-2 and Bcl-X(L) correlated with that of BAG-1 in breast normal and carcinoma cell lines but not tissues. Our study suggests that BAG-1 isoforms may serve as a molecular marker, independent of Bcl-2 and Bcl-X(L), for human breast cancer.     

Aberrant expression of integrin and erbB subunits in breast cancer cell lines

Integrin and growth factor receptors play an important role in cell functions and their aberrant expressions are implicated in breast cancer malignancy. Recent studies have shown that integrins physically and functionally associate with growth factor receptors suggesting the cooperative regulation of these two signals. We studied the expression of integrin and erbB subunits by flow cytometer in human normal mammary epithelial (HME) cell, non-metastatic (MCF-7, ZR-75-1, MDA-MB453) and metastatic tumor cell lines (MDA-MB231, MDA-MB435). Compared with HME cells, all of non-metastatic and metastatic cell lines showed decreased expressions of alpha2 and beta4 integrin subunits. Two metastatic cell lines, but not three non-metastatic tumor cell lines, expressed alpha5 and alpha6 comparable to HME cells. There was no correlation of erbB2 expression with integrin expressions. We isolated MDA-MB435 subpopulations expressing lower amount of alpha6 integrin and found that alpha5, but not alpha2 and alphav integrins, was concomitantly decreased while erbB family was not affected. Then we transfected erbB2 gene into MDA-MB435 and found the induction of erbB3 expression but not erbB1 and erbB4. However, erbB2 transfection had no effect on the expression of alpha6 and beta4 integrin subunits. These data suggest that the expression of alpha5 and alpha6 integrins may contribute to metastasis, and that the regulation of erbB2 and alpha6 integrin expressions is independent in breast cancer cells.