Investigating the contribution of FGF receptors in breast tumorigenesis using models of mammary cancer

Breast cancer, the main cancer in women, occurs in approximately 27% of all yearly diagnosed cancer cases. It is estimated that one out of eight women will develop breast cancer in her lifetime. Therefore it is not surprising that this disease ranks second as a cause of cancer death in women, after lung cancer (American Cancer Society, Cancer Facts and Figures 2009). Based on these numbers, many studies have been undertaken in the area of breast cancer and since 1990, death rates from breast cancer have been decreasing, mainly due to earlier detection and improved treatments. Since the 1970s, hormonal therapies targeting the estrogen receptor have been very successful for treatment of estrogen receptor positive (ER+) breast cancers that consist of about 60% of the cases. More recently, therapies targeting the ErbB2 receptor tyrosine kinase that is overexpressed in about 20% of breast cancer have shown to be of benefit for this subset of breast cancer patients. However, not every patient responds to these treatments or patients become resistant, thus for both therapies there is a high risk of relapse. Consequently, novel therapies are required and will likely arise from an improved understanding of the disease biology. Recent studies have shown that members of the fibroblast growth factor receptor (FGFR) family of tyrosine kinase receptors are deregulated in breast cancer. Indeed FGFR1 gene is amplified in 9% of breast cancers and single nucleotide polymorphisms in FGFR2 gene are strongly associated with an increased probability to develop breast cancer. Using models of breast carcinoma (4T1, 4TO7, 168FARN and 67NR cell lines), our study aimed at a better understanding of how FGFRs contribute to breast tumorigenesis. Furthermore we analysed the effects of blocking the activity of these receptors in cell lines and primary tumors. In vitro, we showed that 4T1, 4TO7, 168FARN and 67NR cell lines co-express FGF ligands and FGFRs, suggesting an autocrine loop activating FGFRs which leads to the constitutive basal activation of downstream signaling pathways, mainly MAPK and PI3K/AKT. Using TKI258, a tyrosine kinase inhibitor blocking FGFRs, we inhibited the basal activity of FGFRs. This inhibition resulted in a decrease of ERK1/2 and AKT activity, confirming that FGFR signaling maintains the activity of MAPK and PI3K/AKT pathways. Furthermore, interfering with autocrine FGFR signaling dramatically impaired proliferation of the four cell lines and apoptotic cell death was observed in 4T1 and 4TO7. Using constitutively active mutants, we demonstrated that Ras and AKT contribute to cell survival downstream of FGFRs and that 4T1 cells expressing these constructs are partially rescued from the effects of TKI258. Interestingly, when we i Summary ii combined inhibitors of MEK (UO126) or PI3K (LY294002) with TKI258, we increased the sensitivity of 4T1 cells to TKI258 induced cell death. In vivo, we describe that TKI258 treatment inhibits FGFR signaling in mice bearing 4T1-induced tumors. In addition, daily oral treatment of mice bearing 4T1- or 67NR- induced tumors with TKI258 over 14 days, significantly reduces tumor outgrowth and decreases 4T1 lung metastasis, showing that blockade of FGFR has strong anti-tumor and anti-metastatic activities. Two microarray analyses performed on treated 4T1 cells or 4T1 tumors, led to the identification of genes that were regulated after TKI258 treatment. Detailed analyses showed that some of these genes were known to contribute to the metastatic process (matrix metalloproteinases and extracellular matrix proteins) or to cell cycle progression (cyclins and E2F transcription factors). Comparison of these TKI258-regulated genes with publicly available databases of breast cancer patients identified a cohort of patients showing overexpression of genes down-regulated upon TKI258 treatment, and these patients have a higher probability of metastatic disease compared to the other patients. These highly expressed genes might therefore reflect activation of receptor tyrosine kinase signaling pathways like FGFR in 4T1 tumors. In summary our results show that targeting FGFRs using a TKI has an impact on various biological characteristics of FGFR driven models of breast cancer, including proliferation and survival. In addition, our observations show that blockade of FGFR signaling can be achieved in vivo and this leads to reduction of tumor outgrowth as well as decreased metastases formation. Finally, our meta-analysis on genes that are changed in 4T1 treated cells and tumors provides evidences that results obtained using animal models of a diseases are meaningful in terms of prognostic and can be translated to breast cancer patients

Coadministration of NVP-AEW541 and Dasatinib induces mitochondrial-mediated apoptosis through Bax activation in Malignant Human Glioma Cell Lines

Despite recent advances in treatment, glioblastoma multiforme (GBM) represents an incurable disease for which development of new therapies is required. We report the effect of NVP-AEW541, a small molecule inhibitor of insulin-like growth factor-I receptor (IGF-IR) kinase activity on growth and signaling in a panel of glioma cell lines. NVPAEW541 blocked phosphorylation of IGF-IR in a dose- and time-dependent manner and inhibited proliferation and clonogenicity with median effective concentrations (IC50) in the range of 2.5 to 10 µM. NVPAEW541 also induced loss of mitochondrial membrane potential and release of cytochrome c and apoptosis-inducing factor (AIF) from mitochondria. However, the concentrations of NVP-AEW541 required to significantly inhibit glioma cell viability and downstream signaling also had some inhibitory activity on non-neoplastic astrocytes, which led us to question whether therapeutic efficacy could be enhanced by combination of lower concentrations of NVP-AEW541 with inhibition of other tyrosine kinases dysregulated in gliomas. Dasatinib was selected as a combination agent based on its distinct profile of inhibiting other relevant signaling targets in gliomas, including EGFR and PDGFR. Combined treatment of glioma cells with NVP-AEW541 and dasatinib induced significantly more apoptosis than either agent alone in glioma cells, but not non-neoplastic astrocytes, as well as synergistically inhibiting clonogenic survival. Mechanistic studies indicated that the combination of NVP-AEW541 and dasatinib resulted in a significant reduction of pERK and pAkt and provoked a marked increase in AIF release to the nucleus, Bax oligomerization and loss of mitochondrial potential compared to each agent alone. Overexpression of BCL-2 and Akt significantly attenuated NVP-AEW541 and dasatinib-Bax activation and cell death. Our data indicate that activation of BAX plays a critical role in mediating NVP-AEW541 and dasatinib-induced apoptotic cascades. These data suggest the potential value of combining IGFR inhibition with other classes of tyrosine kinase inhibitors as a way to potentiate therapeutic efficacy

Efficacy of RAD001 (everolimus) in peritoneal dissemination of gastric cancer

Peritoneal dissemination occurs frequently in patients with unresectable advanced stage gastric cancer. In this study, we tested mTOR inhibitor RAD001 (everolimus) for efficacy on peritoneal dissemination of gastric cancer. Using the two cell lines 58As1, a highly peritoneal metastatic cell line, and its parental HSC58, a human scirrhous gastric cancer cell line, we first examined the growth inhibition activity of everolimus in vitro. Methylene blue assay demonstrated a moderate inhibitory effect on both cell lines under normal culture condition. When cells were maintained in hypoxic (1% O2) conditions, growth inhibition by everolimus was greatly reduced in HSC58, whereas the reduction was much smaller in 58As1. In western blotting, phosphorylation of mTOR, and its down-stream signaling molecules, P70S6K and 4E-BP1, were decreased under hypoxic conditions in HSC58. However, in 58As1, phospho-P70S6K and -4E-BP1 remained active state in hypoxic conditions and was suppressed by treatment with everolimus. Cell-cycle analysis showed that the hypoxia-induced G1 arrest was not manifested in 58As1 cells as compared to HSC58 cells. Separately, an in vivo orthotopic mouse model of 58As1 revealed that everolimus significantly reduces peritoneal dissemination as evaluated by quantitative photon counting method. Taken together, our results suggest that everolimus may have activity against gastric cancer, particularly in cases with peritoneal dissemination

Targeting fibroblast growth factor receptors blocks PI3K/AKT signaling, induces apoptosis, and impairs mammary tumor outgrowth and metastasis

Members of the fibroblast growth factor receptor (FGFR) family have essential roles in normal physiology and in cancer where they control diverse processes. FGFRs have been associated with breast cancer development and models to study their role in cancer and their targeting potential are desirable. Here, we present an in vitro and in vivo analysis of FGFRs in the mammary cancer cell lines 67NR and 4T1. We show that both cell lines co-express FGFRs and ligands and display autocrine FGFR signaling activity. Fibroblast growth factor receptor substrate 2 (FRS2), a downstream mediator of FGFR is constitutively tyrosine-phosphorylated and multiple signaling pathways are active. Treatment of 67NR and 4T1 cultures with TKI258, a selective FGFR tyrosine kinase inhibitor caused a rapid decrease in FRS2 phosphorylation, decreased the activity of ERK1/2, AKT and PLC and blocked proliferation and induced 4T1 apoptotic cell death via blockade of the PI3K/AKT pathway. In vivo, one dose of TKI258 rapidly lowered FRS2 phosphorylation and ERK1/2 and AKT activity in mammary tumors. Long-term treatment of 4T1 and 67NR tumor-bearing mice had a significant impact on primary tumor outgrowth and 4T1 tumor induced lung metastases. A microarray analyses was carried out to identify prognostic markers in human breast tumors and targets with roles in TKI258- anti-tumor activity. Of interest are the down-regulated matrix metalloproteases (MMP), in particular MMP-9, which is essential for metastatic spread of 4T1 tumors

Somatostatin analogue inhibits mobility of prostate carcinoma cells and SSTR2a is a prognostic and therapeutic biomarker for prostate carcinoma

The somatostatin analogue (SA), Octreotide, has been used as a therapeutic reagent for somatostatin receptor type 2a (SSTR2a)-positive cancers. The purpose of this study is to detect SSTR2a in human prostate carcinomas and to elucidate the effects of SA on SSTR2a-positive prostate carcinoma cells to determine the potential of this drug as a new therapeutic method for advanced prostate carcinoma. Material and Methods: Immunohistochemical study of SSTR2a was performed on 108 prostate carcinoma cases. RT-PCR analysis was performed on 11 needle biopsy and 3 metastatic cases. Proliferation, migration and invasion assays were performed by using SSTR2a-positive prostate cancer cells, DU145 and PC3, treated with SA and/or Rho-kinase inhibitor Y-27632. Morphological changes of prostate carcinoma cells were also examined by staining for vinculin, rhodamine phalloidin, and alpha tubulin. Results: The study showed expression of SSTR2a in 13 of the 108 cases (12%); the histological grade (Gleason) and tumor stage of the prostate carcinoma were directly related to SSTR2a expression. Among the seven cases of lymph node (LN) metastasis, SSTR2a expression was markedly higher. SSTR2 mRNA was detected by RT-PCR in only 4 of 11 needle biopsy cases and 1 of 3 cases of LN metastasis. Migration and invasion abilities of DU145 and PC3 cells were inhibited by SA in a dose-dependent manner. This inhibition was reversed by Y-27632. Morphological changes of the prostate cancer cells treated with SA and Y27632 corroborate the migration and invasion assays, although SA had no effect on proliferation of DU145 and PC3 cells. Conclusion: SSTR2a is a potential prognostic factor for prostatic carcinoma. SA may be beneficial for patients with advanced prostate carcinoma or to protect from distal metastasis if they are positive for SSTR2a

Effects of vatalanib on tumor growth can be potentiated by mTOR blockade in vivo

Background: Vatalanib is a selective inhibitor of vascular endothelial growth factor (VEGF) receptors 1-3. Vatalanib increases pro-angiogenic serum VEGF in response to VEGF receptor blockade which is considered to limit vatalinib´s anti-tumor activity. Everolimus is an inhibitor of mammalian target of rapamycin (mTOR) signaling and decreases VEGF-secretion of cells. It was hypothesized that inhibition of VEGF production by everolimus potentiates the anti-tumor activity of vatalanib. Methods: In vitro, the effects of vatalanib and everolimus on gastric cancer cell proliferation, cell cycle, apoptosis and signal transduction were studied. Effects on angiogenesis were mimicked using tube formation assays of cultured human umbilical vein endothelial cells (HUVECs). In vivo, the anti-tumor effect of compounds was studied using a gastric cancer xenograft nude mouse model. VEGF production from the murine tumor host and human gastric cancer cells was analyzed by species specific ELISAs. Tumor vascularization and proliferation were studied by immunohistochemistry. Results: In vitro, everolimus but not vatalanib decreased gastric cancer proliferation without affecting apoptosis. Vatalanib completely abolished endothelial cell tube formation, whereas inhibition of tube formation by everolimus was incomplete. In vivo, the combination of vatalanib with everolimus was superior to single agent treatment. Treatment with vatalanib + everolimus significantly decreased mVEGF levels but not hVEGF. Treatment with everolimus decreased hVEGF production significantly. There was a trend for lower vascular density and proliferation for combination treatment. Conclusion: We conclude that anti-tumor activity of vatalanib can be augmented by everolimus in a preclinical model of gastric cancer, an effect potentially mediated by suppression of mVEGF production in the tumor microenvironment

Effects of vatalanib on tumor growth can be potentiated by mTOR blockade in vivo

.The vascular endothelial growth factor (VEGF) is a central mediator of tumor-induced angiogenesis. Everolimus, a mammalian target of rapamycin (mTOR) inhibitor, decreases VEGF-secretion of cancer cells. Vatalanib is a selective inhibitor of VEGF receptors 1-3. In the present study it was hypothesized that dual inhibition of VEGF signaling by inhibition of VEGF production and VEGF receptor signaling leads to synergistic anti-tumor effects. In vitro, effects of vatalanib and everolimus on cell proliferation, cell cycle, apoptosis and signal transduction were examined in three gastric cancer cell lines. Effects on angiogenesis were assessed using tube formation assays of cultured human umbilical vein endothelial cells (HUVECs). In vivo, the antitumor effect of compounds was studied using a gastric cancer xenograft nude mouse model. VEGF of murine origin (mVEGF) and human cancer cell-derived VEGF (hVEGF) were studied separately by specific ELISAs. Tumor vascularization and proliferation were quantified by immunohistochemistry. In vitro, everolimus but not vatalanib decreased gastric cancer proliferation without inducing apoptosis. Vatalanib abolished endothelial cell tube formation, whereas inhibition of tube formation by everolimus was incomplete. In vivo, the combination of vatalanib with everolimus was superior to single agent treatments and reduced tumor size by about 50% relative to everolimus monotherapy (p < 0.005). Pharmacodynamic analysis of VEGF plasma level showed a decrease of hVEGF by everolimus and indicated a trend towards lower mVEGF level only in the combination group. In line, there was a tendency for lower vascular density and proliferation for combination treatment. We conclude that in a preclinical model of gastric cancer the antitumor activity of vatalanib can be augmented by everolimus

Effectiveness and molecular interactions of the clinically active mTORC1 inhibitor everolimus in combination with tamoxifen or letrozole in vitro and in vivo

Strategies to improve the efficacy of endocrine agents in breast cancer (BC) therapy and to delay the onset of resistance include concomitant targeting of the estrogen receptor alpha (ER) and the mammalian target of rapamycin complex 1 (mTORC1), which regulate cell-cycle progression and are supported by recent clinical results

Isoflurane Preconditioning Neuroprotection In Experimental Focal Stroke Is Androgen-Dependent In Male Mice

Isoflurane preconditioning (IsoPC) neuroprotection in experimental stroke is male-specific. The role of androgens in the ischemic sensitivity of IsoPC male brain and whether androgen effects are androgen receptor (AR) dependent were assessed. Male mice were implanted with flutamide (AR antagonist), or castrated (CAST) and implanted with testosterone, dihydroxytestosterone (DHT), flutamide, letrozole (aromatase inhibitor), or vehicle 7-13 days before preconditioning. P450 estrogen aromatase wild-type and knockout mice were also evaluated. All mice were preconditioned for 4 hours with 0% (sham preconditioning) or 1% isoflurane and recovered for 24 hours. Mice then underwent 2 hours of middle cerebral artery occlusion (MCAO) and were evaluated 22 hours later for infarct volume. For neurobehavioral outcomes, preconditioned CAST + DHT groups underwent 1 hour of MCAO followed by 9 days of reperfusion. IsoPC neuroprotection relative to acute infarct volume outcomes were testosterone and DHT dose-specific and AR-dependent. Sensorimotor function improved in IsoPC mice regardless of androgen status while androgen replacement independently improved sensorimotor function. In contrast, IsoPC improved cognitive function in castrates lacking endogenous androgens, but this improvement was absent in androgen replaced mice. Further studies are needed in ischemic male brain to clarify whether androgens have a causal or independent role in IsoPC neuroprotection

Anti-Angiogenic/Vascular Effects of the mTOR Inhibitor Everolimus Are Not Detectable by FDG/FLT-PET1

Noninvasive functional imaging of tumors can provide valuable early-response biomarkers, in particular, for targeted chemotherapy. Using various experimental tumor models, we have investigated the ability of positron emission tomography (PET) measurements of 2-deoxy-2-[18F]fluoro-glucose (FDG) and 3′-deoxy-3′-[18F]fluorothymidine (FLT) to detect response to the allosteric mammalian target of rapamycin (mTOR) inhibitor everolimus. Tumor models were declared sensitive (murine melanoma B16/BL6 and human lung H596) or relatively insensitive (human colon HCT116 and cervical KB31), according to the IC50 values (concentration inhibiting cell growth by 50%) for inhibition of proliferation in vitro (<10 nM and >1 µM, respectively). Everolimus strongly inhibited growth of the sensitive models in vivo but also significantly inhibited growth of the insensitive models, an effect attributable to its known anti-angiogenic/vascular properties. However, although tumor FDG and FLT uptake was significantly reduced in the sensitive models, it was not affected in the insensitive models, suggesting that endothelial-directed effects could not be detected by these PET tracers. Consistent with this hypothesis, in a well-vascularized orthotopic rat mammary tumor model, other antiangiogenic agents also failed to affect FDG uptake, despite inhibiting tumor growth. In contrast, the cytotoxic patupilone, a microtubule stabilizer, blocked tumor growth, and markedly reduced FDG uptake. These results suggest that FDG/FLT-PET may not be a suitable method for early markers of response to antiangiogenic agents and mTOR inhibitors in which anti-angiogenic/vascular effects predominate because the method could provide false-negative responses. These conclusions warrant clinical testing