The presence of cancer stem cells (CSCs) is linked to preexisting or acquired drug resistance and tumor relapse. Therefore, targeting both differentiated tumor cells and CSCs was suggested as an effective approach for non-small cell lung cancer (NSCLC) treatment. After screening of chemotherapeutic agents, tyrosine kinase inhibitors (TKIs) or monoclonal antibody in combination with the putative stem cell killer Salinomycin (SAL), we found Metformin (METF), which modestly exerted a growth inhibitory effect on monolayer cells and alveospheres/CSCs of 5 NSCLC cell lines regardless of their EGFR, KRAS, EML4/ALK and LKB1 status, interacted synergistically with SAL to effectively promote cell death. Inhibition of EGFR (AKT, ERK1/2) and mTOR (p70 s6k) signaling with the combination of METF and SAL can be augmented beyond that achieved using each agent individually. Phospho-kinase assay further suggested the multiple roles of this combination in reducing oncogenic effects of modules, such as ß-catenin, Src family kinases (Src, Lyn, Yes), Chk-2 and FAK. Remarkably, significant reduction of sphere formation was seen under combinatorial treatment in all investigated NSCLC cell lines. In conclusion, METF in combination with SAL could be a promising treatment option for patients with advanced NSCLC irrespective of their EGFR, KRAS, EML4/ALK and LKB1 status.
Tumors are often greatly dependent on signaling cascades promoting cell growth or survival and may become hypersensitive to inactivation of key components within these signaling pathways. Ras and RAF mutations found in human cancer confer constitutive activity to these signaling molecules thereby converting them into an oncogenic state. RAF dimerization is required for normal Ras-dependent RAF activation and is required for the oncogenic potential of mutant RAFs. Here we describe a new mouse model for lung tumor development to investigate the role of B-RAF in oncogenic C-RAF-mediated adenoma initiation and growth. Conditional elimination of B-RAF in C-RAF BxB-expressing embryonic alveolar epithelial type II cells did not block adenoma formation. However, loss of B-RAF led to significantly reduced tumor growth. The diminished tumor growth upon B-RAF inactivation was due to reduced cell proliferation in absence of senescence and increased apoptosis. Furthermore, B-RAF elimination inhibited C-RAF BxB-mediated activation of the mitogenic cascade. In line with these data, mutation of Ser-621 in C-RAF BxB abrogated in vitro the dimerization with B-RAF and blocked the ability to activate the MAPK cascade. Taken together these data indicate that B-RAF is an important factor in oncogenic C-RAF-mediated tumorigenesis.
Effective pharmacological intervention of advanced hepatocellular carcinoma (HCC) is currently lacking. Despite the use of tyrosine kinase inhibitors (TKIs) for the targeted therapy of several malignancies, no agent has been developed to specifically interfere with the oncogenic tyrosine kinase signaling aberrations found in HCC. Therefore, we adopted an orthogonal biological phenotypic screening approach to uncover candidate compounds: based on a potent cytotoxicity toward HCC-derived cell lines, and minimal toxicity toward normal liver cells. Given the success of indolinone as a chemical scaffold in deriving potent multi-kinase inhibitors (e.g. sunitinib), we screened a group of newly synthesized benzylidene-indolinones. Among the candidates, E/Z 6-Chloro-3-(3-trifluoromethyl-benzyliden)-1,3-dihydroindol-2-one (compound 47) exhibited potent anti-proliferative, anti-migratory, pro-apoptotic properties and good safety profile as compared to known multi-targeted tyrosine kinase inhibitors sunitinib and sorafenib. Additionally, an accompanying suppression of alpha-fetoprotein (AFP) transcription, an HCC tumor marker, implies a favorable selectivity and efficacy on HCC. The in vivo efficacy was demonstrated in an HCC xenograft where 47 was administered once weekly (60 mg/kg) and suppressed tumor burden to the same extent as sorafenib (30 mg/kg daily). A receptor tyrosine kinase (RTK) array study revealed promising inhibition of multiple tyrosine kinases such as IGF-1R, Tyro3 and EphA2 phosphorylation. Gene silencing of these targets ameliorated the cytotoxic potential of 47 on the HuH7 cell line, thereby implicating their contribution to the tumorigenicity of HCC. Hence, 47 exhibits potent anti-cancer effects on HCC cell lines, and is a suitable lead for developing multi-targeted kinase inhibitors of relevance to HCC.
Activating mutations in the epidermal growth factor receptor (EGFR) have been identified in a subset of non-small cell lung cancer (NSCLC), which is one of the leading cancer types worldwide. Application of EGFR tyrosine kinase inhibitors leads to acquired resistance by secondary EGFR mutations or by amplification of the hepatocyte growth factor receptor (c-Met) gene. Although several EGFR and c-Met inhibitors have been reported, potent dual EGFR/c-Met inhibitors, which can overcome this latter resistance mechanism, have hitherto not been published and have not reached clinical trials. In the present study we have identified dual EGFR/c-Met inhibitors and designed novel N-[4-(quinolin-4-yloxy)-phenyl]-biarylsulfonamide derivatives, which inhibit the c-Met receptor and both the wild-type and the activating mutant EGFR kinases in nanomolar range. We have demonstrated by Western blot analysis that compound 10 inhibits EGFR and c-Met phosphorylation at cellular level and effectively inhibits viability of the NSCLC cell lines.
The Axl receptor tyrosine kinase (RTK) has been established as a strong candidate for targeted therapy of cancer. However, the benefits of targeted therapies are limited due to acquired resistance and activation of alternative RTKs. Therefore, we asked if cancer cells are able to overcome targeted Axl therapies. Here, we demonstrate that inhibition of Axl by short interfering RNA or the tyrosine kinase inhibitor (TKI) BMS777607 induces the expression of human epidermal growth factor receptor 3 (HER3) and the neuregulin 1(NRG1)-dependent phosphorylation of HER3 in MDA-MB231 and Ovcar8 cells. Moreover, analysis of 20 Axl-expressing cancer cell lines of different tissue origin indicates a low basal phosphorylation of RAC-? serine/threonine-protein kinase (AKT) as a general requirement for HER3 activation on Axl inhibition. Consequently, phosphorylation of AKT arises as an independent biomarker for Axl treatment. Additionally, we introduce phosphorylation of HER3 as an independent pharmacodynamic biomarker for monitoring of anti-Axl therapy response. Inhibition of cell viability by BMS777607 could be rescued by NRG1-dependent activation of HER3, suggesting an escape mechanism by tumor microenvironment. The Axl-TKI MPCD84111 simultaneously blocked Axl and HER2/3 signaling and thereby prohibited HER3 feedback activation. Furthermore, dual inhibition of Axl and HER2/3 using BMS777607 and lapatinib led to a significant inhibition of cell viability in Axl-expressing MDA-MB231 and Ovcar8 cells. Therefore, we conclude that, in patient cohorts with expression of Axl and low basal activity of AKT, a combined inhibition of Axl and HER2/3 kinase would be beneficial to overcome acquired resistance to Axl-targeted therapies.
The advent of effective targeted therapeutics has led to increasing emphasis on precise biomarkers for accurate patient stratification. Here, we describe the role of ACK1, a non-receptor tyrosine kinase in abrogating migration and invasion in KRAS mutant lung adenocarcinoma. Bosutinib, which inhibits ACK1 at 2.7 nM IC50, was found to inhibit cell migration and invasion but not viability in a panel of non-small cell lung cancer (NSCLC) cell lines. Knockdown of ACK1 abrogated bosutinib-induced inhibition of cell migration and invasion specifically in KRAS mutant cells. This finding was further confirmed in an in vivo zebrafish metastatic model. Tissue microarray data on 210 Singaporean lung adenocarcinomas indicate that cytoplasmic ACK1 was significantly over-expressed relative to paired adjacent non-tumor tissue. Interestingly, ACK1 expression in "normal" tissue adjacent to tumour, but not tumour, was independently associated with poor overall and relapse-free survival. In conclusion, inhibition of ACK1 with bosutinib attenuates migration and invasion in the context of KRAS mutant NSCLC and may fulfil a therapeutic niche through combinatorial treatment approaches.
Tumour metastasis is the primary cause of mortality in cancer patients and remains the key challenge for cancer therapy. New therapeutic approaches to block inhibitory pathways of the immune system have renewed hopes for the utility of such therapies. Here we show that genetic deletion of the E3 ubiquitin ligase Cbl-b (casitas B-lineage lymphoma-b) or targeted inactivation of its E3 ligase activity licenses natural killer (NK) cells to spontaneously reject metastatic tumours. The TAM tyrosine kinase receptors Tyro3, Axl and Mer (also known as Mertk) were identified as ubiquitylation substrates for Cbl-b. Treatment of wild-type NK cells with a newly developed small molecule TAM kinase inhibitor conferred therapeutic potential, efficiently enhancing anti-metastatic NK cell activity in vivo. Oral or intraperitoneal administration using this TAM inhibitor markedly reduced murine mammary cancer and melanoma metastases dependent on NK cells. We further report that the anticoagulant warfarin exerts anti-metastatic activity in mice via Cbl-b/TAM receptors in NK cells, providing a molecular explanation for a 50-year-old puzzle in cancer biology. This novel TAM/Cbl-b inhibitory pathway shows that it might be possible to develop a 'pill' that awakens the innate immune system to kill cancer metastases.
Biomarkers predicting resistance to particular chemotherapy regimens could play a key role in optimally individualized treatment concepts. PTK7 (protein tyrosine kinase 7) belongs to the receptor tyrosine kinase family involved in several physiological, but also malignant, cell behaviors. Recent studies in acute myeloid leukemia have associated PTK7 expression with resistance to anthracycline therapy. PTK7 mRNA expression in primary tumor tissue (PTT) and corresponding lymph node tissue (LNT) were retrospectively measured in 117 patients with early breast cancer; PTK7 expression was available in 103 PTT and 108 LNT samples. Median age was 60 years (range, 27-87 years). At a median follow-up of 28.5 months, 6 deaths and 16 recurrences had occurred. PTK7 expression correlations with clinicopathological features were computed and PTK7 expression effects on patient outcome were analyzed in three cohorts defined by adjuvant treatment: anthracycline-based treatment, other chemotherapy regimens (including taxane or other substances), or no chemotherapy. Association of PTK7 expression with clinicopathological features was seen only for age in PTT and nodal stage in LNT. High LN PTK7 was associated with poorer disease-free survival (DFS) in the total population (3-year DFS: low [81.7%] versus high [70.4%]; P=0.016) and in patients without adjuvant chemotherapy (3-year DFS: low [91.7%] versus high [22.3%]; P<0.001), but not in patients receiving adjuvant chemotherapy (P=0.552). DFS stratified by PTK7 expression was compared in treatment cohorts: In patients with low LN PTK7 expression, neither chemotherapy cohort showed significantly better survival than the no-chemotherapy cohort. In patients with high LN PTK7 expression, those receiving chemotherapy, including substances other than anthracyclines, but not those receiving only anthracycline-based chemotherapy, showed significantly better DFS than those receiving no chemotherapy (P=0.001). Our results support earlier findings that PTK7 may be a prognostic and predictive marker associated with resistance to anthracycline-based chemotherapy. Further investigations are needed to validate these findings in breast cancer.
Protein Tyrosin Kinase 7 (PTK7) is upregulated in several human cancers; however, its clinical implication in breast cancer (BC) and lymph node (LN) is still unclear. In order to investigate the function of PTK7 in mediating BC cell motility and invasivity, PTK7 expression in BC cell lines was determined. PTK7 signaling in highly invasive breast cancer cells was inhibited by a dominant-negative PTK7 mutant, an antibody against the extracellular domain of PTK7, and siRNA knockdown of PTK7. This resulted in decreased motility and invasivity of BC cells. We further examined PTK7 expression in BC and LN tissue of 128 BC patients by RT-PCR and its correlation with BC related genes like HER2, HER3, PAI1, MMP1, K19, and CD44. Expression profiling in BC cell lines and primary tumors showed association of PTK7 with ER/PR/HER2-negative (TNBC-triple negative BC) cancer. Oncomine data analysis confirmed this observation and classified PTK7 in a cluster with genes associated with agressive behavior of primary BC. Furthermore PTK7 expression was significantly different with respect to tumor size (ANOVA, p?=?0.033) in BC and nodal involvement (ANOVA, p?=?0.007) in LN. PTK7 expression in metastatic LN was related to shorter DFS (Cox Regression, p?=?0.041). Our observations confirmed the transforming potential of PTK7, as well as its involvement in motility and invasivity of BC cells. PTK7 is highly expressed in TNBC cell lines. It represents a novel prognostic marker for BC patients and has potential therapeutic significance.
NEK9 is known to play a role in spindle assembly and in the control of centrosome separation, but the consequences of NEK9 targeting in cancer cells remain to be elucidated. In this study, we used siRNA to investigate the consequences of targeting NEK9 in glioblastoma and kidney cancer cells as a first step in assessing its potential as an anti-cancer therapeutic target. Live cell imaging revealed that NEK9 depletion of U1242 glioblastoma and Caki2 kidney carcinoma cells resulted in failure of cytokinesis. Interestingly, NEK9-depleted Caki2 cells overrode mitosis under incorrect chromosome alignment and were converted to a micronucleated phenotype, leading to cell death. Whereas, the RPE1 normal epithelium cell line was refractory to abnormal mitosis upon NEK9 knockdown. Nocodazole-induced mitotic arrest was compromised after NEK9 depletion, indicating that NEK9 has an important role in mitotic checkpoint system. Taken together, we propose that NEK9 inhibition represents a novel anti-cancer strategy by induction of mitotic catastrophe via impairment of spindle dynamics, cytokinesis and mitotic checkpoint control.
Tumor-associated macrophages (TAM) promote malignant progression, yet the repertoire of oncogenic factors secreted by TAM has not been clearly defined. We sought to analyze which EGFR- and STAT3-activating factors are secreted by monocytes/macrophages exposed to tumor cell-secreted factors.
The fibroblast growth factor receptor 4 (FGFR4)-R388 single-nucleotide polymorphism has been associated with cancer risk and prognosis. Here we show that the FGFR4-R388 allele yields a receptor variant that preferentially promotes STAT3/5 signaling. This STAT activation transcriptionally induces Grb14 in pancreatic endocrine cells to promote insulin secretion. Knockin mice with the FGFR4 variant allele develop pancreatic islets that secrete more insulin, a feature that is reversed through Grb14 deletion and enhanced with FGF19 administration. We also show in humans that the FGFR4-R388 allele enhances islet function and may protect against type 2 diabetes. These data support a common genetic link underlying cancer and hyperinsulinemia.
Abnormal accumulation and dysregulation of the epidermal growth factor receptor family member HER3 is associated with the development of various human cancers including those of the breast, lung, and ovary. We have previously shown that in melanoma HER3 is frequently overexpressed and is associated with poor prognosis. However, the importance of HER3 in colon cancer and its putative prognostic significance is still unknown.
Pituitary tumors are common intracranial neoplasms, yet few germline abnormalities have been implicated in their pathogenesis. Here we show that a single nucleotide germline polymorphism (SNP) substituting an arginine (R) for glycine (G) in the FGFR4 transmembrane domain can alter pituitary cell growth and hormone production. Compared with FGFR4-G388 mammosomatotroph cells that support prolactin (PRL) production, FGFR4-R388 cells express predominantly growth hormone (GH). Growth promoting effects of FGFR4-R388 as evidenced by enhanced colony formation was ascribed to Src activation and mitochondrial serine phosphorylation of STAT3 (pS-STAT3). In contrast, diminished pY-STAT3 mediated by FGFR4-R388 relieved GH inhibition leading to hormone excess. Using a knock-in mouse model, we demonstrate the ability of FGFR4-R385 to promote GH pituitary tumorigenesis. In patients with acromegaly, pituitary tumor size correlated with hormone excess in the presence of the FGFR4-R388 but not the FGFR4-G388 allele. Our findings establish a new role for the FGFR4-G388R polymorphism in pituitary oncogenesis, providing a rationale for targeting Src and STAT3 in the personalized treatment of associated disorders.
Prostate disease incidence is expected to rise among developing nations secondary to increased prevalence of obesity and the elderly. Although many case-control studies have associated obesity to prostate cancer aggressiveness, few have correlated markers of prostate pathology to biomarkers of visceral obesity and insulin resistance, using an apparently healthy cohort. This study aims to fill this gap.
Activated Cdc42-associated Kinase, ACK1, is a non-receptor tyrosine kinase with numerous interacting partners, including Cdc42 and EGFR. Gene amplification and overexpression of ACK1 were found in many cancer types such as those of the lung and prostate. Previously, we identified both somatic- and germ line missense mutations in the ACK1 coding sequence, by surveying 261 cancer cell lines and 15 control tissues. Here, we verified and characterized the non-synonymous mutation, ACK-S985 N, located in the ubiquitin association domain of the protein. Both overexpression and silencing experiments in MCF7 and A498 cells, respectively, demonstrated a role of the ACK1 S985 N mutation in enhancing cell proliferation, migration and anchorage-independent growth as well as the epithelial-mesenchymal transition. Further, we showed that the ACK1 S985 N mutant is unable to bind ubiquitin, unlike the wild type kinase. This contributed to ACK1 protein stability and stabilized EGFR after EGF stimulation, thereby prolonging mitogenic signaling in cancer cells. In addition, the ACK1 S985 N-EGFR interaction is enhanced, but not the ubiquitination of the receptor. Intriguingly, silencing of ACK1 in A498 cells sensitized the renal carcinoma cells to gefitinib, against which they are otherwise resistant. The work demonstrates that other than gene amplification, a single somatic mutation in ACK1 can result in extended protein stability enabling the oncoprotein to exert its oncogenic function in tumor progression. It also provides a rationale to target ACK1 in combination with other chemotherapeutic drugs, such as EGFR inhibitors, to potentiate therapeutic action against resistant tumors.
In the growth factor receptor gene FGFR4 the presence of the common single nucleotide polymorphism Arg388 has been associated with progression of various types of cancer including breast cancer. However, a causative relationship is not readily assigned due to genetic heterogeneity in different patient cohorts. To address this issue, we compared the effects of this allele on malignant progression in the WAP-TGFalpha transgenic mouse model of breast cancer. A knock-in strain was generated to introduce an analogous Arg385 allele into the murine FGFR4 gene. Mouse embryonic fibroblasts derived from this strain displayed accelerated cell transformation, with transformed cells exhibiting greater motility and invasive behavior. In the in vivo context of TGFalpha-induced mammary carcinogenesis, tumor development and progression was significantly advanced in tumor mass, size, and onset of pulmonary metastases. Our findings definitively identify the FGFR4 Arg388 allele as a functional prognostic marker for breast cancer progression.
The mitogen-inducible gene-6 (mig-6) is a multi-adaptor protein implicated in the regulation of the HER family of receptor tyrosine kinases. We have reported recently that mig-6 is a negative regulator of epidermal growth factor receptor (EGFR)-dependent skin morphogenesis and tumor formation in vivo. In the liver, ablation of mig-6 leads to an increase in EGFR protein levels, suggesting that mig-6 is a negative regulator of EGFR function. In line with this observation, primary hepatocytes isolated from mig-6 knockout and wild-type control mice display sustained mitogenic signaling in response to EGF. In order to explore the role of mig-6 in the liver in vivo, we analyzed liver regeneration in mig-6 knockout and wild-type control mice. Interestingly, mig-6 knockout mice display enhanced hepatocyte proliferation in the initial phases after partial hepatectomy. This phenotype correlates with activation of endogenous EGFR signaling, predominantly through the protein kinase B pathway. In addition, mig-6 is an endogenous inhibitor of EGFR signaling and EGF-induced tumor cell migration in human liver cancer cell lines. Moreover, mig-6 is down-regulated in human hepatocellular carcinoma and this correlates with increased EGFR expression. Conclusion: Our data implicate mig-6 as a regulator of EGFR activity in hepatocytes and as a suppressor of EGFR signaling in human liver cancer.
Exposure to extensive ultraviolet (UV) rays is a major cause of skin cancer, which is thought to be initiated by DNA mutations. Members of the epidermal growth factor receptor (EGFR) family are important in various pathophysiologic processes like cancer and are shown to be phosphorylated upon UV exposure. Here we show that EGFR phosphorylation by modest UV doses is dependent on metalloprotease activity and resultant epidermal growth factor (EGF) family proligand shedding. This proligand cleavage releases the mature ligand, which then binds to and activates EGFR. We show that UV induced EGFR phosphorylation in transformed cell lines of melanocyte and keratinocyte origin, which was reduced upon preincubation with a broad-spectrum metalloprotease inhibitor, BB94. UV also activated EGFR downstream signaling via Erk and Akt pathways in a BB94-sensitive manner. Furthermore, using neutralizing antibodies we found that proligand amphiregulin was required for UV-induced EGFR activation in SCC-9 cells. Using RNAi this EGFR activation was further shown to depend on the metalloproteases ADAM9 and ADAM17 in SCC-9 cells. cDNA array hybridization and RT-PCR analysis showed overexpression of a Disintegrin and a Metalloproteases (ADAMs) and EGF family proligands in melanoma cell lines. Additionally, blocking EGFR signal transactivation by BB94 led to increased apoptosis in UV-irradiated cells. EGFR signal transactivation also led to increased stability of the DNA repair protein, PARP, under UV stress. Thus, both antiapoptotic and DNA repair pathways are activated simultaneously by EGFR signal transactivation. Together, our data provide novel insights into the mechanism of UV-induced EGFR activation, suggesting broad relevance of the UV-ADAM-proligand-EGFR-Erk/Akt pathway and its significance in skin cancer.
Protein tyrosine phosphatases (PTPs) are key regulators of different processes during development of the central nervous system. However, expression patterns and potential roles of PTPs in the developing superior colliculus remain poorly investigated. In this study, a degenerate primer-based reverse transcription-polymerase chain reaction (RT-PCR) approach was used to isolate seven different intracellular PTPs and nine different receptor-type PTPs (RPTPs) from embryonic E15 mouse superior colliculus. Subsequently, the expression patterns of 11 PTPs (TC-PTP, PTP1C, PTP1D, PTP-MEG2, PTP-PEST, RPTPJ, RPTP?, RPTPRR, RPTP?, RPTP? and RPTP?) were further analyzed in detail in superior colliculus from embryonic E13 to postnatal P20 stages by quantitative real-time RT-PCR, Western blotting and immunohistochemistry. Each of the 11 PTPs exhibits distinct spatiotemporal regulation of mRNAs and proteins in the developing superior colliculus suggesting their versatile roles in genesis of neuronal and glial cells and retinocollicular topographic mapping. At E13, additional double-immunohistochemical analysis revealed the expression of PTPs in collicular nestin-positive neural progenitor cells and RC-2-immunoreactive radial glia cells, indicating the potential functional importance of PTPs in neurogenesis and gliogenesis.
We analyzed the G-actin-regulated transcriptome by gene expression analysis using previously characterized actin-binding drugs. We found many known MAL/MRTF-dependent target genes of serum response factor (SRF), as well as additional directly regulated genes. Surprisingly, several putative antiproliferative target genes were identified, including mig6/errfi-1, a negative regulator of the EGFR family. Mig6 induction occurred through actin-MAL-SRF signaling, and MAL was inducibly recruited to and activated a mig6 promoter element. Upregulation of Mig6 by lipid agonists such as LPA and S1P or actin drugs involved MAL and correlated with decreased activation of EGFR, MAPK/Erk, and c-fos. Mig6 depletion restored EGFR signaling and provided a proliferative advantage. Overexpression of MAL exhibited strong antiproliferative effects requiring the domains for SRF binding and transactivation, which supports antagonistic functions of MAL on growth-promoting signals. Our results show the existence of negatively acting transcriptional networks between pro- and antiproliferative signaling pathways toward SRF.
FGFR4, a member of the fibroblast growth factor receptor family, has been recently associated with progression of melanoma, breast and head and neck carcinoma. Given its uniquely high expression in the liver, we investigated its contributory role to hepatocellular carcinoma (HCC).
Establishment of antiapoptotic signaling pathways in tumor cells is a major cause for the failure of chemotherapy against cancer. To investigate the underlying mechanisms, we developed an experimental approach that is based on the genetic plasticity of cancer cells and the selection for cell survival on treatment with chemotherapeutic agents.
Current clinical protocols favor a combination of antiangiogenic/antivascular compounds with classical chemotherapy. However, it remains unclear to what extent an antiangiogenic/antivascular therapy influences the delivery of chemotherapy. Therefore, the aim of the present study was to characterize the effects of the antiangiogenic tyrosine kinase inhibitor sunitinib on tumor microhemodynamics and delivery of chemotherapy. SF126 tumor cells were implanted subcutaneously into nude mice and were analyzed repeatedly by intravital microscopy. Treatment with sunitinib was initiated 7 days after implantation. To assess the effects of sunitinib on tumor vasculature and hemodynamics, we analyzed total and functional vessel densities, microvascular diameter, and microvascular blood flow rate. To study the delivery of chemotherapy, autofluorescent doxorubicin was systemically administered and its vascular delivery to the tumor tissue was quantified. Histological analysis included endothelial cell proliferation, pericyte coverage of tumor vessels, and tumor cell proliferation. Sunitinib significantly suppressed tumor growth by both antivascular and antiangiogenic effects. However, a number of tumor vessels escaped antiangiogenic therapy. Interestingly, in these surviving blood vessels sunitinib treatment resulted in an increased microvascular blood flow rate resulting in an improved delivery of chemotherapy via these blood vessels. Besides its potent antiangiogenic and antivascular efficacy, sunitinib treatment results in improved microhemodynamics and blood flow in tumor blood vessels that escape therapy leading to an improved vascular delivery of chemotherapy. These results provide the basis for a potential chemosensitizing effect of sunitinib.
Elevated serum or tissue levels of lectin galactoside-binding soluble 3 binding protein (LGALS3BP) have been associated with short survival and development of metastasis in a variety of human cancers. However, the role of LGALS3BP, particularly in the context of tumor-host relationships, is still missing. Here, we show that LGALS3BP knockdown in MDA-MB-231 human breast cancer cells leads to a decreased adhesion to fibronectin, a reduced transendothelial migration and, more importantly, a reduced expression of vascular endothelial growth factor (VEGF). Production of VEGF, that was restored by exposure of silenced cells to recombinant LGALS3BP, required an intact PI3k/Akt signaling. Furthermore, we show that LGALS3BP was able to directly stimulate HUVEC tubulogenesis in a VEGF-independent, galectin-3-dependent manner. Immunohistochemical analysis of human breast cancer tissues revealed a correlation among LGALS3BP expression, VEGF expression, and blood vessel density. We propose that in addition to its prometastatic role, LGALS3BP secreted by breast cancer cells functions critically as a pro-angiogenic factor through a dual mechanism, i.e by induction of tumor VEGF and stimulation of endothelial cell tubulogenesis.
Resistance to chemotherapy is a serious problem for the successful treatment of ovarian cancer patients but signalling pathways that contribute to this chemoinsensitivity are largely unknown. We demonstrate that the chemotherapeutic drug doxorubicin induces activation of the HER3-PI3K-AKT signalling cascade in ovarian cancer cells. We further show that the induction of this anti-apoptotic signalling pathway is based on upregulated expression of HER3 ligands, their shedding by the metalloprotease ADAM17, and is dependent on the HER2 receptor. The doxorubicin-mediated activation of this important survival cascade can be blocked by the kinase inhibitors lapatinib or erlotinib as well as by the therapeutic monoclonal antibody trastuzumab. Inhibition of the doxorubicin-induced activation of HER3-PI3K-AKT signalling significantly increased apoptosis of ovarian cancer cells. Besides doxorubicin, treatment of cells with cisplatin resulted in activation of the HER3 receptor whereas other chemotherapeutics did not show this effect. The increase in HER3 phosphorylation was detected in well-established ovarian cancer cell lines which originate from patients previously treated with these chemotherapeutic drugs. Based on these results, we postulate that activation of the HER3-PI3K-AKT cascade represents a major mechanism of chemoresistance in ovarian cancer.
A major obstacle in the successful treatment of cancer is the occurrence of chemoresistance. Cancer cells surviving chemotherapy and giving rise to a recurrence of the tumor are termed cancer stem cells and can be identified by elevated levels of certain stem cell markers. Eradication of this cell population is a priority objective in cancer therapy. Here, we report elevated levels of stem cell markers in MCF-7 mammospheres. Likewise, an upregulation of HER2 and its differential expression within individual cells of mammospheres was observed. Sorting for HER2(high) and HER2(low) cells revealed an upregulation of stem cell markers NANOG, OCT4 and SOX2 in the HER2(low) cell fraction. Accordingly, HER2(low) cells also showed reduced proliferation, ductal-like outgrowths and an increased number of colonies in matrigel. Xenografts from subcutaneously injected HER2(low) sorted cells exihibited earlier onset but slower growth of tumors and an increase in stem cell markers compared to tumors developed from the HER2(high) fraction. Treatment of mammospheres with salinomycin reduced the expression of SOX2 indicating a selective targeting of cancer stem cells. Trastuzumab however, did not reduce the expression of SOX2 in mammospheres. Furthermore, a combinatorial treatment of mammospheres with trastuzumab and salinomycin was superior to single treatment with each drug. Thus, targeting HER2 expressing tumors with anti-HER2 therapies will not necessarily eliminate cancer stem cells and may lead to a more aggressive cancer cell phenotype. Our study demonstrates efficient killing of both HER2 positive cells and cancer stem cells, hence opening a possibility for a new combinatorial treatment strategy.
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