Fascin Activates ?-Catenin Signaling and Promotes Breast Cancer Stem Cell Function Mainly Through Focal Adhesion Kinase (FAK): Relation With Disease Progression.
ABSTRACT: Cancer stem cells (CSCs), a rare population of tumor cells with high self-renewability potential, have gained increasing attention due to their contribution to chemoresistance and metastasis. We have previously demonstrated a critical role for the actin-bundling protein (fascin) in mediating breast cancer chemoresistance through activation of focal adhesion kinase (FAK). The latter is known to trigger the ?-catenin signaling pathway. Whether fascin activation of FAK would ultimately trigger ?-catenin signaling pathway has not been elucidated. Here, we assessed the effect of fascin manipulation in breast cancer cells on triggering ?-catenin downstream targets and its dependence on FAK. Gain and loss of fascin expression showed its direct effect on the constitutive expression of ?-catenin downstream targets and enhancement of self-renewability. In addition, fascin was essential for glycogen synthase kinase 3? inhibitor-mediated inducible expression and function of the ?-catenin downstream targets. Importantly, fascin-mediated constitutive and inducible expression of ?-catenin downstream targets, as well as its subsequent effect on CSC function, was at least partially FAK dependent. To assess the clinical relevance of the in vitro findings, we evaluated the consequence of fascin, FAK, and ?-catenin downstream target coexpression on the outcome of breast cancer patient survival. Patients with coexpression of fascinhigh and FAKhigh or high ?-catenin downstream targets showed the worst survival outcome, whereas in fascinlow, patient coexpression of FAKhigh or high ?-catenin targets had less significant effect on the survival. Altogether, our data demonstrated the critical role of fascin-mediated ?-catenin activation and its dependence on intact FAK signaling to promote breast CSC function. These findings suggest that targeting of fascin-FAK-?-catenin axis may provide a novel therapeutic approach for eradication of breast cancer from the root.
Project description:Breast cancer remains the second cause of tumor-related mortality in women worldwide mainly due to chemoresistance and metastasis. The chemoresistance and metastasis are attributed to a rare subpopulation with enriched stem-like characteristics, thus called Cancer Stem Cells (CSCs). We have previously reported aberrant expression of the actin-bundling protein (fascin) in breast cancer cells, which enhances their chemoresistance, metastasis and enriches CSC population. The intracellular mechanisms that link fascin with its downstream effectors are not fully elucidated. Here, loss and gain of function approaches in two different breast cancer models were used to understand how fascin promotes disease progression. Importantly, findings were aligned with expression data from actual breast cancer patients. Expression profiling of a large breast cancer dataset (TCGA, 530 patients) showed statistically significant correlation between fascin expression and a key adherence molecule, ?1 integrin (ITGB1). In vitro manipulation of fascin expression in breast cancer cells exhibited its direct effect on ITGB1 expression. Fascin-mediated regulation of ITGB1 was critical for several breast cancer cell functions including adhesion to different extracellular matrix, self-renewability and chemoresistance. Importantly, there was a significant relationship between fascin and ITGB1 co-expression and short disease-free as well as overall survival in chemo-treated breast cancer patients. This novel role of fascin effect on ITGB1 expression and its outcome on cell self-renewability and chemoresistance strongly encourages for dual targeting of fascin-ITGB1 axis as a therapeutic approach to halt breast cancer progression and eradicate it from the root.
Project description:BACKGROUND:Recent advancements in cancer biology field suggest that glucose metabolism is a potential target for cancer treatment. However, little if anything is known about the metabolic profile of cancer stem cells (CSCs) and the related underlying mechanisms. METHODS:The metabolic phenotype in lung CSC was first investigated. The role of collagen XVII, a putative stem cell or CSC candidate marker, in regulating metabolic reprogramming in lung CSC was subsequently studied. Through screening the genes involved in glycolysis, we identified the downstream targets of collagen XVII that were involved in metabolic reprogramming of lung CSCs. Collagen XVII and its downstream targets were then used to predict the prognosis of lung cancer patients. RESULTS:We showed that an aberrant upregulation of glycolysis and oxidative phosphorylation in lung CSCs is associated with the maintenance of CSC-like features, since blocking glycolysis and oxidative phosphorylation reduces sphere formation, chemoresistance, and tumorigenicity. We also showed that the Oct4-hexokinase 2 (HK2) pathway activated by collagen XVII-laminin-332 through FAK-PI3K/AKT-GSB3?/?-catenin activation induced the upregulation of glycolysis and maintenance of CSC-like features. Finally, we showed that collagen XVII, Oct4, and HK2 could be valuable markers to predict the prognosis of lung cancer patients. CONCULSIONS:These data suggest the Oct4-HK2 pathway regulated by collagen XVII plays an important role in metabolic reprogramming and maintenance of CSC-like features in lung CSCs, which may aid in the development of new strategies in cancer treatment.
Project description:BACKGROUND: A major therapeutic challenge for breast cancer is the ability of cancer cells to evade killing of conventional chemotherapeutic agents. We have recently reported the actin-bundling protein (fascin) as a major regulator of breast cancer metastasis and survival. METHODS: Survival of breast cancer patients that received chemotherapy and xenograft tumour model was used to assess the effect of chemotherapy on fascin-positive and -negative breast cancer cells. Molecular and cellular assays were used to gain in-depth understanding of the relationship between fascin and chemoresistance. RESULTS: We showed a significant correlation between fascin expression and shorter survival in breast cancer patients who received chemotherapy. In xenograft experiments, fascin-positive cancer cells displayed significantly more resistance to chemotherapy-mediated apoptotic cell death than fascin-negative counterparts. This increased chemoresistance was at least partially mediated through PI3K/Akt signalling, and was paralleled by increased FAK phosphorylation, enhanced expression of the inhibitor of apoptosis proteins (XIAP and Livin) and suppression of the proapoptotic markers (caspase 9, caspase 3 and PARP). CONCLUSIONS: This is the first report to demonstrate fascin involvement in breast cancer chemotherapeutic resistance, supporting the development of fascin-targeting drugs for better treatment of chemoresistance breast cancer.
Project description:Cancer stem-like cells (CSC) contribute to therapy resistance and recurrence. Focal adhesion kinase (FAK) has a role in CSC regulation. We determined the effect of FAK inhibition on breast CSC activity alone and in combination with adjuvant therapies. FAK inhibition reduced CSC activity and self-renewal across all molecular subtypes in primary human breast cancer samples. Combined FAK and paclitaxel reduced self-renewal in triple negative cell lines. An invasive breast cancer cohort confirmed high FAK expression correlated with increased risk of recurrence and reduced survival. Co-expression of FAK and CSC markers was associated with the poorest prognosis, identifying a high-risk patient population. Combined FAK and paclitaxel treatment reduced tumour size, Ki67, ex-vivo mammospheres and ALDH+ expression in two triple negative patient derived Xenograft (PDX) models. Combined treatment reduced tumour initiation in a limiting dilution re-implantation PDX model. Combined FAK inhibition with adjuvant therapy has the potential to improve breast cancer survival.
Project description:Transmembrane 4 L6 family proteins have been known to promote cancer. In this study, we demonstrated that transmembrane 4 L6 family member 4 (TM4SF4), which is induced by ?-radiation in non-small cell lung cancer (NSCLC) cells, is involved in epithelial-to-mesenchymal transition (EMT) and cancer stem cell (CSC) properties of NSCLC through the regulation of osteopontin (OPN). Forced TM4SF4 overexpression in A549 cells increased the secretion of OPN, which activates CD44 or integrin signaling and thus maintains EMT-associated CSC-like properties. OPN, known as a downstream target of ?-catenin/T-cell factor 4 (TCF-4), was induced by up-regulated ?-catenin via TM4SF4-driven phosphorylation of glycogen synthase kinase 3b (GSK3?). TCF4 complexed to promoter regions of OPN in TM4SF4-overexpressing A549 cells was also confirmed by chromatin immunoprecipitation. Knockout of either ?-catenin or TCF4-suppressed OPN expression, demonstrating that both factors are essential for OPN expression in NSCLC cells. OPN secreted by TM4SF4/GSK3?/?-catenin signaling activated the JAK2/STAT3 or FAK/STAT3 pathway, which also up-regulates OPN expression in an autocrine manner and consequently maintains the self-renewal and metastatic capacity of cancer cells. Neutralizing antibody to OPN blocked the autocrine activation of OPN expression, consequently weakened the metastatic and self-renewal capacity of cancer cells. Collectively, our findings indicate that TM4SF4-triggered OPN expression is involved in the persistent reinforcement of EMT or cancer stemness by creating a positive feedback autocrine loop with JAK2/STAT3 or FAK/STAT3 pathways.
Project description:Fascin-1, an actin-bundling protein, plays an important role in cancer cell migration and invasion; however, the underlying mechanism remains unclear. On the basis of a 12-O-tetradecanoylphorbol 13-acetate (TPA)-induced cell migration model, it was shown that TPA increased fascin-1 mRNA and protein expression and fascin-1-dependent cell migration. TPA dose- and time-dependently increased PKC? and STAT3? activation and GSK3? phosphorylation; up-regulated Wnt-1, ?-catenin, and STAT3? expression; and increased the nuclear translocation of ?-catenin and STAT3?. Rottlerin, a PKC? inhibitor, abrogated the increases in STAT3? activation and ?-catenin and fascin-1 expression. WP1066, a STAT3 inhibitor, suppressed TPA-induced STAT3? DNA binding activity and ?-catenin expression. Knockdown of ?-catenin attenuated TPA-induced fascin-1 and STAT3? expression as well as cell migration. In addition to MCF-7, migration of Hs578T breast cancer cells was inhibited by silencing fascin-1, ?-catenin, and STAT3? expression as well. TPA also induced Wnt-1 expression and secretion, and blocking Wnt-1 signaling abrogated ?-catenin induction. DHA pretreatment attenuated TPA-induced cell migration, PKC? and STAT3? activation, GSK3? phosphorylation, and Wnt-1, ?-catenin, STAT3?, and fascin-1 expression. Our results demonstrated that TPA-induced migration is likely associated with the PKC? and Wnt-1 pathways, which lead to STAT3? activation, GSK3? inactivation, and ?-catenin increase and up-regulation of fascin-1 expression. Moreover, the anti-metastatic potential of DHA is partly attributed to its suppression of TPA-activated PKC? and Wnt-1 signaling.
Project description:Gene copy number changes, cancer stem cell (CSC) increases, and platinum chemotherapy resistance contribute to poor prognosis in patients with recurrent high grade serous ovarian cancer (HGSOC). CSC phenotypes involving Wnt-b-catenin and aldehyde dehydrogenase activities, platinum resistance, and tumor initiating frequency are here associated with spontaneous genetic gains, including genes encoding KRAS, MYC and FAK, in a new murine model of ovarian cancer (KMF). Noncanonical FAK signaling was sufficient to sustain human and KMF tumorsphere proliferation, CSC survival, and platinum resistance. Increased FAK tyrosine phosphorylation occurred in HGSOC patient tumors surviving neo-adjuvant platinum and paclitaxel chemotherapy and platinum resistant tumorspheres acquired FAK dependence for growth. Importantly, combining a pharmacologic FAK inhibitor with platinum overcame chemoresistance and triggered apoptosis in vitro and in vivo. Knockout, rescue, genomic and transcriptomic analyses collectively identified more than 400 genes regulated along a FAK/b-catenin/Myc axis impacting stemness and DNA repair in HGSOC, with 66 genes gained in a majority of Cancer Genome Atlas samples. Together, these results support combinatorial testing of FAK inhibitors for the treatment of recurrent ovarian cancer. Overall design: mRNA profiles of KMF parental, KMF clone KT13 (FAK-/-), KT13 + GFP-FAK, KT13 + GFP-FAK R454, and KT13 + deltaGSK beta-catenin cells were generated in triplicate using an Illumina NovaSeq 6000 Sequencing System
Project description:Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype that promotes a higher risk of metastasis and cancer reoccurrence. Cisplatin is one of the potential anticancer drugs for treating TNBC. However, the occurrence of cisplatin resistance still remains one of the challenges in fully eradicating TNBC. The presence of cancer stem cells (CSCs) has been proposed as one of the factors contributing to the development of cisplatin resistance. In this study, we aimed to characterize the cellular properties and reveal the corresponding putative target genes involved in cisplatin resistance associated with CSCs using the TNBC cell line (MDA-MB-231). CSC-like cells were isolated from parental cells and the therapeutic effect of cisplatin on CSC-like cells was compared to that of the parental cells via cell characterization bioassays. A PCR array was then conducted to study the expression of cellular mRNA for each subpopulation. As compared to treated parental cells, treated CSCs displayed lower events of late apoptosis/necrosis and G2/M phase cell arrest, with higher mammosphere formation capacity. Furthermore, a distinct set of putative target genes correlated to the Hedgehog pathway and angiogenesis were dysregulated solely in CSC-like cells after cisplatin treatment, which were closely related to the regulation of chemoresistance and self-renewability in breast cancer. In summary, both cellular and gene expression studies suggest the attenuated cytotoxicity of cisplatin in CSC-like cells as compared to parental cells. Understanding the role of dysregulated putative target genes induced by cisplatin in CSCs may aid in the potential development of therapeutic targets for cisplatin-resistant breast cancer.
Project description:Gene copy number changes, cancer stem cell (CSC) increases, and platinum chemotherapy resistance contribute to poor prognosis in patients with recurrent high grade serous ovarian cancer (HGSOC). CSC phenotypes involving Wnt-b-catenin and aldehyde dehydrogenase activities, platinum resistance, and tumor initiating frequency are here associated with spontaneous genetic gains, including genes encoding KRAS, MYC and FAK, in a new murine model of ovarian cancer (KMF). Noncanonical FAK signaling was sufficient to sustain human and KMF tumorsphere proliferation, CSC survival, and platinum resistance. Increased FAK tyrosine phosphorylation occurred in HGSOC patient tumors surviving neo-adjuvant platinum and paclitaxel chemotherapy and platinum resistant tumorspheres acquired FAK dependence for growth. Importantly, combining a pharmacologic FAK inhibitor with platinum overcame chemoresistance and triggered apoptosis in vitro and in vivo. Knockout, rescue, genomic and transcriptomic analyses collectively identified more than 400 genes regulated along a FAK/b-catenin/Myc axis impacting stemness and DNA repair in HGSOC, with 66 genes gained in a majority of Cancer Genome Atlas samples. Together, these results support combinatorial testing of FAK inhibitors for the treatment of recurrent ovarian cancer. Overall design: mRNA profiles of ID8 and ID8-IP / KMF cells were generated in triplicate using an Illumina NovaSeq 6000 Sequencing System
Project description:The abnormal activation of Wnt/β-catenin signaling plays a critical role in the development of lung cancer, which is also important in the generation and maintenance of lung cancer stem cell (CSC). CSCs have unique capabilities to resist anticancer therapy, seed recurrent tumors, and disseminate to and colonize distant tissues. Apatinib, a small-molecule VEGFR2-tyrosine kinase inhibitor, shows highly efficient antitumor activity in heavily treated, chemoresistant, and metastatic lung cancer. We speculated that inhibition of Wnt/β-catenin signaling and targeting lung CSCs could be one of the anti-tumor mechanisms of apatinib. In the present study we demonstrated that apatinib repressed lung CSC-like traits by hindering sphere formation ability, lung CSC-related marker expression and decreasing chemoresistance derived stemness. Mechanistically, apatinib exerted its anti-CSC effects by inhibiting β-catenin and its downstream targets. Moreover, apatinib induced the production of reactive oxyen species (ROS), which participated in the inhibitory effects of apatinib on lung CSCs. It was found that β-catenin regulated apatinib-induced production of ROS. Inhibition or promotion of ROS production with N-acetyl-L-cysteine or H<sub>2</sub>O<sub>2</sub> not only upregulated or downregulated β-catenin expression, but also prevented or promoted DNA damage, rescued or impeded sphere formation, respectively. Collectively, our findings reveal that apatinib directly inhibits β-catenin signaling and promotes ROS generation to suppress lung CSC-like characteristics. A clearer understanding of the anti-cancer mechanisms of apatinib is required for its better application in combating advanced and refractory/recurrent lung cancer when combined with conventional chemotherapy.