Evaluation of Cancer Stem Cell Markers CD133, CD44, CD24: Association with AKT Isoforms and Radiation Resistance in Colon Cancer Cells

The cell surface proteins CD133, CD24 and CD44 are putative markers for cancer stem cell populations in colon cancer, associated with aggressive cancer types and poor prognosis. It is important to understand how these markers may predict treatment outcomes, determined by factors such as radioresistance. The scope of this study was to assess the connection between EGFR, CD133, CD24, and CD44 (including isoforms) expression levels and radiation sensitivity, and furthermore analyze the influence of AKT isoforms on the expression patterns of these markers, to better understand the underlying molecular mechanisms in the cell. Three colon cancer cell-lines were used, HT-29, DLD-1, and HCT116, together with DLD-1 isogenic AKT knock-out cell-lines. All three cell-lines (HT-29, HCT116 and DLD-1) expressed varying amounts of CD133, CD24 and CD44 and the top ten percent of CD133 and CD44 expressing cells (CD133^high/CD44^high) were more resistant to gamma radiation than the ten percent with lowest expression (CD133^low/CD44^low). The AKT expression was lower in the fraction of cells with low CD133/CD44. Depletion of AKT1 or AKT2 using knock out cells showed for the first time that CD133 expression was associated with AKT1 but not AKT2, whereas the CD44 expression was influenced by the presence of either AKT1 or AKT2. There were several genes in the cell adhesion pathway which had significantly higher expression in the AKT2 KO cell-line compared to the AKT1 KO cell-line; however important genes in the epithelial to mesenchymal transition pathway (CDH1, VIM, TWIST1, SNAI1, SNAI2, ZEB1, ZEB2, FN1, FOXC2 and CDH2) did not differ. Our results demonstrate that CD133^high/CD44^high expressing colon cancer cells are associated with AKT and increased radiation resistance, and that different AKT isoforms have varying effects on the expression of cancer stem cell markers, which is an important consideration when targeting AKT in a clinical setting.     

Breast Tumors with Elevated Expression of 1q Candidate Genes Confer Poor Clinical Outcome and Sensitivity to Ras/PI3K Inhibition

Genomic aberrations are common in cancers and the long arm of chromosome 1 is known for its frequent amplifications in breast cancer. However, the key candidate genes of 1q, and their contribution in breast cancer pathogenesis remain unexplored. We have analyzed the gene expression profiles of 1635 breast tumor samples using meta-analysis based approach and identified clinically significant candidates from chromosome 1q. Seven candidate genes including exonuclease 1 (EXO1) are consistently over expressed in breast tumors, specifically in high grade and aggressive breast tumors with poor clinical outcome. We derived a EXO1 co-expression module from the mRNA profiles of breast tumors which comprises 1q candidate genes and their co-expressed genes. By integrative functional genomics investigation, we identified the involvement of EGFR, RAS, PI3K / AKT, MYC, E2F signaling in the regulation of these selected 1q genes in breast tumors and breast cancer cell lines. Expression of EXO1 module was found as indicative of elevated cell proliferation, genomic instability, activated RAS/AKT/MYC/E2F1 signaling pathways and loss of p53 activity in breast tumors. mRNA–drug connectivity analysis indicates inhibition of RAS/PI3K as a possible targeted therapeutic approach for the patients with activated EXO1 module in breast tumors. Thus, we identified seven 1q candidate genes strongly associated with the poor survival of breast cancer patients and identified the possibility of targeting them with EGFR/RAS/PI3K inhibitors.     

EGFR Signal-Network Reconstruction Demonstrates Metabolic Crosstalk in EMT

Epithelial to mesenchymal transition (EMT) is an important event during development and cancer metastasis. There is limited understanding of the metabolic alterations that give rise to and take place during EMT. Dysregulation of signalling pathways that impact metabolism, including epidermal growth factor receptor (EGFR), are however a hallmark of EMT and metastasis. In this study, we report the investigation into EGFR signalling and metabolic crosstalk of EMT through constraint-based modelling and analysis of the breast epithelial EMT cell model D492 and its mesenchymal counterpart D492M. We built an EGFR signalling network for EMT based on stoichiometric coefficients and constrained the network with gene expression data to build epithelial (EGFR_E) and mesenchymal (EGFR_M) networks. Metabolic alterations arising from differential expression of EGFR genes was derived from a literature review of AKT regulated metabolic genes. Signaling flux differences between EGFR_E and EGFR_M models subsequently allowed metabolism in D492 and D492M cells to be assessed. Higher flux within AKT pathway in the D492 cells compared to D492M suggested higher glycolytic activity in D492 that we confirmed experimentally through measurements of glucose uptake and lactate secretion rates. The signaling genes from the AKT, RAS/MAPK and CaM pathways were predicted to revert D492M to D492 phenotype. Follow-up analysis of EGFR signaling metabolic crosstalk in three additional breast epithelial cell lines highlighted variability in in vitro cell models of EMT. This study shows that the metabolic phenotype may be predicted by in silico analyses of gene expression data of EGFR signaling genes, but this phenomenon is cell-specific and does not follow a simple trend.     

TWIST1 a new determinant of epithelial to mesenchymal transition in EGFR mutated lung adenocarcinoma

Metastasis is a multistep process and the main cause of mortality in lung cancer patients. We previously showed that EGFR mutations were associated with a copy number gain at a locus encompassing the TWIST1 gene on chromosome 7. TWIST1 is a highly conserved developmental gene involved in embryogenesis that may be reactivated in cancers promoting both malignant conversion and cancer progression through an epithelial to mesenchymal transition (EMT). The aim of this study was to investigate the possible implication of TWIST1 reactivation on the acquisition of a mesenchymal phenotype in EGFR mutated lung cancer. We studied a series of consecutive lung adenocarcinoma from Caucasian non-smokers for which surgical frozen samples were available (n = 33) and showed that TWIST1 expression was linked to EGFR mutations (P<0.001), to low CDH1 expression (P<0.05) and low disease free survival (P = 0.044). To validate that TWIST1 is a driver of EMT in EGFR mutated lung cancer, we used five human lung cancer cell lines and demonstrated that EMT and the associated cell mobility were dependent upon TWIST1 expression in cells with EGFR mutation. Moreover a decrease of EGFR pathway stimulation through EGF retrieval or an inhibition of TWIST1 expression by small RNA technology reversed the phenomenon. Collectively, our in vivo and in vitro findings support that TWIST1 collaborates with the EGF pathway in promoting EMT in EGFR mutated lung adenocarcinoma and that large series of EGFR mutated lung cancer patients are needed to further define the prognostic role of TWIST1 reactivation in this subgroup.     

Signaling through the Phosphatidylinositol 3-Kinase (PI3K)/Mammalian Target of Rapamycin (mTOR) Axis Is Responsible for Aerobic Glycolysis mediated by Glucose Transporter in Epidermal Growth Factor Receptor (EGFR)-mutated Lung Adenocarcinoma

Oncogenic epidermal growth factor receptor (EGFR) signaling plays an important role in regulating global metabolic pathways, including aerobic glycolysis, the pentose phosphate pathway (PPP), and pyrimidine biosynthesis. However, the molecular mechanism by which EGFR signaling regulates cancer cell metabolism is still unclear. To elucidate how EGFR signaling is linked to metabolic activity, we investigated the involvement of the RAS/MEK/ERK and PI3K/AKT/mammalian target of rapamycin (mTOR) pathways on metabolic alteration in lung adenocarcinoma (LAD) cell lines with activating EGFR mutations. Although MEK inhibition did not alter lactate production and the extracellular acidification rate, PI3K/mTOR inhibitors significantly suppressed glycolysis in EGFR-mutant LAD cells. Moreover, a comprehensive metabolomics analysis revealed that the levels of glucose 6-phosphate and 6-phosphogluconate as early metabolites in glycolysis and PPP were decreased after inhibition of the PI3K/AKT/mTOR pathway, suggesting a link between PI3K signaling and the proper function of glucose transporters or hexokinases in glycolysis. Indeed, PI3K/mTOR inhibition effectively suppressed membrane localization of facilitative glucose transporter 1 (GLUT1), which, instead, accumulated in the cytoplasm. Finally, aerobic glycolysis and cell proliferation were down-regulated when GLUT1 gene expression was suppressed by RNAi. Taken together, these results suggest that PI3K/AKT/mTOR signaling is indispensable for the regulation of aerobic glycolysis in EGFR-mutated LAD cells.     

Fluid shear stress induces Runx‐2 expression via upregulation of PIEZO1 in MC3T3‐E1 cells

Mechanically induced biological responses in bone cells involve a complex biophysical process. Although various mechanosensors have been identified, the precise mechanotransduction pathway remains poorly understood. PIEZO1 is a newly discovered mechanically activated ion channel in bone cells. This study aimed to explore the involvement of PIEZO1 in mechanical loading (fluid shear stress)‐induced signaling cascades that control osteogenesis. The results showed that fluid shear stress increased PIEZO1 expression in MC3T3‐E1 cells. The fluid shear stress elicited the key osteoblastic gene Runx‐2 expression; however, PIEZO1 silencing using small interference RNA blocked these effects. The AKT/GSK‐3β/β‐catenin pathway was activated in this process. PIEZO1 silencing impaired mechanically induced activation of the AKT/GSK‐3β/β‐catenin pathway. Therefore, the results demonstrated that MC3T3‐E1 osteoblasts required PIEZO1 to adapt to the external mechanical fluid shear stress, thereby inducing osteoblastic Runx‐2 gene expression, partly through the AKT/GSK‐3β/β‐catenin pathway.     

Differential roles of ERRFI1 in EGFR and AKT pathway regulation affect cancer proliferation

AKT signaling is modulated by a complex network of regulatory proteins and is commonly deregulated in cancer. Here, we present a dual mechanism of AKT regulation by the ERBB receptor feedback inhibitor 1 (ERRFI1). We show that in cells expressing high levels of EGFR, ERRF1 inhibits growth and enhances responses to chemotherapy. This is mediated in part through the negative regulation of AKT signaling by direct ERRFI1‐dependent inhibition of EGFR. In cells expressing low levels of EGFR, ERRFI1 positively modulates AKT signaling by interfering with the interaction of the inactivating phosphatase PHLPP with AKT, thereby promoting cell growth and chemotherapy desensitization. These observations broaden our understanding of chemotherapy response and have important implications for the selection of targeted therapies in a cell context‐dependent manner. EGFR inhibition can only sensitize EGFR‐high cells for chemotherapy, while AKT inhibition increases chemosensitivity in EGFR‐low cells. By understanding these mechanisms, we can take advantage of the cellular context to individualize antineoplastic therapy. Finally, our data also suggest targeting of EFFRI1 in EGFR‐low cancer as a promising therapeutic approach.     

Blockade of Hedgehog Signaling Synergistically Increases Sensitivity to Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitors in Non-Small-Cell Lung Cancer Cell Lines

Aberrant activation of the hedgehog (Hh) signaling pathway has been implicated in the epithelial-to-mesenchymal transition (EMT) and cancer stem-like cell (CSC) maintenance; both processes can result in tumor progression and treatment resistance in several types of human cancer. Hh cooperates with the epidermal growth factor receptor (EGFR) signaling pathway in embryogenesis. We found that the Hh signaling pathway was silenced in EGFR-TKI-sensitive non-small-cell lung cancer (NSCLC) cells, while it was inappropriately activated in EGFR-TKI-resistant NSCLC cells, accompanied by EMT induction and ABCG2 overexpression. Upregulation of Hh signaling through extrinsic SHH exposure downregulated E-cadherin expression and elevated Snail and ABCG2 expression, resulting in gefitinib tolerance (P < 0.001) in EGFR-TKI-sensitive cells. Blockade of the Hh signaling pathway using the SMO antagonist SANT-1 restored E-cadherin expression and downregulate Snail and ABCG2 in EGFR-TKI-resistant cells. A combination of SANT-1 and gefitinib markedly inhibited tumorigenesis and proliferation in EGFR-TKI-resistant cells (P < 0.001). These findings indicate that hyperactivity of Hh signaling resulted in EGFR-TKI resistance, by EMT introduction and ABCG2 upregulation, and blockade of Hh signaling synergistically increased sensitivity to EGFR-TKIs in primary and secondary resistant NSCLC cells. E-cadherin expression may be a potential biomarker of the suitability of the combined application of an Hh inhibitor and EGFR-TKIs in EGFR-TKI-resistant NSCLCs.     

AKT isoforms modulate Th1‐like Treg generation and function in human autoimmune disease

Foxp3⁺ regulatory T cells (Tregs) exhibit plasticity, which dictates their function. Secretion of the inflammatory cytokine IFNγ, together with the acquisition of a T helper 1 (Th1)‐like effector phenotype as observed in cancer, infection, and autoimmune diseases, is associated with loss of Treg suppressor function through an unknown mechanism. Here, we describe the signaling events driving the generation of human Th1‐Tregs. Using a genome‐wide gene expression approach and pathway analysis, we identify the PI3K/AKT/Foxo1/3 signaling cascade as the major pathway involved in IFNγ secretion by human Tregs. Furthermore, we describe the opposing roles of AKT isoforms in Th1‐Treg generation ex vivo. Finally, we employ multiple sclerosis as an in vivo model with increased but functionally defective Th1‐Tregs. We show that the PI3K/AKT/Foxo1/3 pathway is activated in ex vivo‐isolated Tregs from untreated relapsing–remitting MS patients and that blockade of the pathway inhibits IFNγ secretion and restores the immune suppressive function of Tregs. These data define a fundamental pathway regulating the function of human Tregs and suggest a novel treatment paradigm for autoimmune diseases.     

Dissecting the EGFR-PI3K-AKT pathway in oral cancer highlights the role of the EGFR variant III and its clinical relevance

Background

Dysregulated epidermal growth factor receptor (EGFR)-phosphoinositide-3-kinase (PI3K)-AKT signaling is considered pivotal for oral cancer, and the pathway is a potential candidate for therapeutic targeting.

Results

A total of 108 archival samples which were from surgically resected oral cancer were examined. Immunohistochemical staining showed the protein expression of membranous wild-type EGFR and cytoplasmic phosphorylated AKT was detected in 63.9% and 86.9% of the specimens, respectively. In 49.1% of the samples, no phosphatase and tensin homolog (PTEN) expression was detected. With regard to the EGFR variant III (EGFRvIII), 75.0% of the samples showed positive expression for moderate to severe staining, 31.5% of which had high expression levels. Real-time polymerase chain reaction assays for gene copy number assessment of PIK3CA revealed that 24.8% of the samples had alterations, and of EGFR showed that 49.0% had amplification. Direct sequencing of PIK3CA gene showed 2.3% of the samples had a hotspot point mutation. Statistical assessment showed the expression of the EGFRvIII correlated with the T classification and TNM stage. The Kaplan-Meier analyses for patient survival showed that the individual status of phosphorylated AKT and EGFRvIII led to significant differences in survival outcome. The multivariate analysis indicated that phosphorylated AKT, EGFRvIII expression and disease stage were patient survival determinants.

Conclusions

Aberrations in the EGFR-PI3K-AKT pathway were frequently found in oral cancers. EGFRvIII and phosphorylated AKT were predictors for the patient survival and clinical outcome.     

HER2+ Cancer Cell Dependence on PI3K vs. MAPK Signaling Axes Is Determined by Expression of EGFR,ERBB3 and CDKN1B

Understanding the molecular pathways by which oncogenes drive cancerous cell growth, and how dependence on such pathways varies between tumors could be highly valuable for the design of anti-cancer treatment strategies. In this work we study how dependence upon the canonical PI3K and MAPK cascades varies across HER2+ cancers, and define biomarkers predictive of pathway dependencies. A panel of 18 HER2+ (ERBB2-amplified) cell lines representing a variety of indications was used to characterize the functional and molecular diversity within this oncogene-defined cancer. PI3K and MAPK-pathway dependencies were quantified by measuring in vitro cell growth responses to combinations of AKT (MK2206) and MEK (GSK1120212; trametinib) inhibitors, in the presence and absence of the ERBB3 ligand heregulin (NRG1). A combination of three protein measurements comprising the receptors EGFR, ERBB3 (HER3), and the cyclin-dependent kinase inhibitor p27 (CDKN1B) was found to accurately predict dependence on PI3K/AKT vs. MAPK/ERK signaling axes. Notably, this multivariate classifier outperformed the more intuitive and clinically employed metrics, such as expression of phospho-AKT and phospho-ERK, and PI3K pathway mutations (PIK3CA, PTEN, and PIK3R1). In both cell lines and primary patient samples, we observed consistent expression patterns of these biomarkers varies by cancer indication, such that ERBB3 and CDKN1B expression are relatively high in breast tumors while EGFR expression is relatively high in other indications. The predictability of the three protein biomarkers for differentiating PI3K/AKT vs. MAPK dependence in HER2+ cancers was confirmed using external datasets (Project Achilles and GDSC), again out-performing clinically used genetic markers. Measurement of this minimal set of three protein biomarkers could thus inform treatment, and predict mechanisms of drug resistance in HER2+ cancers. More generally, our results show a single oncogenic transformation can have differing effects on cell signaling and growth, contingent upon the molecular and cellular context.     

Garcinone C suppresses colon tumorigenesis through the Gli1-dependent hedgehog signaling pathway

BackgroundAlthough garcinone C, a natural xanthone derivative identified in the pericarp of Garcinia mangostana, has been demonstrated to exert different health beneficial activities in oxidative stress and β-amyloid aggregation, the role of garcinone C in colon tumorigenesis has not been investigated. In addition, aberrant Hedgehog (Hh) signaling activation is associated with tumorigenesis including colon cancer. Here, we hypothesized that garcinone C can prevent colon tumorigenesis through regulating the Hh signaling pathway.MethodColony formation assay and flow cytometry were used to evaluate the effect of garcinone C on the proliferation and cell cycle progression of colon cancer cells. Protein expression of cell cycle related markers and Hh/Gli1 signaling mediators were determined. The regulatory effect of orally administered garcinone C on the Hh/Gli1 signaling pathway and colon tumorigenesis was evaluated in an azoxymethane (AOM)/dextran sulfate sodium (DSS)-induced colon cancer animal model.ResultsGarcinone C suppressed the proliferation of colon cancer cells, induced G0/G1 cell cycle arrest, as well as regulated the expression of cell cycle-related markers such as cyclin D1, cyclin E, CDK6, and p21. Garcinone C inhibited the expression of Gli1, a key mediator of Hedgehog signaling, and protein kinase B (AKT) phosphorylation in Smo-independent colon cancer cells. In the AOM/DSS-induced colon tumorigenesis model, garcinone C significantly inhibited tumor development, regulated the expression of cell cycle markers and Gli1, and reduced AKT phosphorylation in colon tumor tissues, which is consistent with our in vitro results.ConclusionGarcinone C can suppress colon tumorigenesis in vitro and in vivo through Gli1-dependent non-canonical Hedgehog signaling, suggesting that it may serve as a potent chemopreventive agent against colon tumorigenesis.     

FGF9/FGFR1 promotes cell proliferation,epithelial-mesenchymal transition,M2 macrophage infiltration and liver metastasis of lung cancer

Fibroblast growth factors 9 (FGF9) modulates cell proliferation, differentiation and motility for development and repair in normal cells. Abnormal activation of FGF9 signaling is associated with tumor progression in many cancers. Also, FGF9 may be an unfavorable prognostic indicator for non-small cell lung cancer patients. However, the effects and mechanisms of FGF9 in lung cancer remain elusive. In this study, we investigated the FGF9-induced effects and signal activation profiles in mouse Lewis lung carcinoma (LLC) in vitro and in vivo. Our results demonstrated that FGF9 significantly induced cell proliferation and epithelial-to-mesenchymal transition (EMT) phenomena (migration and invasion) in LLC cells. Mechanism-wise, FGF9 interacted with FGFR1 and activated FAK, AKT, and ERK/MAPK signal pathways, induced the expression of EMT key proteins (N-cadherin, vimentin, snail, MMP2, MMP3 and MMP13), and reduced the expression of E-cadherin. Moreover, in the allograft mouse model, intratumor injection of FGF9 to LLC-tumor bearing C57BL/6 mice enhanced LLC tumor growth which were the results of increased Ki67 expression and decreased cleaved caspase-3 expression compared to control groups. Furthermore, we have a novel finding that FGF9 promoted liver metastasis of subcutaneous inoculated LLC tumor with angiogenesis, EMT and M2-macrophage infiltration in the tumor microenvironment. In conclusion, FGF9 activated FAK, AKT, and ERK signaling through FGFR1 with induction of EMT to stimulate LLC tumorigenesis and hepatic metastasis. This novel FGF9/LLC allograft animal model may therefore be useful to study the mechanism of liver metastasis which is the worst prognostic factor for lung cancer patients with distant organ metastasis.     

BX-795 inhibits neuroblastoma growth and enhances sensitivity towards chemotherapy

High-risk neuroblastoma (NB) represents a major clinical challenge in pediatric oncology due to relapse of metastatic, drug-resistant disease, and treatment-related toxicities. An analysis of 1235 primary NB patient dataset revealed significant increase in AKT1 and AKT2 gene expression with cancer stage progression. Additionally, Both AKT1 and AKT2 expression inversely correlate with poor overall survival of NB patients. AKT1 and AKT2 genes code for AKT that drive a major oncogenic cell signaling pathway known in many cancers, including NB. To inhibit AKT pathway, we repurposed an antiviral inhibitor BX-795 that inhibits PDK1, an upstream activator of AKT. BX-795 potently inhibits NB cell proliferation and colony growth in a dose-dependent manner. BX-795 significantly enhances apoptosis and blocks cell cycle progression at mitosis phase in NB. Additionally, BX-795 potently inhibits tumor formation and growth in a NB spheroid tumor model. We further tested dual therapeutic approaches by combining BX-795 with either doxorubicin or crizotinib and found synergistic and significant inhibition of NB growth, in contrast to either drug alone. Overall, our data demonstrate that BX-795 inhibits AKT pathway to inhibit NB growth, and combining BX-795 with current therapies is an effective and clinically tractable therapeutic approach for NB.