FOXP3 functions not only as the master regulator in regulatory T cells but also as an X-linked tumor suppressor. The tumor suppressive activity of FOXP3 has been observed in tumor initiation, but its role during tumor progression remains controversial. Moreover, the mechanism of FOXP3-mediated tumor suppressive activity remains largely unknown. Using chromatin immunoprecipitation sequencing, we identified a series of potential FOXP3-targeted microRNAs (miRs) in MCF7 cells. Notably, FOXP3 significantly induced the expression of miR-146a/b. In vitro, FOXP3-induced miR-146a/b prevented tumor cell proliferation and enhanced apoptosis. Functional analyses in vitro and in vivo revealed that FOXP3-induced miR-146a/b negatively regulate NF-κB activation by inhibiting the expression of IRAK1 and TRAF6. In chromatin immunoprecipitation assays, FOXP3 directly bound the promoter region of miR-146a but not of miR-146b, and FOXP3 interacted directly with NF-κB p65 to regulate an miR-146-NF-κB negative feedback regulation loop in normal breast epithelial and tumor cells, as demonstrated with luciferase reporter assays. Although FOXP3 significantly inhibited breast tumor growth and migration in vitro and metastasis in vivo, FOXP3-induced miR-146a/b contributed only to the inhibition of breast tumor growth. These data suggest that miR-146a/b contribute to FOXP3-mediated tumor suppression during tumor growth by triggering apoptosis. The identification of a FOXP3-miR-146-NF-κB axis provides an underlying mechanism for disruption of miR-146 family member expression and constitutive NF-κB activation in breast cancer cells. Linking the tumor suppressor function of FOXP3 to NF-κB activation reveals a potential therapeutic approach for cancers with FOXP3 defects.
FOXP3; microRNA; NF-κB; breast cancer
The tumor suppressive activity of FOXP3 has been observed in tumor initiation, but the underlying mechanism still remains largely unknown. Here, we identified a FOXP3-microRNA-146 (miR-146)-NF-κB axis in vitro and in vivo in prostate cancer cells. We observed that FOXP3 dramatically induced the expression of miR-146a/b, which contributed to transcriptional inhibition of IRAK1 and TRAF6, in prostate cancer cell lines. Tissue-specific deletion of Foxp3 in mouse prostate caused a significant reduction of miR-146a and upregulation of NF-κB activation. In addition, prostatic intraepithelial neoplasia lesions were observed in miR-146a mutant mice as well as in Foxp3 mutant mice. Notably, the NF-κB inhibitor bortezomib inhibited cell proliferation and induced apoptosis in prostate epithelial cells, attenuating prostatic intraepithelial neoplasia formation in Foxp3 mutant mice. Our data suggest that the FOXP3-miR-146-NF-κB axis has a functional role during tumor initiation in prostate cancer. Targeting the miR-146-NF-κB axis may provide a new therapeutic approach for prostate cancers with FOXP3 defects.
FOXP3; microRNA; NF-κB; prostate cancer; therapy
MicroRNA-128 (miR-128) is reduced in prostate cancer (PCa) relative to normal/benign prostate tissues but causal roles are obscure. Here we show that exogenously introduced miR-128 suppresses tumor regeneration in multiple PCa xenograft models. Cancer stem-like cell (CSC) associated properties were blocked, including holoclone and sphere formation as well as clonogenic survival. Using a miR-128 sensor to distinguish cells on the basis of miR-128 expression, we found that miR-128-lo cells possessed higher clonal, clonogenic and tumorigenic activities than miR-128-hi cells. miR-128 targets the stem cell regulatory factors BMI-1, NANOG, and TGFBR1, the expression of which we found to vary inversely with miR-128 expression in PCa stem/progenitor cell populations. In particular, we defined BMI-1 as a direct and functionally relevant target of miR-128 in PCa cells, where these genes were reciprocally expressed and exhibited opposing biological functions. Our results define a tumor suppressor function for miR-128 in PCa by limiting CSC properties mediated by BMI-1 and other central stem cell regulators, with potential implications for PCa gene therapy.
microRNA; prostate cancer; miR-128; BMI-1; cancer stem cells; tumor suppressor
Drug resistance is a major cause of failure in cancer chemotherapy. Therefore, identification and combined use of adjuvant compounds that can overcome drug resistance may improve the efficacy of cancer therapy. We screened extracts of Verticillium sp-infected mushrooms for anti-tumor compounds and identified the compound Verticillin A as an inducer of hepatoma cell apoptosis in vitro and an inhibitor of tumor xenograft growth in vivo. Verticillin A exhibited a potent apoptosis sensitizing activity in human colon carcinoma cells exposed to TRAIL or Fas in vitro. Furthermore, Verticillin A effectively sensitized metastatic human colon carcinoma xenograft to TRAIL-mediated growth inhibition in vivo. At the molecular level, we observed that Verticillin A induces cell cycle arrest in the G2 phase of the cell cycle in human colon carcinoma cells, markedly upregulating BNIP3 in both hepatoma and colon carcinoma cells. Notably, silencing BNIP3 decreased the sensitivity of tumor cells to Verticillin A-induced apoptosis in the absence or presence of TRAIL. We found that the BNIP3 promoter are methylated in both human hepatoma and colon carcinoma cells and tumor specimens. Verticillin A upregulated the expression of a panel of genes known to be regulated at the level of DNA methylation, in support of the concept that Verticillin A may act by demethylating the BNIP3 promoter to upregulate BNIP3 expression. Taken together, our findings identify Verticillin A as a potent apoptosis sensitizer with great promise for further development as an adjuvant agent to overcome drug resistance in human cancer therapy.
Verticillin A; Drug Resistance; TRAIL; Fas; BNIP3
Trastuzumab shows remarkable efficacy in treatment of ErbB2-positive breast cancers when used alone or in combination with other chemotherapeutics. However, acquired resistance develops in most treated patients, necessitating alternate treatment strategies. Increased aerobic glycolysis is a hallmark of cancer and inhibition of glycolysis may offer a promising strategy to preferentially kill cancer cells. In this study, we investigated the antitumor effects of trastuzumab in combination with glycolysis inhibitors in ErbB2-positive breast cancer. We found that trastuzumab inhibits glycolysis via downregulation of heat shock factor 1 (HSF1) and lactate dehydrogenase A (LDH-A) in ErbB2-positive cancer cells, resulting in tumor growth inhibition. Moreover, increased glycolysis via HSF1 and LDH-A contributes to trastuzumab resistance. Importantly, we found that combining trastuzumab with glycolysis inhibition synergistically inhibited trastuzumab-sensitive and -resistant breast cancers in vitro and in vivo, due to more efficient inhibition of glycolysis. Taken together, our findings show how glycolysis inhibition can dramatically enhance the therapeutic efficacy of trastuzumab in ErbB2-positive breast cancers, potentially useful as a strategy to overcome trastuzumab resistance.
Warburg effect; glycolysis; HSF1; LDH-A; trastuzumab; ErbB2; resistance
Transforming growth factor beta (TGF-β) receptors are centrally involved in TGF-β-mediated cell growth and differentiation and are frequently inactivated in non-small cell lung cancer (NSCLC). Constitutively decreased type I TGF-β receptor (TGFBR1) expression is emerging as a novel tumor-predisposing phenotype. The association of TGFBR1 haplotypes with risk for NSCLC has not yet been studied. We tested the hypothesis that single nucleotide polymorphisms (SNPs) and/or TGFBR1 haplotypes are associated with risk of NSCLC. We genotyped six TGFBR1 haplotype tagging SNPs (htSNPs) by PCR-restriction fragment length polymorphism (PCR-RFLP) assays and one htSNP by PCR-single strand conformation polymorphism (PCR-SSCP) assay in two case-control studies. Case-control study 1 included 102 NSCLC patients and 104 healthy controls from Suzhou. Case-control study 2 included 131 patients with NSCLC and 133 healthy controls from Wuxi. Individuals included in both case-control studies were Han Chinese. Haplotypes were reconstructed according to the genotyping data and linkage disequilibrium (LD) status of these seven htSNPs. None of the htSNP was associated with NSCLC risk in either study. However, a four-marker haplotype CTGC was significantly more common among controls than among cases in both studies (P=0.014 and P=0.010, respectively) indicating that this haplotype is associated with decreased NSCLC risk (adjusted OR, 0.09; 95% CI, 0.01-0.61 and adjusted OR, 0.11; 95% CI, 0.02-0.59, respectively). Combined analysis of both studies shows a strong association of this four-marker haplotype with decreased NSCLC risk (adjusted OR, 0.11; 95% CI, 0.03-0.39). This is the first evidence of an association between a TGFBR1 haplotype and risk for NSCLC.
NSCLC; TGFBR1; Polymorphisms; Haplotypes; Risk
Resistance to currently available targeted therapies significantly hampers the survival of prostate cancer (PCa) patients with bone metastasis. Here we demonstrate an important resistance mechanism initiated from tumor-induced bone. Studies using an osteogenic patient-derived xenograft, MDA-PCa-118b, revealed that tumor cells resistant to cabozantinib, a Met and VEGFR-2 inhibitor, reside in a "resistance niche" adjacent to PCa-induced bone. We performed secretome analysis of the conditioned medium from tumor-induced bone to identify proteins (termed "osteocrines") found within this resistance niche. In accordance with previous reports demonstrating that activation of integrin signaling pathways confers therapeutic resistance, 27 of the 90 osteocrines identified were integrin ligands. We found that following cabozantinib treatment, only tumor cells positioned adjacent to newly-formed woven bone remained viable and expressed high levels of pFAK-Y397 and pTalin-S425, mediators of integrin signaling. Accordingly, treatment of C4-2B4 cells with integrin ligands resulted in increased pFAK-Y397 expression and cell survival, whereas targeting integrins with FAK inhibitors PF-562271 or defactinib inhibited FAK phosphorylation and reduced the survival of PC3-mm2 cells. Moreover, treatment of MDA-PCa-118b tumors with PF-562271 led to decreased tumor growth, irrespective of initial tumor size. Finally, we show that upon treatment cessation, the combination of PF-562271 and cabozantinib delayed tumor recurrence in contrast to cabozantinib treatment alone. Our studies suggest that identifying paracrine de novo resistance mechanisms may significantly contribute to the generation of a broader set of potent therapeutic tools that act combinatorially to inhibit metastatic PCa.
prostate cancer; therapy resistance; cabozantinib; tumor-induced bone; bone metastasis; osteocrines; osteoblast
With the goal to remove the roots of cancer, eliminate metastatic seeds, and overcome therapy resistance, the 2014 inaugural International Cancer Stem Cell (CSC) Conference at Cleveland, OH, convened together over 320 investigators, including 55 invited world-class speakers, 25 short oral presenters, and 100 poster presenters, to gain an in-depth understanding of CSCs and explore therapeutic opportunities targeting CSCs. The meeting enabled intriguing discussions on several topics including: genetics and epigenetics; cancer origin and evolution; microenvironment and exosomes; metabolism and inflammation; metastasis and therapy resistance; single cell and heterogeneity; plasticity and reprogramming; as well as other new concepts. Reports of clinical trials targeting CSCs emphasized the urgent need for strategically designing combinational CSC-targeting therapies against cancer.
Patient-derived human-in-mouse xenograft models of breast cancer (PDX models) that exhibit spontaneous lung metastases offer a potentially powerful model of cancer metastasis. In this study, we evaluated the malignant character of lung micro-metastases that emerge in such models after orthotopic implantation of human breast tumor cells into the mouse mammary fat pad. Interestingly, relative to the parental primary breast tumors, the lung metastasis (met)-derived mammary tumors exhibited a slower growth rate and a reduced metastatic potential with a more differentiated epithelial status. Epigenetic correlates were determined by gene array analyses. Lung met-derived tumors displayed differential expression of negative regulators of cell proliferation and metabolism and positive regulators of mammary epithelial differentiation. Clinically, this signature correlated with breast tumor subtypes. We identified microRNA-138 as a novel regulator of invasion and epithelial-mesenchymal transition in breast cancer cells, acting by directly targeting the polycomb epigenetic regulator EZH2. Mechanistic investigations showed that GATA3 transcriptionally controlled miR-138 levels in lung metastases. Notably, the miR-138 activity signature served as a novel independent prognostic marker for patient survival beyond traditional pathologic variables, intrinsic subtypes or a proliferation gene signature. Our results highlight the loss of malignant character in some lung micro-metastatic lesions and the epigenetic regulation of this phenotype.
breast cancer; pulmonary metastasis; microRNA; EZH2; malignancy
Blocking the MDM2-p53 protein-protein interaction has long been considered to offer a broad cancer therapeutic strategy, despite the potential risks of selecting tumors harboring p53 mutations that escape MDM2 control. In this study, we report a novel small molecule inhibitor of the MDM2-p53 interaction, SAR405838 (MI-77301) that has been advanced into Phase I clinical trials. SAR405838 binds to MDM2 with Ki = 0.88 nM and has high specificity over other proteins. A co-crystal structure of the SAR405838:MDM2 complex shows that in addition to mimicking three key p53 amino acid residues, the inhibitor captures additional interactions not observed in the p53-MDM2 complex and induces refolding of the short, unstructured MDM2 N-terminal region to achieve its high affinity. SAR405838 effectively activates wild-type p53 in vitro and in xenograft tumor tissue of leukemia and solid tumors, leading to p53-dependent cell cycle arrest and/or apoptosis. At well-tolerated dose schedules, SAR405838 achieves either durable tumor regression or complete tumor growth inhibition in mouse xenograft models of SJSA-1 osteosarcoma, RS4;11 acute leukemia, LNCaP prostate cancer and HCT-116 colon cancer. Remarkably, a single oral dose of SAR405838 is sufficient to achieve complete tumor regression in the SJSA-1 model. Mechanistically, robust transcriptional up-regulation of PUMA induced by SAR405838 results in strong apoptosis in tumor tissue, leading to complete tumor regression. Our findings provide a preclinical basis upon which to evaluate SAR405838 as a therapeutic agent in patients whose tumors retain wild-type p53.
MDM2; p53; small-molecule inhibitor; protein-protein interaction
Notch pathway plays an important role in the growth of high-grade serous ovarian (HGS-OvCa) and other cancers, but its clinical and biological mechanisms are not well understood. Here, we found that the Notch pathway alterations are prevalent and significantly related to poor clinical outcome in patients with ovarian cancer. Particularly, Notch3 alterations, including amplification and upregulation, were highly associated with poor patient survival. Targeting Notch3 inhibited OvCa growth and induced apoptosis. Importantly, we found that DNM-mediated endocytosis was required for selectively activating Jagged-1-mediated Notch3 signaling. Cleaved Notch3 expression was the critical determinant of response to Notch-targeted therapy. Collectively, these data identify previously unknown mechanisms underlying Notch3 signaling and identify new, biomarker-driven approaches for therapy.
HMGA2, a high-mobility-group AT-hook protein, is an oncogene involved in tumorigenesis of many malignant neoplasms. HMGA2 overexpression is common in both early and later stage of high grade ovarian serous papillary carcinoma. To test whether HMGA2 participates in the initiation of ovarian cancer and promotion of aggressive tumor growth, we examined the oncogenic properties of HMGA2 in ovarian surface epithelial (OSE) cell lines. We found that introduction of HMGA2 overexpression was sufficient to induce OSE transformation in vitro. HMGA2-mediated OSE transformation resulted in tumor formation in xenografts of nude mice. By silencing HMGA2 in HMGA2 overexpressing OSE and ovarian cancer cell lines, the aggressiveness of tumor cell growth behaviors was partially suppressed. Global gene profiling analyses revealed that HMGA2-mediated tumorigenesis was associated with expression changes of target genes and microRNAs that are involved in epithelial-to-mesenchymal transition (EMT). Lumican (LUM), a tumor suppressor that inhibits EMT, was found to be transcriptionally repressed by HMGA2 and was frequently lost in human high-grade serous papillary carcinoma.
HMGA2; ovarian cancer; miRNA; oncogene; EMT
miRNAs are essential for self-renewal and differentiation of normal and malignant stem cells by regulating the expression of key stem cell regulatory genes. Here, we report evidence implicating the miR100 in self-renewal of cancer stem-like cells (CSC). We found that miR100 expression levels relate to the cellular differentiation state, with lowest expression in cells displaying stem cell markers. Utilizing a tetracycline-inducible lentivirus to elevate expression of miR100 in human cells, we found that increasing miR100 levels decreased the production of breast CSCs. This effect was correlated with an inhibition of cancer cell proliferation in vitro and in mouse tumor xenografts due to attenuated expression of the CSC regulatory genes SMARCA5, SMARCD1, and BMPR2. Furthermore, miR100 induction in breast CSCs immediately upon their orthotopic implantation or intracardiac injection completely blocked tumor growth and metastasis formation. Clinically, we observed a significant association between miR100 expression in breast cancer specimens and patient survival. Our results suggest that miR100 is required to direct CSC self-renewal and differentiation.
CD22 is a transmembrane glycoprotein expressed by mature B cells. It inhibits signal transduction by the B cell receptor and its co-receptor CD19. Recently it was reported that most human lung cancer cells and cell lines express CD22 making it an important new lung cancer therapeutic target (Can Res 72:5556, 2012). The objective of our studies was to independently validate these results with the goal of testing the efficacy of our CD22 immunotoxins on lung cancer cell lines. As determined by qRT-PCR analysis, we found that levels of CD22 mRNA in a panel of human lung cancer cell lines were 200–60,000- fold lower than those observed in the human CD22+ Burkitt’s lymphoma cells, Daudi. Using flow cytometry with a panel of CD22 monoclonal antibodies and Western blot analyses, we could not detect surface or intracellular expression of CD22 protein in a panel of lung cancer cell lines. In addition, the in vitro proliferation of the lung tumor cell lines was not affected by CD22 antibodies or our highly potent anti-CD22 immunotoxin. By contrast, CD22+ Daudi cells expressed high levels of CD22 mRNA and protein and were sensitive to our CD22 immunotoxin. Importantly, primary non-small cell lung cancers from over 250 patient specimens did not express detectable levels of CD22 protein as assessed by immunohistochemistry. We conclude that CD22 is not expressed at measurable levels on the surface of lung cancer cells and that these cells can not be killed by anti-CD22 immunotoxins.
lung cancer; CD22 expression
Sexual transmission of human papillomavirus, particularly HPV16, has been associated with an increasing incidence of oropharyngeal squamous cell carcinoma (OPC). Telomere shortening results in chromosomal instability, subsequently leading to cancer development. Given that HPV16 can affect telomerase activity and telomere length (TL), we conjectured that TL in peripheral blood lymphocytes (PBLs) may affect the risk of HPV16-associated OPC and tumor HPV16 status in patients. TL in PBLs and HPV16 serological status were measured in peripheral blood samples in 188 patients with OPC, 137 patients with oral cavity cancer (OCC) and 335 controls of non-Hispanic whites. Tumor HPV status was determined in 349 OPC cases. Odds ratios and 95% confidence intervals were calculated in univariate and multivariable logistic regression models. Overall, compared with long TL, short TL was associated significantly with a moderately increased risk of OPC but no increased risk of OCC. When we stratified the data by HPV16 serological status, using long TL and HPV16 seronegativity as the reference group, we found that the risk associated with HPV16 seropositivity was higher among OPC patients with short TL. Notably, such risk was particularly pronounced in never smokers, never drinkers and those >50 years of age. Furthermore, short TL was also associated significantly with tumor HPV-positive OPC. Together, our findings suggest that TL in PBLs may be associated with higher risk of HPV16-associated OPC and tumor HPV16 status, particularly in certain patient subgroups. Larger studies are needed to validate these findings.
telomere length; HPV; molecular epidemiology; oropharyngeal cancer
FPR2 (Fpr2 in mouse) is a G protein-coupled receptor interacting with bacterial and host-derived chemotactic agonists. Fpr2 supports innate and adaptive immune responses as illustrated by the reduction in severity of allergic airway inflammation in Fpr2-KO mice, due to impaired trafficking of antigen presenting dendritic cells (DCs). The aim of this study is to examine the role of Fpr2 in host anti-tumor responses. We found that Fpr2-KO mice bearing subcutaneously implanted Lewis lung carcinoma (LLC) cells exhibited significantly shortened survival than normal mice due to more rapidly growing tumors. In contrast, in Fpr2-transgenic mice over-expressing Fpr2, subcutaneously implanted LLC tumors grew more slowly than those in wild type (WT) littermates. Investigation of tumor tissues revealed an increased number of macrophages associated with tumors grown in Fpr2-KO mice. Macrophages derived from Fpr2-KO mice showed a more potent chemotactic response to LLC-derived supernatant (LLC Sup), which could be neutralized by an anti-CCL2 antibody. The increased chemotaxis of Fpr2-KO mouse macrophages in response to LLC Sup was due to their higher level expression of CCR4, a chemokine receptor that also recognizes the ligand CCL2. Furthermore, macrophages from Fpr2-KO mice acquired an M2 phenotype after stimulation with LLC Sup. These results suggest that Fpr2 plays an important role in host defense against implanted LLC by sustaining macrophages in an M1 phenotype with more potent anti-tumor activities.
Fpr2; macrophages; chemotaxis; LLC; supernatant
A truncated version of retinoid X receptor-α, tRXRα, promotes cancer cell survival by activating the PI3K/AKT pathway. However, targeting the tRXRα-mediated survival pathway for cancer treatment remains to be explored. We report here our identification of a new natural product molecule, CF31, a xanthone isolated from Cratoxylum formosum ssp. Pruniflorum, and the biological evaluation of its regulation of the tRXRα-mediated PI3K/AKT pathway. CF31 binds RXRα and its binding results in inhibition of RXRα transactivation. Through RXRα mutational analysis and computational studies, we show that Arg316 of RXRα, known to form salt bridges with certain RXRα ligands such as 9-cis-retinoic acid (9-cis-RA), is not required for the antagonist effect of CF31, demonstrating a distinct binding mode. Evaluation of several CF31 analogs suggests that the antagonist effect is mainly attributed to an interference with Leu451 of helix H12 in RXRα. CF31 is a potent inhibitor of AKT activation in various cancer cell lines. When combined with TNFα, it suppresses TNFα activation of AKT by inhibiting TNFα-induced tRXRα interaction with the p85α regulatory subunit of PI3K. CF31 inhibition of TNFα activation of AKT also results in TNFα-dependent activation of caspase-8 and apoptosis. Together, our results demonstrate that CF31 is an effective converter of TNFα signaling from survival to death by targeting tRXRα in a unique mode and suggest that identification of a natural product that targets an RXR-mediated cell survival pathway that regulates PI3K/Akt may offer a new therapeutic strategy to kill cancer cells.
retinoid X receptor; CF31; AKT; TNFα; cancer; apoptosis
Gastric cancer is the most common cancer in Asia and most developing countries. Despite the use of multimodality therapeutics, it remains the second leading cause of cancer death in the world. To identify the molecular underpinnings of gastric cancer in the Asian population, we applied an RNA-sequencing approach to gastric tumor and noncancerous specimens, generating 680 million informative short reads to quantitatively characterize the entire transcriptome of gastric cancer (including mRNAs and microRNAs). A multi-layer analysis was then developed to identify multiple types of transcriptional aberrations associated with different stages of gastric cancer, including differentially expressed mRNAs, recurrent somatic mutations and key differentially expressed microRNAs. Through this approach, we identified the central metabolic regulator AMPK-α as a potential functional target in Asian gastric cancer. Further, we experimentally demonstrated the translational relevance of this gene as a potential therapeutic target for early-stage gastric cancer in Asian patients. Together, our findings not only provide a valuable information resource for identifying and elucidating the molecular mechanisms of Asian gastric cancer, but also represent a general integrative framework to develop more effective therapeutic targets.
RNA-sequencing; integrative analysis; microRNA; AMPK; gastric cancer
mTOR is a highly conserved serine/threonine protein kinase that serves as a central regulator of cell growth, survival and autophagy. Deregulation of the PI3K/Akt/mTOR signaling pathway occurs commonly in cancer and numerous inhibitors targeting the ATP-binding site of these kinases are currently undergoing clinical evaluation. Here we report the characterization of Torin2, a second generation ATP-competitive inhibitor that is potent and selective for mTOR with a superior pharmacokinetic profile to previous inhibitors. Torin2 inhibited mTORC1-dependent T389 phosphorylation on S6K (RPS6KB1) with an EC50 of 250 pM with approximately 800-fold selectivity for cellular mTOR versus PI3K. Torin2 also exhibited potent biochemical and cellular activity against PIKK family kinases including ATM (EC50 28 nM), ATR (EC50 35 nM) and DNA-PK (EC50 118 nM) (PRKDC), the inhibition of which sensitized cells to Irradiation. Similar to the earlier generation compound Torin1 and in contrast to other reported mTOR inhibitors, Torin2 inhibited mTOR kinase and mTORC1 signaling activities in a sustained manner suggestive of a slow dissociation from the kinase. Cancer cell treatment with Torin2 for 24 hours resulted in a prolonged block in negative feedback and consequent T308 phosphorylation on Akt. These effects were associated with strong growth inhibition in vitro. Single agent treatment with Torin2 in vivo did not yield significant efficacy against KRAS-driven lung tumors, but the combination of Torin2 with MEK inhibitor AZD6244 yielded a significant growth inhibition. Taken together, our findings establish Torin2 as a strong candidate for clinical evaluation in a broad number of oncological settings where mTOR signaling has a pathogenic role.
mTOR; ATM; ATR; lung cancer; kinase inhibitors
Somatic mutation of the tumor suppressor gene LKB1 occurs frequently in lung cancer where it causes tumor progression and metastasis, but the underlying mechanisms remain mainly unknown. Here, we show that the oncogene NEDD9 is an important downstream mediator of lung cancer progression evoked by LKB1 loss. In de novo mouse models, RNAi-mediated silencing of Nedd9 inhibited lung tumor progression, whereas ectopic NEDD9 expression accelerated this process. Mechanistically, LKB1 negatively regulated NEDD9 transcription by promoting cytosolic translocation of CRTC1 from the nucleus. Notably, ectopic expression of either NEDD9 or CRTC1 partially reversed the inhibitory function of LKB1 on metastasis of lung cancer cells. In clinical specimens, elevated expression of NEDD9 was associated with malignant progression and metastasis. Collectively, our results decipher the mechanism through which LKB1 deficiency promotes lung cancer progression and metastasis, and provide a mechanistic rationale for therapeutic attack of these processes.
The mitochondrial transcription factor A (TFAM) is required for mitochondrial DNA (mtDNA) replication and transcription. Disruption of TFAM results in heart failure and premature aging in mice. But very little is known about the role of TFAM in cancer development. Here, we report the identification of frequent frameshift mutations in the coding mononucleotide repeat of TFAM in sporadic colorectal cancer (CRC) cell lines and in primary tumors with microsatellite instability (MSI), but not in microsatellite stable (MSS) CRC cell lines and tumors. The presence of the TFAM truncating mutation, in CRC cells with MSI, reduced the TFAM protein level in vivo and in vitro and correlated with mtDNA depletion. Furthermore, forced overexpression of wild-type TFAM in RKO cells carrying a TFAM truncating mutation suppressed cell proliferation and inhibited RKO cell-induced xenograft tumor growth. Moreover, these cells showed more susceptibility to cisplatin-induced apoptosis due to an increase of cytochrome b (Cyt b) expression and its release from mitochondria. An interaction assay between TFAM and the heavy-strand promoter (HSP) of mitochondria revealed that mutant TFAM exhibited reduced binding to HSP, leading to reduction in Cyt b transcription. Collectively, these data provide evidence that a high incidence of TFAM truncating mutations leads to mitochondrial copy number reduction and mitochondrial instability, distinguishing most CRC with MSI from MSS CRC. These mutations may play an important role in tumorigenesis and cisplatin-induced apoptotic resistance of most microsatellite-unstable CRCs.
Tumor hypoxia is an inherent impediment to cancer treatment that is both clinically significant and problematic. In this study, we performed a cell-based screen to identify small molecules that could reverse the apoptotic resistance of hypoxic cancer cells. Among the compounds we identified were a structurally-related group that sensitized hypoxic cancer cells to apoptosis by inhibiting the kinases GSK-3β and CDK1. Combinatorial inhibition of these proteins in hypoxic cancer cells and tumors increased levels of c-Myc and decreased expression of c-IAP2 and the central hypoxia response regulator Hif-1α. In mice, these compounds augmented the hypoxic tumor cell death induced by cytotoxic chemotherapy, blocking angiogenesis and tumor growth. Taken together, our findings suggest that combinatorial inhibition of GSK-3β and CDK1 augment the apoptotic sensitivity of hypoxic tumors, and they offer preclinical validation of a novel and readily translatable strategy to improve cancer therapy.
GSK-3β; CDK1; c-Myc; Hif-1α; c-IAP2; hypoxia; apoptosis; drug screen; drug resistance
Nuclear receptors (NRs) comprise a superfamily of ligand-activated transcription factors that play important roles in both physiology and diseases including cancer. The technologies of Chromatin ImmunoPrecipitation followed by array hybridization (ChIP-chip) or massively parallel sequencing (ChIP-seq) has been used to map, at an unprecedented rate, the in vivo genome-wide binding (cistrome) of NRs in both normal and cancer cells. We developed a curated database of 88 NR cistrome datasets and other associated high-throughput datasets, including 121 collaborating factor cistromes, 94 epigenomes and 319 transcriptomes. Through integrative analysis of the curated NR ChIP-chip/seq datasets, we discovered novel factor-specific noncanonical motifs that may have important regulatory roles. We also revealed a common feature of NR pioneering factors to recognize relatively short and AT-rich motifs. Most NRs bind predominantly to introns and distal intergenetic regions, and binding sites closer to transcription start sites (TSSs) were found to be neither stronger nor more evolutionarily conserved. Interestingly, while most NRs appear to be predominantly transcriptional activators, our analysis suggests that the binding of ESR1, RARA and RARG has both activating and repressive effects. Through meta-analysis of different omic data of the same cancer cell line model from multiple studies, we generated consensus cistrome and expression profiles. We further made probabilistic predictions of the NR target genes by integrating cistrome and transcriptome data, and validated the predictions using expression data from tumor samples. The final database, with comprehensive cistrome, epigenome, transcriptome datasets, and downstream analysis results, constitutes a valuable resource for the nuclear receptor and cancer community.
IFN regulatory factor 8 (IRF8) is a key transcription factor for myeloid cell differentiation and its expression is frequently lost in hematopoietic cells of human myeloid leukemia patients. IRF8-deficient mice exhibit uncontrolled clonal expansion of undifferentiated myeloid cells that can progress to a fatal blast crisis, thereby resembling human chronic myelogeneous leukemia (CML). Therefore, IRF8 is a myeloid leukemia suppressor. While the understanding of IRF8 function in CML has recently improved, the molecular mechanisms underlying IRF8 function in CML is still largely unknown. In this study, we identified acid ceramidase (A-CDase) as a general transcription target of IRF8. We demonstrated that IRF8 expression is regulated by IRF8 promoter DNA methylation in myeloid leukemia cells. Restoration of IRF8 expression repressed A-CDase expression, resulting in C16 ceramide accumulation and increased sensitivity of CML cells to FasL-induced apoptosis. In myeloid cells derived from IRF8-deficient mice, A-CDase protein level was dramatically increased. Furthermore, we demonstrated that IRF8 directly bind to the A-CDase promoter. At the functional level, inhibition of A-CDase activity, silencing A-CDase expression or application of exogenous C16 ceramide sensitized CML cells to FasL-induced apoptosis, whereas, overexpression of A-CDase decreased CML cells sensitivity to FasL-induced apoptosis. Consequently, restoration of IRF8 expression suppressed CML development in vivo at least partially through a Fas-dependent mechanism. In summary, our findings determine the mechanism of IRF8 downregulation in CML cells and they determine a primary pathway of resistance to Fas-mediated apoptosis and disease progression.
Defective expression of LATS2, a negative regulator of YAP onco-protein, has been reported in cancer of prostate, breast, liver, brain and blood origins. However, no transcriptional regulators for the LATS2 gene have been identified. Defective expression of LATS2, a negative regulator of YAP oncoprotein, has been reported in prostate, breast, liver, brain and blood cancers. However, the basis for LATS2 dysregulation in cancer is undefined. Here we report that spontaneous mutation of the transcription factor FOXP3 reduces expression of the LATS2 gene in mammary epithelial cells. shRNA-mediated silencing of FOXP3 in normal or malignant mammary epithelial cells of mouse and human origin repressed LATS2 expression and increased YAP protein levels. LATS2 induction required binding of FOXP3 to a specific sequence in the LATS2 promoter, and this interaction contributed to FOXP3-mediated growth inhibition of tumor cells. In support of these results, reduced expression and somatic mutations of FOXP3 correlated strongly with defective LATS2 expression in microdissected prostate cancer tissues. Thus, defective expression of LATS2 is attributable to FOXP3 defects and may be a major independent determinant of YAP protein elevation in cancer. Our findings identify a novel mechanism of LATS2 downregulation in cancer and reveal an important tumor suppressor relay between the FOXP3 and HIPPO pathways which are widely implicated in human cancer.
prostate cancer; breast cancer; Hippo pathway; FoxP3; tumor suppressor genes