Cancer cell molecular mimicry of stem cells (SC) imbues neoplastic cells with enhanced proliferative and renewal capacities. In support, numerous mediators of SC self-renewal have been evinced to exhibit oncogenic potential. We have recently reported that shRNA-mediated knockdown of the embryonic stem cell (ESC) self-renewal gene NANOG significantly reduced the clonogenic and tumorigenic capabilities of various cancer cells. In this study, we sought to test the potential pro-tumorigenic functions of NANOG, particularly, in prostate cancer (PCa). Using quantitative RT-PCR, we first confirmed that PCa cells expressed NANOG mRNA primarily from the NANOGP8 locus on chromosome 15q14. We then constructed a lentiviral promoter reporter in which the -3.8 kb NANOGP8 genomic fragment was used to drive the expression of green fluorescence protein (GFP). We observed that NANOGP8-GFP+ PCa cells exhibited cancer stem cell (CSC) characteristics such as enhanced clonal growth and tumor regenerative capacity. To further investigate the functions and mechanisms of NANOG in tumorigenesis, we established tetracycline-inducible NANOG overexpressing cancer cell lines, including both prostate (Du145 and LNCaP) and breast (MCF-7) cancer cells. NANOG induction promoted drug-resistance in MCF-7 cells, tumor regeneration in Du145 cells, and, most importantly, castration-resistant tumor development in LNCaP cells. These pro-tumorigenic effects of NANOG were associated with key molecular changes, including an upregulation of molecules such as CXCR4, IGFBP5, CD133 and ALDH1. The present gain-of-function studies, coupled with our recent loss-of-function work, establish the integral role for NANOG in neoplastic processes and shed light on its mechanisms of action.
Nanog; prostate cancer; cancer stem cells; castration resistance; self-renewal
Chronic inflammation has been associated with a variety of human cancers including prostate cancer. Interleukin-17 (IL-17) is a critical pro-inflammatory cytokine, which has been demonstrated to promote development of prostate cancer, colon cancer, skin cancer, breast cancer, lung cancer, and pancreas cancer. IL-17 promotes prostate adenocarcinoma with a concurrent increase of matrix metalloproteinase 7 (MMP7) expression in mouse prostate. Whether MMP7 mediates IL-17’s action and the underlying mechanisms remain unknown. We generated Mmp7 and Pten double knockout (Mmp7−/− in abbreviation) mouse model and demonstrated that MMP7 promotes prostate adenocarcinoma through induction of epithelial-to-mesenchymal transition (EMT) in Pten-null mice. MMP7 disrupted E-cadherin/β-catenin complex to up-regulate EMT transcription factors in mouse prostate tumors. IL-17 receptor C and Pten double knockout mice recapitulated the weak EMT characteristics observed in Mmp7−/− mice. IL-17 induced MMP7 and EMT in human prostate cancer LNCaP, C4-2B, and PC-3 cell lines, while siRNA knockdown of MMP7 inhibited IL-17-induced EMT. Compound III, a selective MMP7 inhibitor, decreased development of invasive prostate cancer in Pten single knockout mice. In human normal prostates and prostate tumors, IL-17 mRNA levels were positively correlated with MMP7 mRNA levels. These findings demonstrate that MMP7 mediates IL-17’s function in promoting prostate carcinogenesis through induction of EMT, indicating IL-17-MMP7-EMT axis as potential targets for developing new strategies in the prevention and treatment of prostate cancer.
IL-17; MMP7; epithelial-to-mesenchymal transition; prostate cancer
Smad proteins are central mediators in the canonical transforming growth factor-β (TGF-β) signaling pathway in mammalian cells. We report here that bromodomain-containing protein 7 (BRD7) functions as a novel transcription coactivator for Smads in TGF-β signaling. BRD7 forms a TGF-β inducible complex with Smad3/4 through its N-terminal Smad-binding domain. BRD7 simultaneously binds to acetylated histones to promote Smad-chromatin association, and associates with histone acetyltransferase p300 to enhance Smad transcriptional activity. Ectopic expression of BRD7, but not its mutants defective in Smad binding, enhances TGF-β transcriptional, tumor suppressing and epithelial-mesenchymal transition (EMT) responses. Conversely, depletion of BRD7 inhibits TGF-β responses. Thus, our study provides compelling evidence for a new function of BRD7 in fine-tuning TGF-β physiological responses.
BRD7; Transcription coactivator; Transforming growth factor beta; Smad
Chronic lymphocytic leukemia (CLL) is the most common adult leukemia in the western countries and is currently incurable due in part to difficulty in eliminating the leukemia cells protected by stromal microenvironment. Based on previous observations that CLL cells exhibit mitochondrial dysfunction and altered lipid metabolism and that carnitine palmitoyltransferases (CPT) play a major role in transporting fatty acid into mitochondria to support cancer cell metabolism, we tested several clinically relevant inhibitors of lipid metabolism for their ability to eliminate primary CLL cells. We discovered that Perhexiline, an anti-angina agent that inhibits CPT, was highly effective in killing CLL cells in stromal microenvironment at clinically achievable concentrations. These effective concentrations caused low toxicity to normal lymphocytes and normal stromal cells. Mechanistic study revealed that CLL cells expressed high levels of CPT1 and CPT2. Suppression of fatty acid transport into mitochondria by inhibiting CPT using Perhexiline resulted in a depletion of cardiolipin, a key component of mitochondrial membranes, and compromised mitochondrial integrity leading to rapid depolarization and massive CLL cell death. The therapeutic activity of Perhexiline was further demonstrated in vivo using a CLL transgenic mouse model. Perhexiline significantly prolonged the overall animal survival by only 4 drug injections. Our study suggests that targeting CPT using an anti-angina drug is able to effectively eliminate leukemia cells in vivo, and is a novel therapeutic strategy for potential clinical treatment of CLL.
Chronic lymphocytic leukemia; Lipid metabolism; CPT; perhexiline; stromal microenvironment
Non-small cell lung cancer (NSCLC) is the leading cause of cancer death, reflecting the need for better understanding the oncogenesis, and developing new diagnostic and therapeutic targets for the malignancy. Emerging evidence suggests that small nucleolar RNAs (snoRNAs) have malfunctioning roles in tumorigenesis. Our recent study demonstrated that small nucleolar RNA 42 (SNORA42) was overexpressed in lung tumors. Here, we investigate the role of SNORA42 in tumorigenesis of NSCLC. We simultaneously assess genomic dosages and expression levels of SNORA42 and its host gene, KIAA0907, in 10 NSCLC cell lines and a human bronchial epithelial cell line. We then determine in vitro functional significance of SNORA42 in lung cancer cell lines through gain- and loss-of-function analyses. We also inoculate cancer cells with SNORA42-siRNA into mice through either tail vein or subcutaneous injection. We finally evaluate expression level of SNORA42 on frozen surgically resected lung tumor tissues of 64 patients with stage I NSCLC by using quantitative reverse transcriptase PCR assay. Genomic amplification and associated high expression of SNORA42 rather than KIAA0907 are frequently observed in lung cancer cells, suggesting that SNORA42 overexpression is activated by its genomic amplification. SNORA42 knockdown in NSCLC cells inhibits in vitro and in vivo tumorigenicity, whereas enforced SNORA42 expression in bronchial epitheliums increases cell growth and colony formation. Such pleiotropy of SNORA42 suppression could be achieved at least partially through increased apoptosis of NSCLC cells in a p53-dependent manner. SNORA42 expression in lung tumor tissue specimens is inversely correlated with survival of NSCLC patients. Therefore, SNORA42 activation could have an oncogenic role in lung tumorigenesis and provide potential diagnostic and therapeutic targets for the malignancy.
small nucleolar RNA; tumorigenesis; bio-markers; apoptosis; p53
Kaposi sarcoma-associated herpesvirus (KSHV) is an oncogenic virus and the culprit behind the human disease Kaposi sarcoma (KS), an AIDS-defining malignancy. KSHV encodes a viral G-protein-coupled receptor (vGPCR) critical for the initiation and progression of KS. In this study, we identified that YAP/TAZ, two homologous oncoproteins inhibited by the Hippo tumor suppressor pathway, are activated in KSHV infected cells in vitro, KS-like mouse tumors, and clinical human KS specimens. The KSHV-encoded vGPCR acts through Gq/11 and G12/13 to inhibit the Hippo pathway kinases Lats1/2, promoting the activation of YAP/TAZ. Furthermore, depletion of YAP/TAZ blocks vGPCR-induced cell proliferation and tumorigenesis in a xenograft mouse model. The vGPCR-transformed cells are sensitive to pharmacological inhibition of YAP. Our study establishes a pivotal role of the Hippo pathway in mediating the oncogenic activity of KSHV and development of KS, and also suggests a potential of using YAP inhibitors for KS intervention.
Kaposi sarcoma; KSHV; vGPCR; Hippo; YAP; TAZ
STIM1 (stromal interaction molecule 1), an endoplasmic reticulum Ca2+ sensor that triggers the store-operated Ca2+ entry activation, has recently been implicated in cancer progression. However, the role of STIM1 in the progression and metastasis of colorectal cancer (CRC) has not been addressed. In this study, we confirmed increased expression of STIM1 in highly invasive CRC cell lines. Enhanced expression of STIM1 promoted CRC cell metastasis in vitro and in vivo, whereas silencing of STIM1 with small interfering RNA resulted in reduced metastasis. Ectopic expression of STIM1 in CRC cells induced epithelial-to-mesenchymal transition (EMT), whereas silencing of STIM1 had the opposite effect. Furthermore, STIM1 expression was markedly higher in CRC tissues than in adjacent noncancerous tissues. STIM1 overexpression correlated with poor differentiation and higher tumor node metastasis stage. CRC patients with positive STIM1 expression had poorer prognoses than those with negative STIM1 expression. Moreover, STIM1 was found to be a direct target of miR-185, a microRNA (miRNA) that has not previously been reported to be involved in EMT, in both CRC tissues and cell lines. Taken together, these findings demonstrate for the first time that STIM1 promotes metastasis and is associated with cancer progression and poor prognosis in patients with CRC. In addition, we show that expression of STIM1 is regulated by a posttranscriptional regulatory mechanism mediated by a new EMT-related miRNA. This novel miR-185–STIM1 axis promotes CRC metastasis and may be a candidate biomarker for prognosis and a target for new therapies.
Increased CLP36 expression has been found to be closely associated with breast cancer progression. However, whether and how it contributes to malignant behavior of breast cancer cells were not known. We show here that CLP36 is critical for promoting breast cancer cell migration and invasion in vitro and metastasis in vivo, whereas it is dispensable for breast cell proliferation and anchorage independent growth in vitro and tumor growth in vivo. CLP36 interacted with both α-actinin-1 and -4 in breast cancer cells. Depletion of either α-actinin-1 or -4 inhibited breast cancer cell migration. Furthermore, mutations inhibiting the α-actinin-binding activity abolished the ability of CLP36 to promote breast cancer cell migration. Finally, depletion of CLP36 or disruption of the CLP36-α-actinin complex in breast cancer cells substantially inhibited Cdc42 activation, cell polarization and migration. Our results identify CLP36 as an important regulator of breast cancer cell migration and metastasis, and shed light on how increased CLP36 expression contributes to progression of breast cancer.
CLP36; α-actinin; Breast cancer; Cell migration; Invasion; Metastasis
Although members of SOX family have been well documented for their essential roles in embryonic development, cell proliferation and disease, the functional role and molecular mechanism of SOX30 in cancer are largely unexplored. Here, we first identified SRY-box containing gene 30 (SOX30) as a novel preferentially methylated gene using genome-wide methylation screening. SOX30 hypermethylation was detected in 100% of lung cancer cell lines (9/9) and 70.83% (85/120) of primary lung tumor tissues compared with none (0/20) of normal and 8.0% (2/25) of peri-tumoral lung tissues (P<0.01). SOX30 was expressed in normal and peri-tumoral lung tissues in which SOX30 was unmethylated, but was silenced or downregulated in lung cancer cell lines and primary lung tumor tissues harboring a hypermethylated SOX30. De-methylation experiments further confirmed that silence of SOX30 was regulated by its hypermethylation. Ectopic expression of SOX30 induces cancer cell apoptosis with inhibiting proliferation in vitro and represses tumor formation in vivo, whereas knockdown of SOX30 demonstrates a reversed effect both in vitro and in vivo. At the molecular level, the antitumorigenic effect of SOX30 is mediated by directly binding to CACTTTG (+115 to +121) of p53 promoter region and activating p53 transcription, suggesting that SOX30 is a novel transcriptional activating factor of p53. Indeed, blockade of p53 attenuates the tumor inhibition of SOX30. Overall, these findings demonstrate that SOX30 is a novel epigenetic silenced tumor suppressor acting through direct regulation of p53 transcription and expression. This study provides novel insights on the mechanism of tumorigenesis in lung cancer.
The TET (ten–eleven translocation) family of α-ketoglutarate (α-KG)-dependent dioxygenases catalyzes the sequential oxidation of 5-methylcytosine (5mC) to 5-hydroxymethyl-cytosine (5hmC), 5-formylcytosine and 5-carboxylcytosine, leading to eventual DNA demethylation. The TET2 gene is a bona fide tumor suppressor frequently mutated in leukemia, and TET enzyme activity is inhibited in IDH1/2-mutated tumors by the oncometabolite 2-hydroxyglutarate, an antagonist of α-KG, linking 5mC oxidation to cancer development. We report here that the levels of 5hmC are dramatically reduced in human breast, liver, lung, pancreatic and prostate cancers when compared with the matched surrounding normal tissues. Associated with the 5hmC decrease is the substantial reduction of the expression of all three TET genes, revealing a possible mechanism for the reduced 5hmC in cancer cells. The decrease of 5hmC was also observed during tumor development in different genetically engineered mouse models. Together, our results identify 5hmC as a biomarker whose decrease is broadly and tightly associated with tumor development.
TET; 5-hydroxymethylation; DNA methylation; cancer biomarker
Previously, our group identified a novel amplicon at chromosome 9p24 in human esophageal and breast cancers, and cloned the novel gene, GASC1 (gene amplified in squamous cell carcinoma 1, also known as JMJD2C/KDM4C), from this amplicon. GASC1 is a histone demethylase involved in the deregulation of histone methylation in cancer cells. In the current study, we aimed to comprehensively characterize the genes in the 9p24 amplicon in human breast cancer. We performed extensive genomic analyses on a panel of cancer cell lines and narrowed the shortest region of overlap to approximately 2 Mb. Based on statistical analysis of copy number increase and overexpression, the 9p24 amplicon contains six candidate oncogenes. Among these, four genes (GASC1 UHRF2, KIAA1432 and C9orf123) are overexpressed only in the context of gene amplification while two genes (ERMP1 and IL33) are overexpressed independent of the copy number increase. We then focused our studies on the UHRF2 gene, which has a potential involvement in both DNA methylation and histone modification. Knocking down UHRF2 expression inhibited the growth of breast cancer cells specifically with 9p24 amplification. Conversely, ectopic overexpression of UHRF2 in non-tumorigenic MCF10A cells promoted cell proliferation. Furthermore, we demonstrated that UHRF2 has the ability to suppress the expression of key cell-cycle inhibitors, such as p16INK4a, p21Waf1/Cip1 and p27Kip1. Taken together, our studies support the notion that the 9p24 amplicon contains multiple oncogenes that may integrate genetic and epigenetic codes and have important roles in human tumorigenesis.
chromosome 9p24; GASC1; UHRF2; gene amplification
Interferon alpha (IFNα) is widely used for treatment of melanoma and certain other malignancies. This cytokine as well as the related IFNβ exerts potent anti-tumorigenic effects; however, their efficacy in patients is often suboptimal. Here we report that inflammatory signaling impedes the effects of IFNα/β. Melanoma cells can secrete pro-inflammatory cytokines that inhibit cellular responses to IFNα/β via activating the ligand-independent pathway for the phosphorylation and subsequent ubiquitination and accelerated degradation of the IFNAR1 chain of Type I IFN receptor. Catalytic activity of the p38 protein kinase was required for IFNAR1 downregulation and inhibition of IFNα/β signaling induced by proinflammatory cytokines such as Interleukin 1 (IL-1). Activation of p38 kinase inversely correlated with protein levels of IFNAR1 in clinical melanoma specimens. Inhibition of p38 kinase augmented the inhibitory effects of IFNα/β on cell viability and growth in vitro and in vivo. The role of inflammation and p38 protein kinase in regulating cellular responses to IFNα/β in normal and tumor cells are discussed.
inflammation; cancer; interferon; receptor; ubiquitin; melanoma
Translational control at the initiation step has been recognized as a major and important regulatory mechanism of gene expression. eIF3a, a putative subunit of eIF3 complex, has recently been shown to play an important role in regulating translation of a subset of mRNAs and found to correlate with prognosis of cancers. In this study, using nasopharyngeal carcinoma (NPC) cells as a model system we tested the hypothesis that eIF3a negatively regulates synthesis of nucleotide excision repair (NER) proteins and, thus, NER activities and cellular response to treatments with DNA damaging agents such as cisplatin. We found that a cisplatin-sensitive subclone S16 isolated from a NPC cell line CNE2 via limited dilution has increased eIF3a expression. Knocking down its expression in S16 cells increased cellular resistance to cisplatin, NER activity, and synthesis of NER proteins XPA, XPC, RAD23B, and RPA32. Altering eIF3a expression also changed cellular response to cisplatin and UV treatment in other NPC cell lines. Taken together, we conclude that eIF3a plays an important role in cisplatin response and NER activity of nasopharyngeal carcinomas by suppressing synthesis of NER proteins.
cisplatin sensitivity; eIF3a; nasopharyngeal carcinoma; nucleotide excision repair; translational control
The overexpression of IRX1 gene correlates with the growth arrest in gastric cancer. Furthermore, overexpression of IRX1 gene suppresses peritoneal spreading and long distance metastasis. To explore the precise mechanisms, we investigated whether restoring IRX1 expression affects the angiogenesis or vasculogenic mimicry (VM). Human umbilical vein endothelial cells (HUVECs) and chick embryo and SGC-7901 gastric cancer cells were used for angiogenesis and VM analysis. Small interfering RNA was used for analyzing the function of BDKRB2, a downstream target gene of IRX1. As results, the remarkable suppression on peritoneal spreading and pulmonary metastasis of SGC-7901 cells by IRX1 transfectant correlates to reduced angiogenesis as well as VM formation. Using the supernatant from SGC-7901/IRX1 cells, we found a strong inhibiting effect on angiogenesis both in vitro and in chick embryo. SGC-7901/IRX1 cells revealed strong inhibiting effect on VM formation too. By gene-specific RNA interference for BDKRB2, or its effector PAK1, we got an effective inhibition on tube formation, cell proliferation, cell migration and invasion in vitro. In conclusion, enforcing IRX1 expression effectively suppresses peritoneal spreading and pulmonary metastasis via anti-angiogenesis and anti-VM mechanisms, in addition to previously found cell growth and invasion. BDKRB2 and its downstream effector might be potential targets for anti-cancer strategy.
gastric carcinoma; IRX1; angiogenesis; vasculogenic mimicry; BDKRB2
The nuclear p68 RNA helicase is a prototypical member of the DEAD box family of RNA helicases. P68 RNA helicase has been implicated in cell proliferation and early organ development and maturation. However, the functional role of p68 RNA helicase in these biological processes at the molecular level is not well understood. We previously reported that tyrosine phosphorylation of p68 RNA helicase mediates the effects of PDGF in induction of EMT by promoting β-catenin nuclear translocation (Yang et.al. Cell 127:139-155 2006). Here we report that phosphorylation of p68 RNA helicase at Y593 up-regulates transcription of the Snail1 gene. The phosphorylated p68 activates transcription of the Snail1 gene by promoting HDAC1 dissociation from the Snail1 promoter. Our results showed that p68 interacted with the nuclear remodeling and deacetylation complex MBD3:Mi-2/NuRD. Thus, our data suggested that a DEAD box RNA unwindase can potentially regulate gene expression by functioning as a protein ‘displacer’ to modulate protein-protein interactions at the chromatin remodeling complex.
P68 RNA helicase; E-cadherin; Snail1; transcription activation; DEAD-box; HDAC1; Mi-2/NuRD
HER2/neu (HER2) and cyclin E are important prognostic indicators in breast cancer. Since both are involved in cell cycle regulation we investigated whether there was a direct interaction between the two. HER2 and cyclin E expression levels were determined in 395 breast cancer patients. Patients with HER2-overexpression and high levels of cyclin E had decreased 5-year disease-specific survival compared with low levels of cyclin E (14% versus 89%, P < .0001) In vitro studies were performed in which HER2-mediated activity in HER2-overexpressing breast cancer cell lines was downregulated by transfection with HER2 siRNA or treatment with trastuzumab. Cyclin E expression levels were determined, and functional effects investigated using kinase assays, MTT assays to assess cell viability as a marker of proliferation, and FACS analysis to determine cell cycle profiles. Decreased HER2-mediated signaling resulted in decreased expression of cyclin E, particularly the low molecular weight (LMW) isoforms. Decreased HER2 and LMW cyclin E expression had functional effects, including decreased cyclin E-associated kinase activity and decreased proliferation, due to increased apoptosis and an increased accumulation of cells in the G1 phase. In vivo studies performed in a HER2-overexpressing breast cancer xenograft model confirmed the effects of trastuzumab on cyclin E expression. Given the relationship between HER2 and cyclin E, in vitro clonogenic assays were performed to assess combination therapy targeting both proteins. Isobologram analysis showed a synergistic interaction between the two agents (trastuzumab targeting HER2 and roscovitine targeting cyclin E). Taken together, these studies demonstrate that HER2-mediated signaling effects LMW cyclin E expression, which in turn effects cell cycle regulation. LMW cyclin E has prognostic and predictive roles in HER2-overexpressing breast cancer, warranting further study of its potential as a therapeutic target.
Breast cancer; HER2/neu; cell cycle regulation; cyclin E
Transforming growth factor-β (TGF-β) regulates epithelial tissue homeostasis by activating processes that control cell cycle arrest, differentiation and apoptosis. Disruption of TGF-β signaling pathway often occurs in colorectal cancers. Previously, we have shown that TGF-β induces apoptosis through the transcription factor Smad3. Affymetrix oligonucleotide microarrays were used to identify TGF-β/Smad3 target genes that regulate apoptosis in rat intestinal epithelial cells (RIE-1). We found that TGF-β repressed the expression of the inhibitor of differentiation (Id) gene family. Knockdown of Id1 and Id2 gene expression induced apoptosis in RIE cells, whereas over-expression of Id2 attenuated TGF-β-induced apoptosis. TranSignal™ Protein/DNA arrays were used to identify hypoxia-inducing factor-1 (HIF-1) as a downstream target of TGF-β. HIF-1 is a bHLH protein, and over-expression of Id2 blocked HIF-1 activation by TGF-β. Furthermore, knockdown of HIF-1 blocked TGF-β-induced apoptosis. Thus, we have identified HIF-1 as a novel mediator downstream of Id2 in the pathway of TGF-β-induced apoptosis.
Apoptosis; Affymetrix oligonucleotide microarrays; Inhibitor of differentiation; TranSignal™; Protein/DNA arrays; Hypoxia-inducing factor
Ebp1, an ErbB3 receptor-binding protein, inhibits cell proliferation and acts as a putative tumor suppressor. Ebp1 translocates into the nucleus and functions as a transcription corepressor for E2F-1. Here, we show that Ebp1 p42 isoform can be sumoylated on both K93 and K298 residues, which mediate its nuclear translocation and is required for its anti-proliferative activity. We find that TLS/FUS, an RNA-binding nuclear protein that is involved in pre- mRNA processing and nucleocytoplasmic shuttling, has Sumo1 E3 ligase activity for Ebp1 p42. Ebp1 directly binds TLS/FUS, which is regulated by genotoxic stress-triggered phosphorylation on Ebp1. Ebp1 sumoylation facilitates its nucleolar distribution and protein stability. Overexpression of TLS enhances Ebp1 sumoylation, while depletion of TLS abolishes Ebp1 sumoylation. Moreover, Unsumoylated Ebp1 mutants fail to suppress E2F-1- regulated transcription, resulting in loss of its anti-proliferation activity. Hence, TLS-mediated sumoylation is required for Ebp1 transcription repressive activity.
Ebp1; TLS/FUS; Sumoylation; Cell proliferation
The removal of DNA interstrand cross-links (ICLs) has proven to be notoriously complicated due to the involvement of multiple pathways of DNA repair, which include the Fanconi anemia/BRCA pathway, homologous recombination, and components of the nucleotide excision and mismatch repair pathways. Members of the SNM1 gene family have also been shown to have a role in mediating cellular resistance to ICLs, although their precise function has remained elusive. Here we show that knockdown of Snm1B/Apollo in human cells results in hypersensitivity to mitomycin C (MMC), but not to IR. We also show that Snm1B-deficient cells exhibit a defective S phase checkpoint in response to MMC, but not to IR, and this finding may account for the specific sensitivity to the cross-linking drug. Interestingly, although previous studies have largely implicated ATR as the major kinase activated in response to ICLs, we show that it is activation of the ATM-mediated checkpoint that is defective in Snm1B-deficient cells. The requirement for Snm1B in ATM checkpoint activation specifically after ICL damage is correlated with its role in promoting double-strand break formation, and thus replication fork collapse. Consistent with this result Snm1B was found to interact directly with Mus81-Eme1 an endonuclease previously implicated in fork collapse. In addition, we also show that Snm1B interacts with the Mre11-Rad50-Nbs1 (MRN) complex and with FancD2 further substantiating its role as a checkpoint/DNA repair protein.
Snm1B/Apollo; interstrand cross-links; cell cycle checkpoint; ATM
HER2 amplification/overexpression (HER2+) frequently co-occurs with PI3K pathway activation in breast tumors. PI3K signaling is most often activated by PIK3CA mutation or PTEN loss, which frequently results in sensitivity to p110α or p110β inhibitors, respectively. To examine the p110 isoform dependence in HER2+, PTEN-deficient tumors, we generated genetic mouse models of breast tumors driven by concurrent Her2-activation and Pten-loss coupled with deletion of p110α or p110β. Ablation of p110α, but not p110β, significantly impaired the development of Her2+/Pten-null tumors in mice. We further show that p110α primarily mediates oncogenic signaling in HER2+/PTEN-deficient human cancers while p110β conditionally mediates PI3K/AKT signaling only upon HER2 inhibition. Combined HER2 and p110α inhibition effectively reduced PI3K/AKT signaling and growth of cancer cells both in vitro and in vivo. Addition of the p110β inhibitor to dual HER2 and p110α inhibition induced tumor regression in a xenograft model of HER2+/PTEN-deficient human cancers. Together, our data suggest that combined inhibition of HER2 and p110α/β may serve as a potent and durable therapeutic regimen for the treatment of HER2+, PTEN-deficient breast tumors.
PI3K; HER2; PTEN; genetic mouse models; breast cancer
Cancer cells are known to execute reprogramed metabolism of glucose, amino acids and lipids. Here, we report a significant role of cholesterol metabolism in cancer metastasis. By using label-free Raman spectromicroscopy, we found an aberrant accumulation of cholesteryl ester in human pancreatic cancer specimens and cell lines, mediated by acyl-CoA cholesterol acyltransferase-1 (ACAT-1) enzyme. Expression of ACAT-1 showed a correlation with poor patient survival. Abrogation of cholesterol esterification, either by an ACAT-1 inhibitor or by shRNA knockdown, significantly suppressed tumor growth and metastasis in an orthotopic mouse model of pancreatic cancer. Mechanically, ACAT-1 inhibition increased intracellular free cholesterol level, which was associated with elevated endoplasmic reticulum stress and caused apoptosis. Collectively, our results demonstrate a new strategy for treating metastatic pancreatic cancer by inhibiting cholesterol esterification.
Cancer cells are known to execute reprogramed metabolism of glucose, amino acids, and lipids. Here, we report a significant role of cholesterol metabolism in cancer metastasis. By employing label-free Raman spectromicroscopy, we found an aberrant accumulation of cholesteryl ester in human pancreatic cancer specimens and cell lines, mediated by acyl-CoA cholesterol acyltransferase-1 (ACAT-1) enzyme. Expression of ACAT-1 showed a correlation with poor patient survival. Abrogation of cholesterol esterification, either by an ACAT-1 inhibitor or by shRNA knockdown, significantly suppressed tumor growth and metastasis in an orthotopic mouse model of pancreatic cancer. Mechanically, ACAT-1 inhibition increased intracellular free cholesterol level, which was associated with elevated endoplasmic reticulum stress and caused apoptosis. Collectively, our results demonstrate a new strategy for treating metastatic pancreatic cancer by inhibiting cholesterol esterification.
cancer metabolism; cholesterol esterification; Raman spectroscopy; pancreatic cancer; metastasis; orthotopic mouse model
Epidemiological studies showed that obesity and its related non-alcoholic fatty liver disease (NAFLD) promote hepatocellular carcinoma (HCC) development. We aimed to uncover the genetic alterations of NAFLD-HCC using whole-exome sequencing. We compared HCC development in genetically obese mice and dietary obese mice with wild-type lean mice fed a normal chow after treatment with diethylnitrosamine. HCC tumor and adjacent normal samples from obese and lean mice were then subjected to whole-exome sequencing. Functional and mechanistic importance of the identified mutations in Carboxyl ester lipase (Cel) gene and Harvey rat sarcoma virus oncogene 1 (Hras) was further elucidated. We demonstrated significantly higher incidences of HCC in both genetic and dietary obese mice with NAFLD development as compared with lean mice without NAFLD. The mutational signatures of NAFLD-HCC and lean HCC were distinct, with <3% overlapped. Eight metabolic or oncogenic pathways were found to be significantly enriched by mutated genes in NAFLD-HCC, but only two of these pathways were dysregulated by mutations in lean HCC. In particular, Cel was mutated significantly more frequently in NAFLD-HCC than in lean HCC. The multiple-site mutations in Cel are loss-of-function mutations, with effects similar to Cel knock-down. Mutant Cel caused accumulation of cholesteryl ester in liver cells, which led to induction of endoplasmic reticulum stress and consequently activated the IRE1α/c-Jun N-terminal kinase (JNK)/c-Jun/activating protein-1 (AP-1) signaling cascade to promote liver cell growth. In addition, single-site mutations in Hras at codon 61 were found in NAFLD-HCC but none in lean HCC. The gain-of-function mutations in Hras (Q61R and Q61K) significantly promoted liver cell growth through activating the mitogen-activated protein kinase (MAPK) and phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)/3-phosphoinositide-dependent protein kinase-1 (PDK1)/Akt pathways. In conclusion, we have identified mutation signature and pathways in NAFLD-associated HCC. Mutations in Cel and Hras have important roles in NAFLD-associated hepatocellular carcinogenesis.
As a transcription factor, localization to the nucleus and the recruitment of co-factors to regulate gene transcription is essential. Nuclear localization and nucleosome remodeling and histone deacetylase (NuRD) complex binding are required for the zinc finger transcription factor CASZ1 to function as neuroblastoma (NB) tumor suppressor. However, the critical amino acids (AAs) that are required for CASZ1 interaction with NuRD complex and the regulation of CASZ1 subcellular localization have not been characterized. Through alanine scanning, immunofluorescence cell staining and co-immunoprecipitation we define a critical region at the CASZ1 N-terminus (AA23-40) that mediates the CASZ1b nuclear localization and NuRD interaction. Furthermore, we identify a nuclear export signal (NES) at the N-terminus (AA176-192) that contributes to CASZ1 nuclear-cytoplasmic shuttling in a chromosomal maintenance 1 (CRM1)-dependent manner. An analysis of CASZ1 protein expression in a primary neuroblastoma tissue microarray shows that high nuclear CASZ1 staining is detected in tumors from NB patients with good prognoses. In contrast, cytoplasmic-restricted CASZ1 staining or low nuclear CASZ1 staining is found in tumors from patients with poor prognoses. These findings provide insight into mechanisms by which CASZ1 regulates transcription, and suggests that regulation of CASZ1 subcellular localization may impact its function in normal development and pathologic conditions such as neuroblastoma tumorigenesis.
CASZ1; NLS; NES; NuRD; Transcription factor; Tumor suppressor