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
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
The retroviral oncoprotein Tax from Human T cell leukemia virus type 1 (HTLV-1), an etiological factor that causes adult T cell leukemia and lymphoma, plays a crucial role in initiating T lymphocyte transformation by inducing oncogenic signaling activation. We here report that Tax is a determining factor for dysregulation of autophagy in HTLV-1-transformed T cells and Tax-immortalized CD4 memory T cells. Tax facilitated autophagic process by activating IκB kinase complex, which subsequently recruited an autophagy molecular complex containing Beclin1 and Bif-1 to the lipid raft microdomains. Tax engaged a crosstalk between IκB kinase complex and autophagic molecule complex by directly interacting with both complexes, promoting assembly of LC3+ autophagosomes. Moreover, expression of lipid raft-targeted Bif-1 or Beclin1 was sufficient to induce formation of LC3+ autophagosomes, suggesting that Tax recruitment of autophagic molecules to lipid rafts is a dominant strategy to deregulate autophagy in the context of HTLV-1 transformation of T cells. Furthermore, depletion of autophagy molecules such as Beclin1 and PI3 kinase class III resulted in impaired growth of HTLV-1-transformed T cells, indicating a critical role of Tax-deregulated autophagy in promoting survival and transformation of virally infected T cells.
HTLV-1 Tax; IKK; autophagy; lipid rafts; Beclin1; Bif-1
Many components of the Wnt/β-catenin signaling pathway have critical functions in mammary gland development and tumor formation, yet the contribution of glycogen synthase kinase-3 (GSK-3α and GSK-3β) to mammopoiesis and oncogenesis is unclear. Here, we report that WAP-Cre-mediated deletion of GSK-3 in the mammary epithelium results in activation of Wnt/β-catenin signaling and induces mammary intraepithelial neoplasia that progresses to squamous transdifferentiation and development of adenosquamous carcinomas at 6 months. To uncover possible β-catenin-independent activities of GSK-3, we generated mammary-specific knockouts of GSK-3 and β-catenin. Squamous transdifferentiation of the mammary epithelium was largely attenuated, however, mammary epithelial cells lost the ability to form mammospheres suggesting perturbation of stem cell properties unrelated to loss of β-catenin alone. At 10 months, adenocarcinomas that developed in glands lacking GSK-3 and β-catenin displayed elevated levels of γ-catenin/plakoglobin as well as activation of the Hedgehog and Notch pathways. Collectively, these results establish the two isoforms of GSK-3 as essential integrators of multiple developmental signals that act to maintain normal mammary gland function and suppress tumorigenesis.
Lineage-restricted transcription factors (TFs) are frequently mutated or overexpressed in cancer and contribute toward malignant behaviors, but the molecular bases of their oncogenic properties are largely unknown. Because TF activities are difficult to inhibit directly with small molecules, the genes and pathways they regulate might represent more tractable targets for drug therapy. We studied GATA6, a TF gene that is frequently amplified or overexpressed in gastric, esophageal, and pancreatic adenocarcinomas. GATA6-overexpressing gastric cancer cell lines cluster in gene expression space, separate from non-overexpressing lines. This expression clustering signifies a shared pathogenic group of genes that GATA6 may regulate through direct cis-element binding. We used chromatin immunoprecipation and sequencing (ChIP-seq) to identify GATA6-bound genes and considered TF occupancy in relation to genes that respond to GATA6 depletion in cell lines and track with GATA6 mRNA (synexpression groups) in primary gastric cancers. Among other cellular functions, GATA6-occupied genes control apoptosis and govern M-phase of the cell cycle. Depletion of GATA6 reduced levels of the latter transcripts and arrested cells in G2 and M phases of the cell cycle. Synexpression in human tumor samples identified likely direct transcriptional targets substantially better than consideration only of transcripts that respond to GATA6 loss in cultured cells. Candidate target genes responded to loss of GATA6 or its homolog GATA4 and even more to depletion of both proteins. Many GATA6-dependent genes lacked nearby binding sites but several strongly dependent, synexpressed, and GATA6-bound genes encode TFs such as MYC, HES1, RARB, and CDX2. Thus, many downstream effects occur indirectly through other TFs and GATA6 activity in gastric cancer is partially redundant with GATA4. This integrative analysis of locus occupancy, gene dependency, and synexpression provides a functional signature of GATA6-overexpressing gastric cancers, revealing both limits and new therapeutic directions for a challenging and frequently fatal disease.
transcriptional control of cancer; synexpression groups; somatic copy number alterations; ChIP-seq; GATA transcription factors
Lung cancer is the most common cause of cancer mortality worldwide. Non-small-cell lung carcinomas (NSCLCs), which represent around 80% of lung tumors, exhibit poor prognosis and are usually refractory to conventional chemotherapy. Elucidating the molecular and cellular mechanisms that are dysregulated in NSCLCs may lead to new possibilities for targeted therapy or enhanced efficacy of current therapies. Here we demonstrate Wnt pathway activation in around 50% of human NSCLC cell lines and primary tumors, through different mechanisms, including autocrine Wnt pathway activation involving upregulation of specific Wnt ligands. Downregulation of activated Wnt signaling inhibited NSCLC proliferation and induced a more differentiated phenotype. Together, our findings establish importance of activated Wnt signaling in human NSCLCs and offer the possibility of targeting upregulated Wnt signaling as a new therapeutic modality for this disease.
autocrine; lung cancer; wnt
The Hippo signaling pathway regulates cellular proliferation and survival, thus exerting profound effects on normal cell fate and tumorigenesis. The pivotal effector of this pathway is YAP, a transcriptional co-activator amplified in mouse and human cancers where it promotes epithelial-to-mesenchymal transition and malignant transformation. Here, we report a novel regulatory mechanism for the YAP oncogenic function via direct interaction with non-receptor tyrosine phosphatase 14 (PTPN14) through the WW domain of YAP and the PPxY domain of PTPN14. We also found that YAP is a direct substrate of PTPN14. In addition, luciferase reporter assay showed that the inhibition of the YAP transcriptional co-activator function by PTPN14 is mediated through their protein interactions and may result from an increase in the inactive cytoplasmic form of YAP. Last, knockdown of PTPN14 induces the nuclear retention of YAP and increases the YAP-dependent cell migration. In summary, our results indicate a potential regulatory role of PTPN14 on YAP and demonstrate a novel mechanism in YAP regulation.
epithelial-to-mesenchymal transition (EMT); Hippo pathway; PTPN14; tumor metastasis; YAP
Proline oxidase (POX) is a novel mitochondrial tumor suppressor, which can suppress proliferation and induce apoptosis through the generation of reactive oxygen species (ROS) and decreasing hypoxia inducible factor (HIF) signaling. Recent studies have demonstrated the absent expression of POX in human cancer tissues, including renal cancer. However, the mechanism for the loss of POX remains obscure. No genetic or epigenetic variation of POX gene was found. Here, we identified the up-regulated miR-23b* in renal cancer as an important regulator of POX. Ectopic overexpression of miR-23b* in normal renal cells resulted in striking down-regulation of POX, while POX expression increased markedly when endogenous miR-23b* was knocked down by its antagomirs in renal cancer cells. Consistent with POX-mediated tumor suppression pathway, these antagomirs induced ROS, inhibited HIF signaling and increased apoptosis. Furthermore, we confirmed the regulation of miR-23b* on POX and its function in the DLD1 Tet-off POX cell system. Using a luciferase reporter system, we verified the direct binding of miR-23b* to POX mRNA 3′UTR. In addition, pairs of human renal carcinoma and normal tissues showed the negative correlation between miR-23b* and POX protein expression, providing its clinical corroboration. Taken together, our results suggested miR-23b*, by targeting POX, could function as an oncogene; decreasing miR-23b* expression may prove to be an effective way of inhibiting kidney tumor growth.
proline oxidase; miR-23b*; renal cancer; ROS; tumor suppressor
Nutrient stress that produces quiescence and catabolism in normal cells
is lethal to cancer cells because oncogenic mutations constitutively drive
anabolism. One driver of biosynthesis in cancer cells is the mTORC1 signaling
complex. Activating mTORC1 by deleting its negative regulator TSC2 leads to
hypersensitivity to glucose deprivation. We have previously shown that ceramide
kills cells in part by triggering nutrient transporter loss and restricting
access to extracellular amino acids and glucose suggesting that TSC2-deficient
cells would be hypersensitive to ceramide. However, murine embryonic fibroblasts
(MEFs) lacking TSC2 were highly resistant to ceramide-induced death. Consistent
with the observation that ceramide limits access to both amino acids and
glucose, TSC2−/− MEFs also had a survival advantage
when extracellular amino acids and glucose were both reduced. As
TSC2−/− MEFs were resistant to nutrient stress
despite sustained mTORC1 activity, we assessed whether mTORC1 signaling might be
beneficial under these conditions. In low amino acid and glucose medium and
following ceramide-induced nutrient transporter loss, elevated mTORC1 activity
significantly enhanced the adaptive up-regulation of new transporter proteins
for amino acids and glucose. Strikingly, the introduction of oncogenic Ras
abrogated the survival advantage of TSC2−/− MEFs upon
ceramide treatment most likely by increasing nutrient demand. These results
suggest that, in the absence of oncogene-driven biosynthetic demand, mTORC1
dependent translation facilitates the adaptive cellular response to nutrient
TSC2; mTOR; nutrient transporters; 4F2hc; GLUT1; ceramide
The phosphatidylinositol-3-kinase (PI3K) pathway is commonly hyperactivated in cancer. One mechanism by which this occurs is by silencing of the phosphatase and tensin homolog (PTEN), a tumor suppressor and major antagonist of the pathway, through genetic, epigenetic or posttranscriptional mechanisms. Here, we used an unbiased siRNA screen in non-small-cell lung cancer cells to identify deubiquitylases (DUBs) that have an impact on PI3K signaling by regulating the abundance of PTEN. We found that PTEN expression was induced by depleting any of three members of the Josephin family DUBs: ataxin 3 (ATXN3), ataxin 3-like (ATXN3L) and Josephin domain containing 1 (JOSD1). However, this effect is not mediated through altered PTEN protein stability. Instead, depletion of each DUB increases expression of both the PTEN transcript and its competing endogenous RNA, PTENP1. In ATXN3-depleted cells, under conditions of transcriptional inhibition, PTEN and PTENP1 mRNAs rapidly decay, suggesting that ATXN3 acts primarily by repressing their transcription. Importantly, the PTEN induction observed in response to ATXN3 siRNA is sufficient to downregulate Akt phosphorylation and hence PI3K signaling. Histone deacetylase inhibitors (HDACi) have been suggested as potential mediators of PTEN transcriptional reactivation in non-small-cell lung cancer. Although PTEN exhibits a very limited response to the broad-spectrum HDACi Vorinostat (SAHA) in A549 cells, we find that combination with ATXN3 depletion enhances PTEN induction in an additive manner. Similarly, these interventions additively decrease cell viability. Thus, ATXN3 provides an autonomous, complementary therapeutic target in cancers with epigenetic downregulation of PTEN.
deubiquitinase; ATXN3; phosphatase and tensin homolog; PTENP1; ceRNA; MJD
Serous ovarian carcinoma is the most lethal gynecological malignancy in Western countries. The molecular events that underlie the development of the disease have been elusive for many years. The recent identification of the fallopian tube secretory epithelial cells (FTSECs) as the cell-of-origin for most cases of this disease has led to studies aimed at elucidating new candidate therapeutic pathways through profiling of normal FTSECs and serous carcinomas. Here, we describe the results of transcriptional profiles that identify the loss of the tumor suppressive transcription factor FOXO3a in a vast majority of high grade serous ovarian carcinomas (HGSOCs). We show that FOXO3a loss is a hallmark of the earliest stages of serous carcinogenesis and occurs both at the DNA, RNA and protein levels. We describe several mechanisms responsible for FOXO3a inactivity, including chromosomal deletion (chromosome 6q21), upregulation of miRNA-182 and destabilization by activated PI3K and MEK. The identification of pathways involved in the pathogenesis of ovarian cancer can advance the management of this disease from being dependant on surgery and cytotoxic chemotherapy alone to the era of targeted therapy. Our data strongly suggest FOXO3a as a possible target for clinical intervention.
Serous ovarian carcinoma; fallopian tube epithelium; FOXO3a; AKT; ERK; miRNA-182
Adult stem cells are multipotent and persist in small numbers in adult tissues throughout the lifespan of an organism. Unlike differentiated cells, adult stem cells are intrinsically resistant to senescence. It is unclear how adult stem cells in solid organs respond to oncogenic stimulation and whether these cells have a role in tumor initiation. We report here that expression of BRAFV600E in human neural crest progenitor cells (hNCPCs) did not induce growth arrest as seen in human melanocytes, but instead, increased their cell proliferation capacity. These cells (hNCPCsV600E) acquired anchorage-independent growth ability and were weakly tumorigenic in vivo. Unlike in human melanocytes, BRAFV600E expression in hNCPCs did not induce p16INK4a expression. BRAFV600E induced elevated expression of CDK2, CDK4, MITF and EST1/2 protein in hNCPCs, and also induced melanocytic differentiation of these cells. Furthermore, overexpression of MITF in hNCPCsV600E dramatically increased their tumorigenicity and resulted in fully transformed tumor cells. These findings indicate that hNCPCs are susceptible to BRAFV600E-induced transformation, and MITF potentiates the oncogenic effect of BRAFV600E in these progenitor cells. These results suggest that the hNCPCs are potential targets for BRAFV600E-induced melanocytic tumor formation.
melanoma; neural crest; transformation; melanocytes; BRAFV600E
Translational control of gene expression has recently been recognized as an important mechanism controlling cell proliferation and oncogenesis and it mainly occurs in the initiation step of protein synthesis that involves multiple eukaryotic initiation factors (eIFs). Many eIFs have been found to have aberrant expression in human tumors and the aberrant expression may contribute to oncogenesis. However, how these previously considered house-keeping proteins are potentially oncogenic remains elusive. In this study, we investigated the expression of eIF3i in human colon cancers, tested its contribution to colon oncogenesis, and determined the mechanism of eIF3i action in colon oncogenesis. We found that eIF3i expression was up-regulated in both human colon adenocarcinoma and adenoma polyps as well as in model inducible colon tumorigenic cell lines. Over-expression of ectopic eIF3i in intestinal epithelial cells causes oncogenesis by directly up-regulating synthesis of COX-2 protein and activates the β-catenin/TCF4 signaling pathway that mediates the oncogenic function of eIF3i. Together, we conclude that eIF3i is a proto-oncogene that drives colon oncogenesis by translationally up-regulating COX-2 and activating β-catenin signaling pathway. These findings imply that protooncogenic eIFs likely exert their tumorigenic function by regulating/altering the synthesis level of down-stream tumor suppressor or oncogenes.
eIF3i; COX-2; β-catenin; translational control; colon cancer; RNA-binding
Polyploid giant cancer cells (PGCCs) have been observed by pathologists for over a century. PGCCs contribute to solid tumor heterogeneity, but their functions are largely undefined. Little attention has been given to these cells, largely because PGCCs have been generally thought to originate from repeated mitosis/cytokinesis failure and have no capacity for long-term survival and cell proliferation. Here we report that we successfully purified and cultured PGCCs from human ovarian cancer cell lines and primary ovarian cancer. These cells are highly resistant to oxygen deprivation and could form through endoreduplication or cell fusion, generating regular-sized cancer cells quickly through budding or burst. They are positive for normal and cancer stem cell markers, divided asymmetrically and cycled slowly. They can differentiate into adipose, cartilage, and bone. A single PGCC formed cancer spheroids in vitro and generated tumors in immunodeficient mice. PGCC-derived tumor gained a mesenchymal phenotype with increased expression of cancer stem cell markers CD44 and CD133 and become more resistant to the treatment of cisplatin. Together, our results reveal that the PGCCs present a resistant form of human cancer generated in response to hypoxia stress and can contribute to generation of cancer stem-like cells and play a fundamental role in regulating tumor heterogeneity, stemness, and chemoresistance in human cancer.
Polyploid giant cancer cells; Cancer stem cells; Cell fusion; Asymmetric cell division; Epithelial-mesenchymal transition
Killing cancer cells through the induction of apoptosis is one of the main mechanisms of chemotherapy. However, numerous cancer cells have primary or acquired apoptosis resistance, resulting in chemoresistance. In this study, using a novel chalcone derivative chalcone-24 (Chal-24), we identified a novel anticancer mechanism through autophagy-mediated necroptosis (RIP1- and RIP3-dependent necrosis). Chal-24 potently killed different cancer cells with induction of necrotic cellular morphology while causing no detectable caspase activation. Blocking the necroptosis pathway with either necrostatin-1 or by knockdown of RIP1 and RIP3 effectively blocked the cytotoxicity of Chal-24, suggesting that Chal-24-induced cell death is associated with necroptosis. Chal-24 robustly activated JNK and ERK and blockage of which effectively suppressed Chal-24-induced cytotoxicity. In addition, Chal-24 strongly induced autophagy that is dependent on JNK-mediated phosphorylation of Bcl-2 and Bcl-xL and dissociation of Bcl-2 or Bcl-xL from Beclin1. Importantly, suppression of autophagy, with either pharmacological inhibitors or siRNAs targeting the essential autophagy components ATG7 and Beclin1, effectively attenuated Chal-24-induced cell death. Furthermore, we found that autophagy activation resulted in c-IAP1 and c-IAP2 degradation and formation of the Ripoptosome that contributes to necroptosis. These results thus establish a novel mechanism for killing cancer cells that involves autophagy-mediated necroptosis, which may be employed for overcoming chemoresistance.
autophagy; necroptosis; RIP1; RIP3; c-IAP; apoptosis
Krüppel-like factor 8 (KLF8) regulates critical gene transcription associated with cancer. The underlying mechanisms, however, remain largely unidentified. We have recently demonstrated that KLF8 expression enhances the activity but not expression of matrix metalloproteinase-2 (MMP2), the target substrate of MMP14. Here, we report a novel KLF8 to MMP14 signaling that promotes human breast cancer invasion and metastasis. Using cell lines for inducible expression and knockdown of KLF8, we demonstrate that KLF8 promotes MMP14 expression at the transcriptional level. Knocking down KLF8 expression inhibited the breast cancer cell invasion both in vitro and in vivo as well as the lung metastasis in mice, which could be rescued by ectopic expression of MMP14. Promoter reporter assays and oligonucleotide and chromatin immunoprecipitations determined that KLF8 activates the human MMP14 gene promoter by both directly acting on the promoter and indirectly via promoting the nuclear translocation of β-catenin, the expression of T cell factor-1 (TCF1) and subsequent activation of the promoter by the β-catenin/TCF1 complex. Inhibition of focal adhesion kinase (FAK) using pharmacological inhibitor, RNA interference or knockout showed that the cell surface presentation of active MMP14 downstream of KLF8 depends upon FAK expression and activity. Taken together, this work identified novel signaling mechanisms by which KLF8 and FAK work together to promote the extracellular activity of MMP14 critical for breast cancer metastasis.
KLF8; FAK; MMP14; β-catenin/TCF1; metastasis; breast cancer