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Molecular Biology of the Cell

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Molecular Cancer Research

Hepatocellular carcinomas (HCC) show resistance to chemotherapy and have blunt response to apoptotic stimuli. HCC cell lines express low levels of the Fas death receptor and are resistant to FasL stimulation, whereas immortalized hepatocytes are sensitive. The variable Fas transcript levels and consistently low Fas protein in HCC cells suggest posttranscriptional regulation of Fas expression. The 3'-untranslated region (UTR) of Fas mRNA was found to interact with the ribonucleoprotein Human Antigen R (HuR) to block mRNA translation. Silencing of HuR in HCC cells increased the levels of cell surface Fas and sensitized HCC cells to FasL. Two AU-rich domains within the 3'-UTR of Fas mRNA were identified as putative HuR-binding sites and were found to mediate the translational regulation in reporter assay. Hydrodynamic transfection of HuR plasmid into mice induced downregulation of Fas expression in livers and established functional resistance to the killing effects of Fas agonist. Human HCC tumor tissues showed significantly higher overall and cytoplasmic HuR staining compared with normal liver tissues, and the high HuR staining score correlated with worse survival of patients with early-stage HCC. Combined, the protumorigenic ribonucleoprotein HuR blocks the translation of Fas mRNA and effectively prevents Fas-mediated apoptosis in HCC, suggesting that targeting HuR would sensitize cells to apoptotic stimuli and reverse tumorigenic properties.

Implications: Demonstrating how death receptor signaling pathways are altered during progression of HCC will enable the development of better methods to restore this potent apoptosis mechanism. Mol Cancer Res; 13(5); 809–18. ©2015 AACR.

Osteolytic bone damage is a major cause of morbidity in several metastatic pathologies. Current therapies using bisphosphonates provide modest improvement, but cytotoxic side effects still occur prompting the need to develop more effective therapies to target aggressive osteoclastogenesis. Increased levels of receptor activator of NF–B ligand (TNFSF11/RANKL), leading to RANKL-RANK signaling, remain the key axis for osteoclast activation and bone resorption. Osteoprotegerin (TNFRSF11B/OPG), a decoy receptor for RANKL, is significantly decreased in patients who present with bone lesions. Despite its potential in inhibiting osteoclast activation, OPG also binds to TNF-related apoptosis-inducing ligand (TNFSF10/TRAIL), making tumor cells resistant to apoptosis. Toward uncoupling the events of TRAIL binding of OPG and to improve its utility for bone remodeling without inducing tumor resistance to apoptosis, OPG mutants were developed by structural homology modeling based on interactive domain identification and by superimposing models of OPG, TRAIL, and its receptor DR5 (TNFRSF10B) to identify regions of OPG for rational design. The OPG mutants were purified and extensively characterized for their ability to decrease osteoclast damage without affecting tumor apoptosis pathway both in vitro and in vivo, confirming their potential in bone remodeling following cancer-induced osteolytic damage.

Implications: OPG variants were developed that lack TRAIL binding, yet retain RANKL binding and suggest new possibilities for therapeutic targeting in osteolytic malignancies. Mol Cancer Res; 13(5); 819–27. ©2015 AACR.

Long noncoding RNAs (lncRNA) are emerging as major regulators of cellular phenotypes and implicated as oncogenes or tumor suppressors. Here, we report a novel tumor-suppressive locus on human chromosome 15q23 that contains two multiexonic lncRNA genes of 100 kb each: DRAIC (LOC145837) and the recently reported PCAT29. The DRAIC lncRNA was identified from RNA-seq data and is downregulated as prostate cancer cells progress from an androgen-dependent (AD) to a castration-resistant (CR) state. Prostate cancers persisting in patients after androgen deprivation therapy (ADT) select for decreased DRAIC expression, and higher levels of DRAIC in prostate cancer are associated with longer disease-free survival (DFS). Androgen induced androgen receptor (AR) binding to the DRAIC locus and repressed DRAIC expression. In contrast, FOXA1 and NKX3-1 are recruited to the DRAIC locus to induce DRAIC, and FOXA1 specifically counters the repression of DRAIC by AR. The decrease of FOXA1 and NKX3-1, and aberrant activation of AR, thus accounts for the decrease of DRAIC during prostate cancer progression to the CR state. Consistent with DRAIC being a good prognostic marker, DRAIC prevents the transformation of cuboidal epithelial cells to fibroblast-like morphology and prevents cellular migration and invasion. A second tumor-suppressive lncRNA PCAT29, located 20 kb downstream of DRAIC, is regulated identically by AR and FOXA1 and also suppresses cellular migration and metastasis. Finally, based on TCGA analysis, DRAIC expression predicts good prognosis in a wide range of malignancies, including bladder cancer, low-grade gliomas, lung adenocarcinoma, stomach adenocarcinoma, renal clear cell carcinoma, hepatocellular carcinoma, skin melanoma, and stomach adenocarcinoma.

Implications: This study reveals a novel tumor-suppressive locus encoding two hormone-regulated lncRNAs, DRAIC and PCAT29, that are prognostic for a wide variety of cancer types. Mol Cancer Res; 13(5); 828–38. ©2015 AACR.

Long noncoding RNAs (lncRNA) have been found to play a role in gene regulation with dysregulated expression in various cancers. The precise role that lncRNA expression plays in the pathogenesis of B-acute lymphoblastic leukemia (B-ALL) is unknown. Therefore, unbiased microarray profiling was performed on human B-ALL specimens, and it was determined that lncRNA expression correlates with cytogenetic abnormalities, which was confirmed by qRT-PCR in a large set of B-ALL cases. Importantly, high expression of BALR-2 correlated with poor overall survival and diminished response to prednisone treatment. In line with a function for this lncRNA in regulating cell survival, BALR-2 knockdown led to reduced proliferation, increased apoptosis, and increased sensitivity to prednisolone treatment. Conversely, overexpression of BALR-2 led to increased cell growth and resistance to prednisone treatment. Interestingly, BALR-2 expression was repressed by prednisolone treatment and its knockdown led to upregulation of the glucocorticoid response pathway in both human and mouse B cells. Together, these findings indicate that BALR-2 plays a functional role in the pathogenesis and/or clinical responsiveness of B-ALL, and that altering the levels of particular lncRNAs may provide a future direction for therapeutic development.

Implications: lncRNA expression has the potential to segregate the common subtypes of B-ALL, predict the cytogenetic subtype, and indicate prognosis. Mol Cancer Res; 13(5); 839–51. ©2015 AACR.

To ensure faithful chromosome segregation, cells use the spindle assembly checkpoint (SAC), which can be activated in aneuploid cancer cells. Targeting the components of SAC machinery required for the growth of aneuploid cells may offer a cancer cell–specific therapeutic approach. In this study, the effects of inhibiting Monopolar spindle 1, MPS1 (TTK), an essential SAC kinase, on the radiosensitization of glioblastoma (GBM) cells were analyzed. Clonogenic survival was used to determine the effects of the MPS1 inhibitor NMS-P715 on radiosensitivity in multiple model systems, including GBM cell lines, a normal astrocyte, and a normal fibroblast cell line. DNA double-strand breaks (DSB) were evaluated using H2AX foci, and cell death was measured by mitotic catastrophe evaluation. Transcriptome analysis was performed via unbiased microarray expression profiling. Tumor xenografts grown from GBM cells were used in tumor growth delay studies. Inhibition of MPS1 activity resulted in reduced GBM cell proliferation. Furthermore, NMS-P715 enhanced the radiosensitivity of GBM cells by decreased repair of DSBs and induction of postradiation mitotic catastrophe. NMS-P715 in combination with fractionated doses of radiation significantly enhanced the tumor growth delay. Molecular profiling of MPS1-silenced GBM cells showed an altered expression of transcripts associated with DNA damage, repair, and replication, including the DNA-dependent protein kinase (PRKDC/DNAPK). Next, inhibition of MPS1 blocked two important DNA repair pathways. In conclusion, these results not only highlight a role for MPS1 kinase in DNA repair and as prognostic marker but also indicate it as a viable option in glioblastoma therapy.

Implications: Inhibition of MPS1 kinase in combination with radiation represents a promising new approach for glioblastoma and for other cancer therapies. Mol Cancer Res; 13(5); 852–62. ©2015 AACR.

Pancreatic cancer is one of the deadliest cancers with poor survival rates and limited therapeutic options. To improve the understanding of this disease's biology, a prerequisite for the generation of novel therapeutics, new platforms for rapid and efficient genetic and therapeutic screening are needed. Therefore, a combined in vitro/in vivo hybrid shRNA assay was developed using isolated murine primary pancreatic ductal cells (PDCs), in which oncogenic KrasG12D could be activated in vitro by genomic recombination through 4OH-tamoxifen–induced nuclear translocation of Cre-ERT2 expressed under control of the ROSA26 promoter. Further genetic manipulation was achieved through selective and stable RNAi against the tumor suppressors p16Ink4a (CDKN2A) or Trp53 (TP53) using lentiviral gene delivery. Treatment of PDCs with 4OH-tamoxifen increased phosphorylation of ERK downstream of KRAS, and subsequent lentiviral transduction resulted in sustained target gene repression. Double-mutant PDCs were then reintroduced into the pancreata of NOD-SCID-gamma (NSG) mice and monitored for tumor growth. Orthotopic implantation of PDCs carrying the activated KrasG12D-allele and shRNA against p16Ink4a or Trp53 resulted in tumor growth, metastasis, and reduced survival of NSG mice. In contrast, KrasG12D alone was not sufficient to induce tumor growth.

Implications: The combinatory in vitro/in vivo approach described in this study allows for rapid and efficient identification of genes involved in carcinogenesis and opens new avenues for the development of therapeutic strategies to improve cancer treatment. Mol Cancer Res; 13(5); 863–9. ©2015 AACR.

Renal cell carcinoma (RCC) is the most common cancer arising from the kidney in adults, with clear cell RCC (ccRCC) representing the majority of all RCCs. Expression of a human HIF1α triple-mutant (P402A, P564A, and N803A) construct in the proximal tubule cells of C57BL/6 mice [TRAnsgenic model of Cancer of the Kidney (TRACK); ref. 1] mimics the histologic changes found in early stage human ccRCC. To better understand the genomic landscape, a high-throughput sequence analysis was performed with cDNA libraries (RNAseq) derived from TRACK transgenic positive (TG+) kidney cortex along with human ccRCC transcripts from the Oncomine and The Cancer Genome Atlas databases. Importantly, the expression profiles of TRACK TG+ kidneys show significant similarities with those observed in human ccRCC, including increased expression of genes involved in glycolysis and the tricarboxylic acid cycle. Some of the transcripts overexpressed in both the TRACK mouse model and human ccRCC include ANKRD37, CA9, EGLN3, HK2, NDUFA4L2, and SLC16A3. These data suggest that constitutive activation of HIF1α in kidney proximal tubule cells transcriptionally reprograms the regulation of metabolic pathways in the kidney and that HIF1α is a major contributor to the altered metabolism observed in human ccRCC.

Implications: TRACK (GGT-HIF1αM3) kidney mRNA profiles show similarities to human ccRCC transcriptome and phenotypes associated with the Warburg effect. Mol Cancer Res; 13(5); 870–8. ©2015 AACR.

Ectodomain cleavage (shedding) of transmembrane proteins by metalloproteases (MMP) generates numerous essential signaling molecules, but its regulation is not totally understood. CD44, a cleaved transmembrane glycoprotein, exerts both antiproliferative or tumor-promoting functions, but whether proteolysis is required for this is not certain. CD44-mediated contact inhibition and cellular proliferation are regulated by counteracting CD44 C-terminal interacting proteins, the tumor suppressor protein merlin (NF2) and ERM proteins (ezrin, radixin, moesin). We show here that activation or overexpression of constitutively active merlin or downregulation of ERMs inhibited 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced [as well as serum, hepatocyte growth factor (HGF), or platelet-derived growth factor (PDGF)] CD44 cleavage by the metalloprotease ADAM10, whereas overexpressed ERM proteins promoted cleavage. Merlin- and ERM-modulated Ras or Rac activity was not required for this function. However, latrunculin (an actin-disrupting toxin) or an ezrin mutant which is unable to link CD44 to actin, inhibited CD44 cleavage, identifying a cytoskeletal C-terminal link as essential for induced CD44 cleavage. Cellular migration, an important tumor property, depended on CD44 and its cleavage and was inhibited by merlin. These data reveal a novel function of merlin and suggest that CD44 cleavage products play a tumor-promoting role. Neuregulin, an EGF ligand released by ADAM17 from its pro-form NRG1, is predominantly involved in regulating cellular differentiation. In contrast to CD44, release of neuregulin from its pro-form was not regulated by merlin or ERM proteins. Disruption of the actin cytoskeleton however, also inhibited NRG1 cleavage. This current study presents one of the first examples of substrate-selective cleavage regulation.

Implications: Investigating transmembrane protein cleavage and their regulatory pathways have provided new molecular insight into their important role in cancer formation and possible treatment. Mol Cancer Res; 13(5); 879–90. ©2015 AACR.

Extracellular vesicles (EV), including exosomes and shed vesicles, have been implicated in intercellular communication; however, their biomarker potential is less clear. Therefore, EVs derived from MCF7 and MCF10A cells were analyzed to identify unique miRNA (miR) profiles that distinguish their origin. One characteristic common to the miR profiles of MCF7 EVs and their parent cells is the high abundance of miR-21, let-7a, miR-100, and miR-125b, and low levels of miR-205. A second characteristic is the high abundance of "miRNA-like" tRNA fragments, which is unique to the MCF7 EVs, and is not found in comparing the cellular profiles. In addition, correlations were examined in the MCF7 cellular expression levels of these five miRs and two tRNA-derived miRNAs, miR-720 and miR-1274b, and compared with the correlations in MCF7 EV levels. Interestingly, correlations in the cellular expression of miR-125b, miR-100, and let-7a are mirrored in the EVs. In contrast, correlations in tRNA-derived miRNA levels are found only in the EVs. The findings suggest that EV miR clusters can be defined based on functional miR interactions related to correlated cellular expression levels or physical miR interactions, for example, aggregation due to comparable binding affinities to common targets.

Implications: These results point to using high levels of tRNA-derived small RNA fragments in combination with known miR signatures of tumors to distinguish tumor-derived EVs in circulation from EVs derived from other cell sources. Such biomarkers would be unique to the EVs where high abundances of tRNA fragments are amplified with respect to their cellular levels. Mol Cancer Res; 13(5); 891–901. ©2015 AACR.

The WW domain containing oxidoreductase (WWOX) has recently been shown to inhibit of the Wnt/β-catenin pathway by preventing the nuclear import of disheveled 2 (DVL2) in human breast cancer cells. Here, it is revealed that WWOX also interacts with the BCL9-2, a cofactor of the Wnt/β-catenin pathway, to enhance the activity of the β-catenin–TCF/LEF (T-cell factor/lymphoid enhancer factors family) transcription factor complexes. By using both a luciferase assay in MCF-7 cells and a Xenopus secondary axis induction assay, it was demonstrated that WWOX inhibits the BCL9-2 function in Wnt/β-catenin signaling. WWOX does not affect the BCL9-2–β-catenin association and colocalizes with BCL9-2 and β-catenin in the nucleus of the MCF-7 cells. Moreover, WWOX inhibits the β-catenin–TCF1 interaction. Further examination found that HDAC3 associates with BCL9-2, enhances the inhibitory effect of WWOX on BCL9-2 transcriptional activity, and promotes the WWOX–BCL9-2 interaction, independent of its deacetylase activity. However, WWOX does not influence the HDAC3–BCL9-2 interaction. Altogether, these results strongly indicate that nuclear WWOX interacts with BCL9-2 associated with β-catenin only when BCL9-2 is in complex with HDAC3 and inhibits its transcriptional activity, in part, by inhibiting the β-catenin–TCF1 interaction. The promotion of the WWOX–BCL9-2 interaction by HDAC3, independent of its deacetylase activity, represents a new mechanism by which this HDAC inhibits transcription.

Implications: The inhibition of the transcriptional activity of BCL9-2 by WWOX and HDAC3 constitutes a new molecular mechanism and provides new insight for a broad range of cancers. Mol Cancer Res; 13(5); 902–12. ©2015 AACR.

NKX3.1 is a prostate-specific homeodomain protein and tumor suppressor whose expression is reduced in the earliest phases of prostatic neoplasia. NKX3.1 expression is not only diminished by genetic loss and methylation, but the protein itself is a target for accelerated degradation caused by inflammation that is common in the aging prostate gland. NKX3.1 degradation is activated by phosphorylation at C-terminal serine residues that mediate ubiquitination and protein turnover. Because NKX3.1 is haploinsufficient, strategies to increase its protein stability could lead to new therapies. Here, a high-throughput screen was developed using an siRNA library for kinases that mediate NKX3.1 degradation. This approach identified several candidates, of which DYRK1B, a kinase that is subject to gene amplification and overexpression in other cancers, had the greatest impact on NKX3.1 half-life. Mechanistically, NKX3.1 and DYRK1B were shown to interact via the DYRK1B kinase domain. In addition, an in vitro kinase assay showed that DYRK1B phosphorylated NKX3.1 at serine 185, a residue critical for NKX3.1 steady-state turnover. Lastly, small-molecule inhibitors of DYRK1B prolonged NKX3.1 half-life. Thus, DYRK1B is a target for enzymatic inhibition in order to increase cellular NKX3.1.

Implications: DYRK1B is a promising and novel kinase target for prostate cancer treatment mediated by enhancing NKX3.1 levels. Mol Cancer Res; 13(5); 913–22. ©2015 AACR.

Cancers exhibit altered metabolism characterized by increased glucose and glutamine uptake. The hexosamine biosynthetic pathway (HBP) uses glucose and glutamine, and directly contributes to O-linked-β-N-acetylglucosamine (O-GlcNAc) modifications on intracellular proteins. Multiple tumor types contain elevated total O-GlcNAcylation, in part, by increasing O-GlcNAc transferase (OGT) levels, the enzyme that catalyzes this modification. Although cancer cells require OGT for oncogenesis, it is not clear how tumor cells regulate OGT expression and O-GlcNAcylation. Here, it is shown that the PI3K–mTOR–MYC signaling pathway is required for elevation of OGT and O-GlcNAcylation in breast cancer cells. Treatment with PI3K and mTOR inhibitors reduced OGT protein expression and decreased levels of overall O-GlcNAcylation. In addition, both AKT and mTOR activation is sufficient to elevate OGT/O-GlcNAcylation. Downstream of mTOR, the oncogenic transcription factor c-MYC is required and sufficient for increased OGT protein expression in an RNA-independent manner and c-MYC regulation of OGT mechanistically requires the expression of c-MYC transcriptional target HSP90A. Finally, mammary tumor epithelial cells derived from MMTV-c-myc transgenic mice contain elevated OGT and O-GlcNAcylation and OGT inhibition in this model induces apoptosis. Thus, OGT and O-GlcNAcylation levels are elevated via activation of an mTOR/MYC cascade.

Implications: Evidence indicates OGT as a therapeutic target in c-MYC–amplified cancers. Mol Cancer Res; 13(5); 923–33. ©2015 AACR.

Epithelial-to-mesenchymal transition (EMT) has been implicated in models of tumor cell migration, invasion, and metastasis. In a search for candidate therapeutic targets to reverse this process, nontumorigenic MCF10A breast epithelial cells were infected with an arrayed lentiviral kinome shRNA library and screened for either suppression or enhancement of a 26-gene EMT RNA signature. No individual kinase gene knockdown was sufficient to induce EMT. In contrast, grouped epithelial markers were induced by knockdown of multiple kinases, including mitogen activated protein kinase 7 (MAPK7). In breast cancer cells, suppression of MAPK7 increased E-cadherin (CDH1) expression and inhibited cell migration. In an orthotopic mouse model, MAPK7 suppression reduced the generation of circulating tumor cells and the appearance of lung metastases. Together, these observations raise the possibility that targeting kinases that maintain mesenchymal cell properties in cancer cells, such as MAPK7, may lessen tumor invasiveness.

Implications: Suppression of MAPK7 induces epithelial markers, reduces generation of circulating tumor cells and appearance of lung metastases. Mol Cancer Res; 13(5); 934–43. ©2015 AACR.

While reviewing chronic lymphocytic leukemia (CLL) bone marrow slides, we identified cytoplasmic lipid vacuoles in CLL cells but not in normal B cells. Because lipoprotein lipase (LPL), which catalyzes hydrolysis of triglycerides into free fatty acids (FFA), is aberrantly expressed in CLL, we investigated whether LPL regulates the oxidative metabolic capacity of CLL cells. We found that unlike normal B cells, CLL cells metabolize FFAs. Because STAT3 is constitutively activated in CLL cells and because we identified putative STAT3 binding sites in the LPL promoter, we sought to determine whether STAT3 drives the aberrant expression of LPL. Transfection of luciferase reporter gene constructs driven by LPL promoter fragments into MM1 cells revealed that STAT3 activates the LPL promoter. In addition, chromatin immunoprecipitation confirmed that STAT3 binds to the LPL promoter. Furthermore, transfection of CLL cells with STAT3-shRNA downregulated LPL transcripts and protein levels, confirming that STAT3 activates the LPL gene. Finally, transfection of CLL cells with LPL-siRNAs decreased the capacity of CLL cells to oxidize FFAs and reduced cell viability.

Implications: Our study suggests that CLL cells adopt their metabolism to oxidize FFA. Activated STAT3 induces LPL, which catalyzes the hydrolysis of triglycerides into FFA. Therefore, inhibition of STAT3 is likely to prevent the capacity of CLL cells to utilize FFA. Mol Cancer Res; 13(5); 944–53. ©2015 AACR.