Telomeres consist of TTAGGG repeats bound by the shelterin complex and end with a 3' overhang. In humans, telomeres shorten at each cell division, unless telomerase (TERT) is expressed and able to add telomeric repeats. For effective telomere maintenance, the DNA strand complementary to that made by telomerase must be synthesized. Recent studies have discovered a link between different activities necessary to process telomeres in the S phase of the cell cycle to reform a proper overhang. Notably, the human CST complex (CTC1/STN1/TEN1), known to interact functionally with the polymerase complex (POLA/primase), was shown to be important for telomere processing. Here, focus was paid to the catalytic (POLA1/p180) and accessory (POLA2/p68) subunits of the polymerase, and their mechanistic roles at telomeres. We were able to detect p68 and p180 at telomeres in S-phase using chromatin immunoprecipitation. We could also show that the CST, shelterin, and polymerase complexes interact, revealing contacts occurring at telomeres. We found that the polymerase complex could associate with telomerase activity. Finally, depletion of p180 by siRNA led to increased overhang amounts at telomeres. These data support a model in which the polymerase complex is important for proper telomeric overhang processing through fill-in synthesis, during S phase. These results shed light on important events necessary for efficient telomere maintenance and protection.
Cancer cell resistance to chemotherapy is a major concern in clinical oncology, resulting in increased tumor growth and decreased patient survival. Manipulation of apoptosis has emerged as a new therapeutic strategy to eliminate cancer cells. The focus of this study resides within a novel approach to target survivin, an integrator of both cell death and mitosis. This protein plays a pivotal role in the resistance of tumors to chemotherapy, especially to paclitaxel. The data herein demonstrate an indirect repression of survivin in both B- and T-cell lymphoma and human NHL by the nontoxic tellurium compound, AS101 [ammonium trichloro(dioxoethylene-o,o')tellurate], via inhibition of tumor autocrine IL10–STAT3–Survivin signaling. As a result of survivin abrogation, sensitization of lymphomas to paclitaxel or to Abraxane, the new albumin-stabilized nanoparticle formulation of paclitaxel, occurs both in vitro and in vivo. Importantly, inhibition of lymphoma cell IL10 secretion is mediated by inactivation of the VLA-4 integrin, recently shown to be an important target of AS101. This activity is followed by inhibition of the PI3K–AKT axis that mediates IL10 suppression. Because a wide variety of lymphomas and other tumor types express VLA-4 and secrete IL10 in an autocrine manner, inhibition of survivin with a small nontoxic agent has vast clinical significance in modulating chemosensitivity in many tumor types.
The dissemination of tumor cells relies on efficient cell adhesion and migration, which in turn depends upon endocytic trafficking of integrins. In the current work, it was found that depletion of the prometastatic protein, NEDD9, in breast cancer cells results in a significant decrease in individual cell migration due to impaired trafficking of ligand-bound integrins. NEDD9 deficiency does not affect the expression or internalization of integrins but heightens caveolae-dependent trafficking of ligand-bound integrins to early endosomes. Increase in mobility of ligand-bound integrins is concomitant with an increase in tyrosine phosphorylation of caveolin-1 (CAV1) and volume of CAV1-vesicles. NEDD9 directly binds to CAV1 and colocalizes within CAV1 vesicles. In the absence of NEDD9, the trafficking of ligand-bound integrins from early to late endosomes is impaired, resulting in a significant decrease in degradation of ligand–integrin complexes and an increase in recycling of ligand-bound integrins from early endosomes back to the plasma membrane without ligand disengagement, thus leading to low adhesion and migration. Reexpression of NEDD9 or decrease in the amount of active, tyrosine 14 phosphorylated (Tyr14) CAV1 in NEDD9-depleted cells rescues the integrin trafficking deficiency and restores cellular adhesion and migration capacity. Collectively, these findings indicate that NEDD9 orchestrates trafficking of ligand-bound integrins through the attenuation of CAV1 activity.
Mucin1 (MUC1) is overexpressed in pancreatic ductal adenocarcinoma (PDA) and is associated with tumor aggressiveness, suggesting that MUC1 is a promising therapeutic target for promoter-driven diphtheria toxin A (DTA). Endogenous MUC1 transcript levels were analyzed by quantitative PCR (qPCR) in multiple PDA cells (Capan1, HPAFII, Su.86.86, Capan2, Hs766T, MiaPaCa2, and Panc1). Expression levels were correlated with luciferase activity and cell death after transfection with MUC1 promoter–driven luciferase and DTA constructs. MUC1-positive (+) cells had significantly elevated MUC1 mRNA expression compared with MUC1-negative (–) cells. Luciferase activity was significantly higher in MUC1+ cells when transfected with MUC1 promoter–driven luciferase and MUC1+ cells underwent enhanced cell death after transfection with a single dose of MUC1 promoter–driven DTA. IFN pretreatment enhanced MUC1 expression in MUC1– cells and induced sensitivity to MUC1–DTA therapy. Matched primary and metastatic tumor lesions from clinical specimens revealed similar MUC1 IHC labeling patterns, and a tissue microarray of human PDA biopsies revealed increased immunolabeling with a combination of MUC1 and mesothelin (MSLN) antibodies, compared with either antibody alone. Combining MUC1 with MSLN-targeted DTA enhanced drug efficacy in an in vitro model of heterogeneous PDA. These data demonstrate that MUC1 promoter–driven DTA preferentially kills MUC1-expressing PDA cells and drugs that enhance MUC1 expression sensitize PDA cells with low MUC1 expression.
The LIN28B->let-7 pathway contributes to regulation of the epithelial–mesenchymal transition (EMT) and stem cell self-renewal. The oncogenic MUC1-C transmembrane protein is aberrantly overexpressed in lung and other carcinomas; however, there is no known association between MUC1-C and the LIN28B->let-7 pathway. Here in non–small cell lung cancer (NSCLC), silencing MUC1-C downregulates the RNA-binding protein LIN28B and coordinately increases the miRNA let-7. Targeting MUC1-C function with a dominant-negative mutant or a peptide inhibitor provided confirming evidence that MUC1-C induces LIN28B->let-7 signaling. Mechanistically, MUC1-C promotes NF-B p65 chromatin occupancy of the LIN28B first intron and activates LIN28B transcription, which is associated with suppression of let-7. Consistent with let-7–mediated inhibition of HMGA2 transcripts, targeting of MUC1-C also decreases HMGA2 expression. HMGA2 has been linked to stemness, and functions as a competing endogenous RNA (ceRNA) of let-7–mediated regulation of the TGFβ coreceptor TGFBR3. Accordingly, targeting MUC1-C suppresses HMGA2 mRNA and protein, which is associated with decreases in TGFBR3, reversal of the EMT phenotype, and inhibition of self-renewal capacity. These findings support a model in which MUC1-C activates the LIN28B->let-7->HMGA2 axis in NSCLC and thereby promotes EMT traits and stemness.
Epigenetic alteration is a hallmark of all cancers. Such alterations lead to modulation of fundamental cancer-related functions, such as proliferation, migration, and invasion. In particular, methylation of Histone H3 Lysine 4 (H3K4), a histone mark generally associated with transcriptional activation, is altered during progression of several human cancers. While the depletion of H3K4 demethylases promotes breast cancer metastasis, the effect of H3K4 methyltransferases on metastasis is not clear. Nevertheless, gene duplications in the human SETD1A (hSETD1A) H3K4 methyltransferase are present in almost half of breast cancers. Herein, expression analysis determined that hSETD1A is upregulated in multiple metastatic human breast cancer cell lines and clinical tumor specimens. Ablation of hSETD1A in breast cancer cells led to a decrease in migration and invasion in vitro and to a decrease in metastasis in nude mice. Furthermore, a group of matrix metalloproteinases (including MMP2, MMP9, MMP12, MMP13, and MMP17) were identified which were downregulated upon depletion of hSETD1A and demonstrated a decrease in H3K4me3 at their proximal promoters based on chromatin immunoprecipitation analysis. These results provide evidence for a functional and mechanistic link among hSETD1A, MMPs, and metastasis in breast cancer, thereby supporting an oncogenic role for hSETD1A in cancer.
In neuroblastoma, MYCN genomic amplification and segmental chromosomal alterations including 1p or 11q loss of heterozygocity and/or 17q gain are associated with progression and poor clinical outcome. Segmental alterations are the strongest predictor of relapse and result from unbalanced translocations attributable to erroneous repair of chromosomal breaks. Although sequence analysis of affected genomic regions suggests that these errors arise by nonhomologous end-joining (NHEJ) of DNA double-strand breaks (DSB), abnormalities in NHEJ have not been implicated in neuroblastoma pathogenesis. On this basis, the hypothesis that an error-prone mechanism of NHEJ is critical for neuroblastoma cell survival was tested. Plasmid-based DSB repair assays demonstrated efficient NHEJ activity in human neuroblastoma cells with repair products that were error-prone relative to nontransformed cells. Neuroblastoma cells derived from tumorigenic neuroblastic phenotypes had differential DNA repair protein expression patterns compared with nontumorigenic cells. Tumorigenic neuroblastoma cells were deficient in DNA ligase IV (Lig4) and Artemis (DCLRE1C), mediators of canonical NHEJ. Conversely, enzymes required for an error-prone alternative NHEJ pathway (alt-NHEJ), DNA Ligase IIIα (Lig3), DNA Ligase I (Lig1), and PARP1 protein were upregulated. Inhibition of Lig3 and Lig1 led to DSB accumulation and cell death, linking alt-NHEJ to cell survival in neuroblastoma. Neuroblastoma cells demonstrated sensitivity to PARP1 inhibition (PARPi) that paralleled PARP1 expression. In a dataset of human neuroblastoma patient tumors, overexpression of genes encoding alt-NHEJ proteins associated with poor survival.
RhoGDI2 (ARHGDIB) suppresses metastasis in a variety of cancers but the mechanism is unclear, thus hampering development of human therapeutics. RhoGDI2 is a guanine nucleotide dissociation inhibitor (GDI) for the Rho family of GTPases thought to primarily bind to Rac1; however, Rac1 activation was not decreased by RhoGDI2 expression in bladder cancer cells. To better understand the GTPase-binding partners for RhoGDI2, a mass spectrometry–based proteomic approach was used in bladder cancer cells. As expected, endogenous RhoGDI2 coimmunoprecipitates with Rac1 and unexpectedly also with RhoC. Further analysis demonstrated that RhoGDI2 negatively regulates RhoC, as knockdown of RhoGDI2 increased RhoC activation in response to serum stimulation. Conversely, overexpression of RhoGDI2 decreased RhoC activation. RhoC promoted bladder cancer cell growth and invasion, as knockdown increased cell doubling time, decreased invasion through Matrigel, and decreased colony formation in soft agar. Importantly, RhoC knockdown reduced in vivo lung colonization by bladder cancer cells following tail vein injection in immunocompromised mice. Finally, unbiased transcriptome analysis revealed a set of genes regulated by RhoGDI2 overexpression and RhoC knockdown in bladder cancer cells.
Lymphocytic infiltration is associated with better prognosis in several epithelial malignancies including breast cancer. The tumor suppressor TP53 is mutated in approximately 30% of breast adenocarcinomas, with varying frequency across molecular subtypes. In this study of 1,420 breast tumors, we tested for interaction between TP53 mutation status and tumor subtype determined by PAM50 and integrative cluster analysis. In integrative cluster 10 (IC10)/basal-like breast cancer, we identify an association between lymphocytic infiltration, determined by an expression score, and retention of wild-type TP53. The expression-derived score agreed with the degree of lymphocytic infiltration assessed by pathologic review, and application of the Nanodissect algorithm was suggestive of this infiltration being primarily of cytotoxic T lymphocytes (CTL). Elevated expression of this CTL signature was associated with longer survival in IC10/Basal-like tumors. These findings identify a new link between the TP53 pathway and the adaptive immune response in estrogen receptor (ER)–negative breast tumors, suggesting a connection between TP53 inactivation and failure of tumor immunosurveillance.
High-grade serous ovarian cancers (HGSOC) are characterized by widespread recurrent regions of copy-number gain and loss. Here, we interrogated 50 genes that are recurrently amplified in HGSOC and essential for cancer proliferation and survival in ovarian cancer cell lines. FRS2 is one of the 50 genes located on chromosomal region 12q15 that is focally amplified in 12.5% of HGSOC. We found that FRS2-amplified cancer cell lines are dependent on FRS2 expression, and that FRS2 overexpression in immortalized human cell lines conferred the ability to grow in an anchorage-independent manner and as tumors in immunodeficient mice. FRS2, an adaptor protein in the FGFR pathway, induces downstream activation of the Ras–MAPK pathway. These observations identify FRS2 as an oncogene in a subset of HGSOC that harbor FRS2 amplifications.
Heparan sulfate endosulfatase-1 and -2 (SULF1 and SULF2) are two important extracellular 6-O-endosulfatases that remove 6-O sulfate groups of N-glucosamine along heparan sulfate (HS) proteoglycan chains often found in the extracellular matrix. The HS sulfation pattern influences signaling events at the cell surface, which are critical for interactions with growth factors and their receptors. SULFs are overexpressed in several types of human tumors, but their role in cancer is still unclear because their molecular mechanism has not been fully explored and understood. To further investigate the functions of these sulfatases in tumorigenesis, stable overexpression models of these genes were generated in the colorectal cancer cells, Caco-2 and HCT-116. Importantly, mimicking overexpression of these sulfatases resulted in increased viability and proliferation, and augmented cell migration. These effects were reverted by shRNA-mediated knockdown of SULF1 or SULF2 and by the addition of unfractionated heparin. Detailed structural analysis of HS from cells overexpressing SULFs showed reduction in the trisulfated disaccharide UA(2S)-GlcNS(6S) and corresponding increase in UA(2S)-GlcNS disaccharide, as well as an unexpected rise in less common disaccharides containing GlcNAc(6S) residues. Moreover, cancer cells transfected with SULFs demonstrated increased Wnt signaling. In summary, SULF1 or SULF2 overexpression contributes to colorectal cancer cell proliferation, migration, and invasion.
Angiogenic remodeling during embryonic development and in adult tissue homeostasis is orchestrated by cooperative signaling between several distinct molecular pathways, which are often exploited by tumors. Indeed, tumors upregulate proangiogenic molecules while simultaneously suppressing angiostatic pathways to recruit blood vessels for growth, survival, and metastatic spread. Understanding how cancers exploit proangiogenic and antiangiogenic signals is a key step in developing new, molecularly targeted antiangiogenic therapies. While EphA2, a receptor tyrosine kinase (RTK), is required for VEGF-induced angiogenesis, the mechanism through which these pathways intersect remains unclear. Slit2 expression is elevated in EphA2-deficient endothelium, and here it is reported that inhibiting Slit activity rescues VEGF-induced angiogenesis in cell culture and in vivo, as well as VEGF-dependent tumor angiogenesis, in EphA2-deficient endothelial cells and animals. Moreover, blocking Slit activity or Slit2 expression in EphA2-deficient endothelial cells restores VEGF-induced activation of Src and Rac, both of which are required for VEGF-mediated angiogenesis. These data suggest that EphA2 suppression of Slit2 expression and Slit angiostatic activity enables VEGF-induced angiogenesis in vitro and in vivo, providing a plausible mechanism for impaired endothelial responses to VEGF in the absence of EphA2 function.
Epithelial cell–cell contacts maintain normal glandular tissue homeostasis, and their breakage can trigger epithelial-to-mesenchymal transition (EMT), a fundamental step in the development of metastatic cancer. Despite the ability of C-type lectin domains (CTLD) to modulate cell–cell adhesion, it is not known if they modulate epithelial adhesion in EMT and tumor progression. Here, the multi-CTLD mannose receptor, Endo180 (MRC2/uPARAP), was shown using the Kaplan–Meier analysis to be predictive of survival outcome in men with early prostate cancer. A proteomic screen of novel interaction partners with the fourth CTLD (CTLD4) in Endo180 revealed that its complex with CD147 is indispensable for the stability of three-dimensional acini formed by nontransformed prostate epithelial cells (PEC). Mechanistic study using knockdown of Endo180 or CD147, and treatment with an Endo180 mAb targeting CTLD4 (clone 39.10), or a dominant-negative GST-CTLD4 chimeric protein, induced scattering of PECs associated with internalization of Endo180 into endosomes, loss of E-cadherin (CDH1/ECAD), and unzipping of cell–cell junctions. These findings are the first to demonstrate that a CTLD acts as a suppressor and regulatory switch for EMT; thus, positing that stabilization of Endo180–CD147 complex is a viable therapeutic strategy to improve rates of prostate cancer survival.
Implications: This study identifies the interaction between CTLD4 in Endo180 and CD147 as an EMT suppressor and indicates that stabilization of this molecular complex improves prostate cancer survival rates.