CITED2 modulates estrogen receptor transcriptional activity in breast cancer cells.

Cbp/p300-interacting transactivator with Glu/Asp-rich carboxy-terminal domain 2 (CITED2) is a member of the CITED family of non-DNA binding transcriptional co-activators of the p300/CBP-mediated transcription complex. Previously, we identified CITED2 as being overexpressed in human breast tumors relative to normal mammary epithelium. Upon further investigation within the estrogen receptor (ER)-positive subset of these breast tumor samples, we found that CITED2 mRNA expression was elevated in those associated with poor survival. In light of this observation, we investigated the effect of elevated CITED2 levels on ER function. While ectopic overexpression of CITED2 in three ER-positive breast cancer cell lines (MCF-7, T47D, and CAMA-1) did not alter cell proliferation in complete media, growth was markedly enhanced in the absence of exogenous estrogen. Correspondingly, cells overexpressing CITED2 demonstrated reduced sensitivity to the growth inhibitory effects of the selective estrogen receptor modulator, 4-hydroxytamoxifen. Subsequent studies revealed that basal ER transcriptional activity was elevated in CITED2-overexpressing cells and was further increased upon the addition of estrogen. Similarly, basal and estrogen-induced expression of the ER-regulated genes trefoil factor 1 (TFF1) and progesterone receptor (PGR) was higher in cells overexpressing CITED2. Concordant with this observation, ChIP analysis revealed higher basal levels of CITED2 localized to the TFF-1 and PGR promoters in cells with ectopic overexpression of CITED2, and these levels were elevated further in response to estrogen stimulation. Taken together, these data indicate that CITED2 functions as a transcriptional co-activator of ER in breast cancer cells and that its increased expression in tumors may result in estrogen-independent ER activation, thereby reducing estrogen dependence and response to anti-estrogen therapy.

Identification of prospective factors promoting osteotropism in breast cancer: a potential role for CITED2.

Breast cancer metastases develop in the bone more frequently than any other site and are a common cause of morbidity in the form of bone pain, pathological fractures, nerve compression and life-threatening hypercalcemia. Despite ongoing research efforts, the molecular and cellular mechanisms that regulate breast cancer cell homing to and colonization of the bone as well as resultant pathological bone alteration remain poorly understood. To identify key mediators promoting breast cancer metastasis to bone, we utilized an immunocompetent, syngeneic murine model of breast cancer metastasis employing the mammary tumor cell line NT2.5. Following intracardiac injection of NT2.5 cells in neu-N mice, metastases developed in the bone, liver and lung, closely mimicking the anatomical distribution of metastases in patients with breast cancer. Using an in vivo selection process, we established NT2.5 sublines demonstrating an enhanced ability to colonize the bone and liver. Genome-wide cDNA microarray analysis comparing gene expression between parental NT2.5 cells and established sublines revealed both known and novel mediators of bone metastasis and osteolysis, including the transcriptional co-activator CITED2. In further studies, we found that expression of CITED2 was elevated in human primary breast tumors and bone metastasis compared to normal mammary epithelium and was highest in breast cancer cell lines that cause osteolytic bone metastasis in animal models. In addition, reducing CITED2 expression in NT2.5 cells inhibited the establishment of bone metastasis and osteolysis in vivo, suggesting a potential role for CITED2 in promoting breast cancer bone metastasis.

Acquired Resistance to FGFR Inhibitor in Diffuse-Type Gastric Cancer through an AKT-Independent PKC-Mediated Phosphorylation of GSK3ß.

Preclinical models of diffuse-type gastric cancer (DGC) that reliably predict clinical activity of novel compounds are lacking. To overcome the problem of poor tumor cellularity in DGC, we used cells from malignant ascites to establish DGC patient-derived xenograft (PDX) models that recapitulate the primary cancer. Cells in PDX model GAGA6 with FGFR2 amplification were sensitive to AZD4547, a potent FGFR inhibitor that is being clinically evaluated for FGFR-aberrant cancer types. Intermittent in vivo treatment of GAGA6 tumors with AZD4547 gave rise to PDX tumors with acquired resistance to AZD4547, GAGA6-R. Surprisingly, there were no mutations in the FGFR2 gene in GAGA6-R, negating gatekeeper mutations as a mechanism of drug resistance. Phosphorylation of FGFR2 and downstream signaling molecules AKT/PKB and MAPK/ERK remained inhibited by AZD4547. Further analysis of signaling pathways identified AKT-independent phosphorylation and inhibition of GSK3ß as a mechanism of drug resistance in GAGA6-R cells. Treatment of GAGA6-R cells with protein kinase C (PKC) inhibitor H7 in combination with AZD4547 led to dephosphorylation and activation of GSK3ß with concomitant downregulation of MCL-1 and BCL-XL. Combined treatment with AZD4547 and H7 in vitro synergistically enhanced cell death in GAGA6-R but not GAGA6 cells. Furthermore, midostaurin, a multikinase inhibitor with PKC-inhibiting activity, in part reversed resistance of GAGA6-R tumor to AZD4547 in vivo Our results suggest that upon challenge with FGFR inhibitors, FGFR2-amplified tumors that are highly dependent on FGFR2 signaling for survival rapidly develop resistance by switching to a PKC-mediated inhibition of GSK3ß to gain a survival advantage. Mol Cancer Ther; 17(1); 232-42. ©2017 AACR.

CITED2 Modulates Breast Cancer Metastatic Ability through Effects on IKKα.

UNLABELLED: Previously, we identified the transcriptional coactivator CITED2 as a potential facilitator of bone metastasis using a murine mammary cancer model. Extending these studies to human breast cancer, it was observed that CITED2 mRNA expression was significantly elevated in patient specimens of metastatic breast cancer relative to primary tumors, with highest levels in metastasis to bone relative to non-bone sites. To further evaluate CITED2 functions in breast cancer metastasis, CITED2 expression was stably reduced in the human breast cancer cell lines MDA-MB-231 and MDA-MB-468, which are metastatic in animal models. While CITED2 knockdown had no effect on cell proliferation, cell migration and invasion were significantly reduced, as was the establishment of metastasis following intracardiac administration in athymic nude mice. To explore the mechanism behind these effects, gene expression following CITED2 knockdown in MDA-MB-231 cells by cDNA microarray was performed. As confirmed at the mRNA and protein levels in both MDA-MB-231 and MDA-MB-468 cells, expression of the NF-κB regulator IKKα was significantly reduced, along with several NF-κB targets with known roles in metastasis (OPN, MMP9, uPA, SPARC, IL11, and IL1ß). Furthermore, ChIP assay revealed recruitment of CITED2 to the promoter of IKKα, indicating a direct role in regulating its expression. Consistent with reduced IKKα expression, CITED2 knockdown inhibited both canonical and noncanonical NF-κB signaling. Finally, restoration of IKKα expression following CITED2 knockdown in MDA-MB-231 and MDA-MB-468 cells rescued their invasive ability. Collectively, these data demonstrate that CITED2 modulates metastatic ability in human breast cancer cells, at least in part, through the regulation of IKKα. IMPLICATIONS: The current study highlights the role of CITED2 in facilitating breast cancer metastasis, partly via regulation of IKKα. Mol Cancer Res; 14(8); 730-9. ©2016 AACR.

A role for RUNX3 in inflammation-induced expression of IL23A in gastric epithelial cells.

RUNX3 functions as a tumor suppressor in the gastric epithelium, where its inactivation is frequently observed during carcinogenesis. We identified IL23A as a RUNX3 target gene in gastric epithelial cells. This was confirmed in a series of in vitro analyses in gastric epithelial cell lines. In elucidating the underlying regulatory network, we uncovered a prominent role for the TNF-α/NF-κB pathway in activating IL23A transcription. Moreover, the activating effect of TNF-α was markedly augmented by the infection of Helicobacter pylori, the primary cause of human gastritis. Of note, H. pylori utilized the CagA/SHP2 pathway to activate IL23A, as well as the induction of the NOD1 pathway by iE-DAP. Importantly, RUNX3 synergized strongly with these physiologically relevant stimuli to induce IL23A. Lastly, we present evidence for the secretion of IL23A by gastric epithelial cells in a form that is distinct from canonical IL-23 (IL23A/IL12B).

CD44v8-10 is a cancer-specific marker for gastric cancer stem cells.

The surface marker CD44 has been identified as one of several markers associated with cancer stem cells (CSC) in solid tumors, but its ubiquitous expression in many cell types, including hematopoietic cells, has hindered its use in targeting CSCs. In this study, 28 paired primary tumor and adjacent nontumor gastric tissue samples were analyzed for cell surface protein expression. Cells that expressed pan-CD44 were found to occur at significantly higher frequency in gastric tumor tissues. We identified CD44v8-10 as the predominant CD44 variant expressed in gastric cancer cells and verified its role as a gastric CSC marker by limiting dilution and serial transplantation assays. Parallel experiments using CD133 failed to enrich for gastric CSCs. Analyses of another 26 primary samples showed significant CD44v8-10 upregulation in gastric tumor sites. Exogenous expression of CD44v8-10 but not CD44 standard (CD44s) increased the frequency of tumor initiation in immunocompromised mice. Reciprocal silencing of total CD44 resulted in reduced tumor-initiating potential of gastric cancer cells that could be rescued by CD44v8-10 but not CD44s expression. Our findings provide important functional evidence that CD44v8-10 marks human gastric CSCs and contributes to tumor initiation, possibly through enhancing oxidative stress defense. In addition, we showed that CD44v8-10 expression is low in normal tissues. Because CD44 also marks CSCs of numerous human cancers, many of which may also overexpress CD44v8-10, CD44v8-10 may provide an avenue to target CSCs in other human cancers.

Enpp1: a potential facilitator of breast cancer bone metastasis.

Bone is the most common site of breast cancer metastasis and once established, it is frequently incurable. Critical to our ability to prevent and treat bone metastasis is the identification of the key factors mediating its establishment and understanding their biological function. To address this issue we previously carried out an in vivo selection process to isolate murine mammary tumor sublines possessing an enhanced ability to colonize the bone. A comparison of gene expression between parental cells and sublines by genome-wide cDNA microarray analysis revealed several potential mediators of bone metastasis, including the pyrophosphate-generating ectoenzyme Enpp1. By qRT-PCR and Western analysis we found that expression of Enpp1 was elevated in human breast cancer cell lines known to produce bone metastasis in animal models compared to non-metastatic and normal mammary epithelial cell lines. Further, in clinical specimens, levels of Enpp1 were significantly elevated in human primary breast tumors relative to normal mammary epithelium, with highest levels observed in breast-bone metastasis as determined by qRT-PCR and immunohistochemical analysis. To examine the potential role of Enpp1 in the development of bone metastasis, Enpp1 expression was stably increased in the breast cancer cell line MDA-MB-231 and the ability to colonize the bone following intracardiac and direct intratibial injection of athymic nude mice was determined. By both routes of administration, increased expression of Enpp1 enhanced the ability of MDA-MB-231 cells to form tumors in the bone relative to cells expressing vector alone, as determined by digital radiography and histological analysis. Taken together, these data suggest a potential role for Enpp1 in the development of breast cancer bone metastasis.

In vitro and in vivo downregulation of MRP1 by antisense oligonucleotides: a potential role in neuroblastoma therapy.

The expression of MRP1 (multidrug resistance protein-1) is associated with chemoresistance and poor prognosis in neuroblastoma. MRP1 antisense oligonucleotides were used in an in vivo mouse-human xenograft model of neuroblastoma to downregulate MRP1. The MRP1 ASO reduced protein levels of MRP1 to an average of 40% of the nil treated controls (p = 0.007). There was significant chemosensitisation to single-agent chemotherapy, VP16 (etoposide), at 1 microg/mL (p = 0.035) and 10 microg/mL (p = 0.02) in comparison to tumours not receiving oligonucleotides. In contrast, MDR1-ASO produced significant chemosensitisation only at 10 microg/mL of VP16 (p = 0.029). No significant chemosensitisation was seen following nonsense oligonucleotides. The downregulation of MRP1 was also associated with an increase in tumour cell death (79% increase in apoptosis index p = 0.0313) and a reduction in cell turnover (42% reduction in mitotic index p = 0.0313), which was not seen with any other oligonucleotide. This new and novel perspective of MRP1 function, which is an apparent involvement in apoptosis and cell cycle progression in neuroblasts, presents a fresh avenue for investigation of the biologic consequences of MRP1 expression that occurs in many tumour cell types. Our work is the first to concurrently explore the effects of downregulation of MRP1 and MDR1 by antisense oligonucleotides in a neuroblastoma xenograft model. It provides rationale for the investigation of therapy adjuvants such as antisense oligonucleotides in the treatment of this malignancy.

Identification and characterization of genes involved in the carcinogenesis of human squamous cell cervical carcinoma.

We utilized RT-PCR differential display and cDNA microarrays to identify cellular genes involved in the multi-step carcinogenesis of squamous cell cervical carcinoma. Thirty-eight cervical cancer patients in various stages of the disease and 5 non-cervical cancer patients were studied. Twenty-five cDNA clones were identified and these were subsequently demonstrated to be consistently over-expressed in squamous cell cervical carcinoma biopsies of various FIGO stages. To further evaluate the possible role that these genes may play in the progression of disease, we performed Northern blot analysis and RNA-RNA in situ hybridization studies using cervical cancer biopsies of various FIGO stages. Of particular interest are the 2 clones G32C4B and G30CC that have been identified to be the NADH dehydrogenase 4 gene and the gene that encodes ribosomal protein S12 respectively when compared to sequences available in the GenBank database. Increased expression of these 2 genes were detected in the matched normal tissues collected together with the late FIGO stages of cervical cancer biopsies. In comparison, upregulation of these 2 genes was not detected in cervical squamous epithelium collected from patients admitted for surgery for non-malignant conditions, suggesting that expression of these 2 genes may have altered in the adjacent histopathologically "normal" cervical squamous epithelial tissue from cervical cancer patients. The ribosomal protein S12 and the NADH dehydrogenase 4 genes may therefore be potentially useful as early pre-transformation diagnostic markers for human cervical cancer.