Aliccia Bollig

Overexpression of cyclin D1 promotes tumor cell growth and confers resistance to cisplatin-mediated apoptosis in an elastase-myc transgene-expressing pancreatic tumor cell line

Purpose: Elevated cyclin D1 in human pancreatic cancer correlates with poor prognosis. Because pancreatic cancer is invariably resistant to chemotherapy, the goal of this study was to examine whether the drug resistance of pancreatic cancer cells is in part attributed to cyclin D1 overexpression. Experimental Design: Stable overexpression and small interfering RNA (siRNA)–mediated knockdown of cyclin D1 were done in the newly established Ela-myc pancreatic tumor cell line. Cisplatin sensitivity of control, overexpressing, and siRNA-transfected cells was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, clonogenic, and apoptotic assays [DNA fragmentation, sub-G1, and poly(ADP-ribose) polymerase cleavage analysis]. The role of nuclear factor-κB and apoptotic proteins in cyclin D1-mediated chemoresistance was examined by EMSA and Western blotting, respectively. Results: Overexpression of cyclin D1 in Ela-myc pancreatic tumor cells promoted cell proliferation and anchorage-independent growth. Moreover, cyclin D1–overexpressing cells exhibited significantly reduced chemosensitivity and a higher survival rate upon cisplatin treatment, as determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and clonogenic assays, respectively. Although overexpression of cyclin D1 rendered cells more resistant to cisplatin-induced apoptosis, siRNA-directed suppression of cyclin D1 expression resulted in enhanced susceptibility to cisplatin-mediated apoptosis. The attenuation of cisplatin-induced cell death in cyclin D1–overexpressing cells was correlated with the up-regulation of nuclear factor-κB activity and maintenance of bcl-2 and bcl-xl protein levels. Conclusions: These results suggest that overexpression of cyclin D1 can contribute to chemoresistance of pancreatic cancer cells because of the dual roles of cyclin D1 in promoting cell proliferation and in inhibiting drug-induced apoptosis.

Expression profile of microRNAs in c-Myc induced mouse mammary tumors

Abstractc-Myc is a transcription factor overexpression of which induces mammary cancer in transgenic mice. To explore whether certain microRNAs (mirRNA) mediate c-Myc induced mammary carcinogenesis, we studied mirRNA expression profile in mammary tumors developed from MMTV-c-myc transgenic mice, and found 50 and 59 mirRNAs showing increased and decreased expression, respectively, compared with lactating mammary glands of wild type mice. Twenty-four of these mirRNAs could be grouped into eight clusters because they had the same chromosomal localizations and might be processed from the same primary RNA transcripts. The increased expression of mir-20a, mir-20b, and mir-9 as well as decreased expression of mir-222 were verified by RT-PCR, real-time RT-PCR, and cDNA sequencing. Moreover, we fortuitously identified a novel non-coding RNA, the level of which was decreased in proliferating mammary glands of MMTV-c-myc mice was further decreased to undetectable level in the mammary tumors. Sequencing of this novel RNA revealed that it was transcribed from a region of mouse chromosome 19 that harbored the metastasis associated lung adenocarcinoma transcript-1 (Malat-1), a non-protein-coding gene. These results suggest that certain mirRNAs and the chromosome 19 derived non-coding RNAs may mediate c-myc induced mammary carcinogenesis.

Aberrant expression of X-linked genes RbAp46, Rsk4, and Cldn2 in breast cancer.

The consequence of activation status or gain/loss of an X-chromosome in terms of the expression of tumor suppressor genes or oncogenes in breast cancer has not been clearly addressed. In this study, we investigated the activation status of the X-chromosomes in a panel of human breast cancer cell lines, human breast carcinoma, and adjacent mammary tissues and a panel of murine mammary epithelial sublines ranging from low to high invasive potentials. Results show that most human breast cancer cell lines were homozygous, but both benign cell lines were heterozygous for highly polymorphic X-loci (IDS and G6PD). On the other hand, 60% of human breast carcinoma cases were heterozygous for either IDS or G6PD markers. Investigation of the activation status of heterozygous cell lines revealed the presence of only one active X-chromosome, whereas most heterozygous human breast carcinoma cases had two active X-chromosomes. Furthermore, we determined whether or not an additional active X-chromosome affects expression levels of tumor suppressor genes and oncogenes. Reverse transcription-PCR data show high expression of putative tumor suppressor genes Rsk4 and RbAp46 in 47% and 79% of breast carcinoma cases, respectively, whereas Cldn2 was down-regulated in 52% of breast cancer cases compared with normal adjacent tissues. Consistent with mRNA expression, immunostaining for these proteins also showed a similar pattern. In conclusion, our data suggest that high expression of RbAp46 is likely to have a role in the development or progression of human breast cancer. The activation status of the X-chromosome may influence the expression levels of X-linked oncogenes or tumor suppressor genes. (Mol Cancer Res 2007;5(2):171–81)

c-Myc-induced chemosensitization is mediated by suppression of cyclin D1 expression and nuclear factor-κB activity in Pancreatic cancer cells

Purpose: Pancreatic cancer is a highly aggressive disease that remains refractory to various chemotherapeutic agents. Because the proto-oncogene c-myc can modulate apoptosis in response to cytotoxic insults and is commonly overexpressed in pancreatic cancer, we investigated the value of c-myc as a potential modulator of cellular response to various chemotherapeutic agents. Experimental Design: Stable overexpression or small interfering RNA (siRNA)–mediated knockdown of c-myc and restoration of cyclin D1 were done in the Ela-myc pancreatic tumor cell line. Cell viability after cisplatin treatment of c-myc–overexpressing, control, and siRNA-transfected cells was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and drug-induced apoptosis was measured by DNA fragmentation, sub-G1, and poly(ADP-ribose) polymerase cleavage analyses. Protein expression profile after cisplatin treatment was determined by Western blotting and DNA binding activity of nuclear factor-κB was examined by electrophoretic mobility shift assay. Results: Ectopic overexpression of c-myc in murine and human pancreatic cancer cell lines, Ela-myc and L3.6pl, respectively, resulted in increased sensitivity to cisplatin and other chemotherapeutic drugs. Increased sensitivity to cisplatin in c-myc–overexpressing cells was due, in part, to the marked increase in cisplatin-induced apoptosis. Conversely, down-regulation of c-myc expression in stable c-myc–overexpressing cells by c-myc siRNA resulted in decreased sensitivity to cisplatin-induced cell death. These results indicate an important role of c-myc in chemosensitivity of pancreatic cancer cells. The c-myc–induced cisplatin sensitivity correlated with inhibition of nuclear factor κB activity, which was partially restored by ectopic cyclin D1 overexpression. Conclusions: Our results suggest that the c-myc–dependent sensitization to chemotherapy-induced apoptosis involves suppression of cyclin D1 expression and nuclear factor κB activity.

Anti-invasive and antimetastatic activities of ribosomal protein S6 kinase 4 in breast cancer cells

Purpose: We have previously shown that p90 ribosomal protein S6 kinase 4 (RSK4), an X-linked gene, is highly up-regulated in mammary tumors of MMTV-c-Myc transgenic mice. In this study, we further investigated whether RSK4 inhibits or promotes breast tumor growth and progression. Experimental Design: Stable overexpression or small interfering RNA–mediated knockdown of RSK4 was done in the MDA-MB-231 cell line. Stable clones were tested for cell proliferation, anchorage-independent growth in soft agar, invasive and metastatic ability of these clones in vitro and tumorigenesis, invasive and metastatic ability in vivo in severe combined immunodeficient mice. Results: Here, we show that exogenous expression of RSK4 resulted in decreased cell proliferation and increased accumulation of cells in G0-G1 phase, which paralleled with enhanced expression of tumor suppressor genes: retinoblastoma protein, retinobl astoma-associated 46 kDa protein, and p21 protein. Overexpression of RSK4 resulted in reduced colony formation in soft agar and suppressed invasive and migratory activities of MDA-MB-231 cells both in vitro and in vivo. Importantly, RSK4-overexpressing cells showed up-regulation of claudin-2 and down-regulation of CXCR4, both of these play roles in invasion and chemotaxis. Conclusions: These results indicate that RSK4 expression may limit the oncogenic, invasive, and metastatic potential of breast cancer cells. Anti-invasive and antimetastatic activities of RSK4 may be, in part, due to its regulation of claudin-2. Increased expression of RSK4 in c-Myc-overexpressing cells and a dose-dependent induction of luciferase reporter gene activity suggest that c-Myc may regulate RSK4 expression.

Synergistic Effect of Cyclin D1 and c-Myc Leads to More Aggressive and Invasive Mammary Tumors in Severe Combined Immunodeficient Mice

Cyclin D1 is one of the most commonly overexpressed oncogenes in breast cancer; yet, it is not clear whether cyclin D1 alone is capable of causing malignant transformation of mammary epithelial cells. Here, we show that ectopic expression of cyclin D1 in benign mouse mammary epithelial cells promotes cell proliferation, anchorage-independent growth in soft agar, and tumorigenesis in severe combined immunodeficient mice. To address the possible interaction of cyclin D1 and c-myc in malignant transformation, we used cyclin D1/c-myc dual-expressing clones, which displayed more aggressive and invasive phenotype than cyclin D1-expressing clones. These data provide evidence that overexpression of cyclin D1 or coexpression with c-myc could cause invasive malignant transformation of benign mouse mammary epithelial cells. Furthermore, microarray analysis of cyclin D1 and cyclin D1/c-myc clones showed that these two tumor-producing clones might use distinct invasive pathways. In summary, overexpression of cyclin D1 may commit mammary epithelia to a tumor-prone phenotype in which cooperation with other genes, such as synergy with c-myc, may lead to a more aggressive phenotype.

Gene expression profiles in primary pancreatic tumors and metastatic lesions of Ela-c-myc transgenic mice

BackgroundPancreatic carcinoma usually is a fatal disease with no cure, mainly due to its invasion and metastasis prior to diagnosis. We analyzed the gene expression profiles of paired primary pancreatic tumors and metastatic lesions from Ela-c-myc transgenic mice in order to identify genes that may be involved in the pancreatic cancer progression. Differentially expressed selected genes were verified by semi-quantitative and quantitative RT-PCR. To further evaluate the relevance of some of the selected differentially expressed genes, we investigated their expression pattern in human pancreatic cancer cell lines with high and low metastatic potentials.ResultsData indicate that genes involved in posttranscriptional regulation were a major functional category of upregulated genes in both primary pancreatic tumors (PT) and liver metastatic lesions (LM) compared to normal pancreas (NP). In particular, differential expression for splicing factors, RNA binding/pre-mRNA processing factors and spliceosome related genes were observed, indicating that RNA processing and editing related events may play critical roles in pancreatic tumor development and progression. High expression of insulin growth factor binding protein-1 (Igfbp1) and Serine proteinase inhibitor A1 (Serpina1), and low levels or absence of Wt1 gene expression were exclusive to liver metastatic lesion samples.ConclusionWe identified Igfbp1, Serpina1 and Wt1 genes that are likely to be clinically useful biomarkers for prognostic or therapeutic purposes in metastatic pancreatic cancer, particularly in pancreatic cancer where c-Myc is overexpressed.

The role of X-linked genes in breast cancer.

While contribution of X chromosome in the susceptibility of prostate and ovarian cancer has been demonstrated, the role of X-linked genes in breast cancinogenesis is not clearly known. This study investigated and compared the X-linked gene expression profiles of MMTV-c-myc transgenic mammary tumor (MT) or MMTV-c-myc/MT-tgf-α double transgenic mouse mammary tumor (DT) to lactating mammary gland. cDNA microarray analysis using the Affymetrix system identified 1081 genes localized on the X chromosome with 174 and 194 genes at ±2-fold change levels in MT and DT samples, respectively. Differentially expressed X-linked genes were predominantly related to chromatin structure/remodeling (e.g., Hdac8, Suv39h1, RbAp46 and Adr1), segregation (e.g., CENP-I and smc111) and, ribosomal biogenesis and translational control (e.g., Dkc1, Rpl44, Rpl39, Eif2s3x, Gspt2 and Rsk4). Confirmation of microarray data by semi-quantitative and quantitative RT-PCR in selected X-linked genes also showed similar pattern. In addition, the expression pattern of two chromosomal regions, XE3 and XF5, suggests that XE3 may have escaped from inactivation and XF5 subjected to inactivation. In conclusion, our data suggest that X-linked genes may play the key regulatory roles in the maintenance of chromatin structure, accurate chromosomal segregation and translational control; hence deregulation of X-linked genes may promote mammary gland tumorigenesis by promoting genetic instability and cell proliferation. Increased understanding of the role of X-linked genes and genetic pathways will provide the strategies to develop the molecular therapeutics to treat and prevent reproductive related cancers.

Identification of the cyclin D1b mRNA variant in mouse

Cyclin D1 plays a key regulatory role during the G1 phase of the cell cycle and its gene is amplified and over-expressed in many cancers. The cyclin D1b mRNA variant was established in human cells and recent functional analyses revealed that its protein product harbors unique activities in human cancer cells. By performing reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) experiments, we identified the cyclin D1b mRNA variant in mouse. Similar to its human counterpart, the mouse cyclin D1b transcript consists of exon 1, 2, 3, 4 and part of intron 4, and contains a long open reading frame (ORF). The predicted peptide from this ORF is 34-amino acid longer than the human cyclin D1b. The expression of this mouse mRNA variant was investigated. It appears to be expressed ubiquitously and differentially in various mouse cell lines and tissues and its level might be proportional to that of the canonical endogenous cyclin D1a mRNA.

HER-2 over expression mediates the transition to aerobic glycolysis and stress/REDOX signaling in progressively transformed human mammary epithelial cells.

Abstract #6032 The goal of this study was to use a computational strategy to identify HER-2 regulated-gene expression networks in an isogenic series of cell lines in which HER-2 functions as a proto-oncogene, as an oncogene driving partial transformation, and as an oncogene driving full malignant transformation. The cells used for these experiments include non-transformed, immortalized MCF10A cells grown in media containing EGF and insulin; MCF10HER2 cells that over express HER-2 and are insulin-independent for growth; and MCF10HER2/E7 cells (also E7 transduced), which are insulin and EGF-independent for growth and fully malignant in vivo. We obtained whole-genome expression data every 3 hours for 45 hours while HER-2 signaling and cell proliferation were blocked by treatment with specific small molecule kinase inhibitors. A dynamic linear finite difference model was used to derive phenomenological gene expression networks from the time-series gene expression data. In these networks each gene is rank-ordered by its degree of connectivity, and hub genes are identified as those with the highest connectivity level. We then compared the hub genes regulated by HER-2 in each cell line, and used Ingenuity Pathway Analysis to identify the biological networks differentially regulated by HER-2. Results show that both normal and transformed gene expression networks have scale-free topology. Using the same connectivity value to define hubs, we found that HER-2 regulated the expression of more hub genes in transformed cells. These findings suggested that the HER-2 signal was integrated differently in progressively transformed cells and led us to map functional association networks for each cell-specific list of hubs. In MCF-10HER-2 cells, which are partially transformed and insulin-independent for growth, two hubs differentially regulated by HER-2 include VAMP8 and PHGDH. These genes influence cell surface expression of glucose transporters, and activate anabolic pathways from glycolysis intermediates, respectively. Thus, HER-2-mediated insulin independence is associated with constitutive glucose transport and the transition to aerobic glycolysis. HER-2/E7 cells are fully transformed, and in these cells, differentially regulated hub genes such as HSP1A1 and GPX4 are part of the coordinated regulation of the stress-protective heat shock response and enzymatic pathways of oxidative defense. In concert with these modular changes, the CITED2 hub gene, a negative regulator of HIF1α, is specifically down-regulated by HER-2 signaling in the MCF10A/HER2 and MCF10A/HER2/E7 cell lines, which results in enhanced HIF1α expression and transcriptional activity. In summary, our systems biology approach resulted in the identification of distinct gene expression and functional association networks that arise when HER-2 transitions from being a proto-oncogene to a fully transforming gene. These results provide evidence regarding the mechanisms by which HER-2 acts as a transforming gene when over expressed. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 6032.