High CCND1 Expression Research Articles

Abstract P6-12-06: Neratinib vs. Trastuzumab plus Ribociclib in ERBB2-positive breast cancer: Preclinical mechanistic efficacy study

Abstract Background: Despite the wide-ranging clinical success of human epidermal growth factor receptor 2 (HER2)-directed therapies, many HER2-positive breast cancer patients eventually progress because of the development of primary or acquired resistance. PIK3CA is found often mutated in breast cancer (>30%, TCGA dataset) and responsible for HER2-directed treatment failure. Purpose: Inhibition of pan-tyrosine kinases of HER-receptors along with the blockade of estrogen receptor (ER) prevents the activation of downstream effectors and crosstalk between HER2 and ER, leading to tumor cell death. Similarly, the combined inhibition of HER2-receptor signaling with cell cycle arrest leads to efficient tumor cell apoptosis. Here, we evaluate the mechanistic efficacy and comparability of neratinib (N) (an irreversible pan-ERBB tyrosine kinase inhibitor) in combination with fulvestrant (F) against ribociclib (R) plus trastuzumab (T) in HER2-amplified breast cancer cells. Methods: HER2+ breast cancer cell lines with Rb- wild-type- BT474 (ER+), SKBR3 (ER-), and MDA-MB453 (ER-, PIK3CA mutated [H1047R]) were used for the study. Cells were treated with N+F (F added in ER+ cell line only) or R or T as a single-agent or combination and assessed for real-time proliferation, 3D ON-TOP assay, changes in mitochondrial potential, and apoptosis. Cells treated with R and/or T were additionally examined for cell cycle arrest and changes in CDK4 mRNA transcription. Baseline CCND1 mRNA transcripts were quantified by RT-qPCR. Immunohistochemistry was used to assess baseline BCL2 expression in HER2+ tumor microarrays (TMA). Western blot was used to evaluate the effects on key downstream signaling proteins in response to the above treatment or combination. Results: High CCND1 expression was found in HER2+ cell lines that show promise for CDK4/6 inhibition. High BCL2 expression was found in HER2+ TMA that confirmed the natural resistance to programmed cell death. BT474 and MDA-MB453 were highly sensitive to N (+F), and high growth inhibition was evident at lower doses (< 20 nM). SKBR3 was comparatively less sensitive to N monotherapy and required dosing >160 nM to induce marked cell death. BT474 and MDA-MB453 had pronounced cytostatic responses to R or T+R, while a moderate response was observed in SKBR3. Substantial reduction in CDK4 transcription was noted following R or R+T treatment in MDA-MB453/BT474 but not in SKBR3. Apoptosis assays confirmed enhanced cell death with the R+T combination in BT474 and MDA-MB453 but not in SKBR3. Inhibition of key downstream oncogenic signaling (p-AKT/p-S6RP/p-ERK) was more evident with N compared to R, T, or R+T combination. Attenuated p-Rb expression (Ser 780 & Ser 807/811) was detected in all cell lines from R administration, indicating interruption in cell cycle progression. Conclusion: Mechanistically, N+F was superior to T+R in terms of inhibition of HER2 and its downstream signaling in the HER2+/ER+ BC model. N+F induced significantly more apoptosis and inhibited cell proliferation compared to T+R. Additionally, N monotherapy was highly effective in HER2-amplified/ER-negative breast cancer cells with PIK3CA mutation. Citation Format: Nischal Koirala, Jennifer Aske, Xiaoqian Lin, Nandini Dey, Pradip De. Neratinib vs. Trastuzumab plus Ribociclib in ERBB2-positive breast cancer: Preclinical mechanistic efficacy study [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P6-12-06.

Cyclin D1 mediated by the nuclear translocation of nuclear factor kappa B exerts an oncogenic role in lung cancer

ABSTRACT The relevance of cyclin D1 (CCND1) has been implicated in lung cancer progression. Nevertheless, the mechanism by which CCND1 supports lung cancer development is yet to be expounded. Here, we established that CCND1 is overexpressed in clinical lung cancer specimens and various lung cancer cells. Importantly, CCND1 overexpression enhanced lung cancer cell proliferation, invasion and migration, and arrested the cell cycle at the S phase. In vivo, overexpression of CCND1 promoted lung cancer growth and metastasis. The nuclear translocation of nuclear factor kappa B (NF-κB) promoted p65 protein expression and CCND1 transcription. Meanwhile, PI3K/AKT pathway activity was significantly reduced when NF-κB nuclear translocation was decreased. PI3K/AKT pathway activity was significantly elevated upon CCND1 overexpression. Inhibition of PI3K/AKT pathway activity or suppression of NF-κB translocation in cells with high CCND1 expression was found to significantly reduce the activity of lung cancer cells in vitro and in vivo. Our data revealed that NF-κB/CCND1/PI3K/AKT axis could act as a prospective diagnostic biomarker and a therapeutic option for lung cancer.

Expression of Cyclin D1 gene in ovarian cancer and effect of silencing its expression on ovarian cancer cells based on the Oncomine database

ABSTRACT We aimed to analyze the expression of Cyclin D1 (CCND1) gene in ovarian cancer and the influence of silencing its expression on ovarian cancer cells based on the Oncomine database. The expression of CCND1 gene in ovarian cancer was analyzed by utilizing the relevant information in different tumors and Oncomine database. The correlation between CCDN1 expression level and prognosis of ovarian cancer was analyzed by the online database Kaplan-Meier (kmplot.com). The expression of CCND1 gene in ovarian cancer and the effect of silencing its expression on cancer cells were analyzed by cell experiments. After mining and comprehensively analyzing 7 studies on the differential expression of CCND1 gene in ovarian cancer tissue and normal ovarian tissue included in the Oncomine database, it was found that the median value of CCND1 gene ranked 218.0 (P = 8.03 × 10−6) among all differentially expressed genes, suggesting that CCND1 gene expression in ovarian cancer tissue was higher than that in normal ovarian tissue. Adib Ovarian, Bonome Ovarian and Hendrix Ovarian microarrays revealed that the expression of CCND1 gene in ovarian cancer tissue was significantly higher than that in normal ovarian tissue (P < 0.05). Kaplan-Meier Plotter database showed that the overall survival and progression-free survival of ovarian cancer patients with high CCND1 expression were significantly shorter than those of patients with low CCND1 expression (P < 0.05). The expression levels of CCND1 gene in normal ovarian epithelial cells and SKOV3 ovarian cancer cells were detected by RT-PCR. The expression of CCND1 gene was significantly higher in SKOV3 group than that in control group (P < 0.01). Flow cytometry revealed that the percentage of cells in G0/G1 phase was significantly higher, while that in S phase was lower in SKOV3 + siCCND1 group than the values of SKOV3 and SKOV3 + siNC groups (P < 0.05). The apoptosis rate of ovarian cancer cells was significantly higher in SKOV3 + siCCND1 group than those of SKOV3 and SKOV3 + siNC groups (P < 0.01). CCND1 gene is highly expressed in ovarian cancer tissue and related to prognosis. Preoperative evaluation of CCND1 gene expression in ovarian cancer patients may benefit the assessment of risk and prognosis.

MLL-AF9 initiates transformation from fast-proliferating myeloid progenitors

Cancer is a hyper-proliferative disease. Whether the proliferative state originates from the cell-of-origin or emerges later remains difficult to resolve. By tracking de novo transformation from normal hematopoietic progenitors expressing an acute myeloid leukemia (AML) oncogene MLL-AF9, we reveal that the cell cycle rate heterogeneity among granulocyte–macrophage progenitors (GMPs) determines their probability of transformation. A fast cell cycle intrinsic to these progenitors provide permissiveness for transformation, with the fastest cycling 3% GMPs acquiring malignancy with near certainty. Molecularly, we propose that MLL-AF9 preserves gene expression of the cellular states in which it is expressed. As such, when expressed in the naturally-existing, rapidly-cycling immature myeloid progenitors, this cell state becomes perpetuated, yielding malignancy. In humans, high CCND1 expression predicts worse prognosis for MLL fusion AMLs. Our work elucidates one of the earliest steps toward malignancy and suggests that modifying the cycling state of the cell-of-origin could be a preventative approach against malignancy.

Selective Targeting BET Family Bdii Bromodomain with Abbv-744 and BCL-2 with Venetoclax (ABT-199) Is Synergistic in Primary Acute Myeloid Leukemia Models

Despite advances in understanding of the biology of acute myeloid leukemia (AML), cure remains elusive for the majority of patients. ABT-199 (Venetoclax) is a small-molecule BH3 mimetic that selectively inhibits BCL-2 causing cell death. First generation BET inhibitor ABBV-075 and Venetoclax were recently shown to be synergistic in AML cell lines (Bui MH,Cancer Res 2017). ABBV-744 is a highly selective inhibitor for the BDII of BET family proteins, exhibiting greater than 300-fold more potent binding affinity to the BDII bromodomain of BRD4 relative to BDI (Warren Kati AACR 2018; Xiaoyu Lin AACR 2018). In this study, we evaluated the anti-leukemia efficacy of the concomitant BCL-2 blockade by venetoclax and of BDII inhibition with ABBV-744 in primary AML samples. Anti-leukemia activity of venetoclax and ABBV-744 was examined in 21 primary AML samples with diverse genomic alterations. The combination significantly enhanced cell death (57.0 ± 6.3%) compared to the single agent treatment (43.9 ± 5.7% in ABT-199 10 nM group, p&amp;lt;0.001 and 23.8 ± 2.9% in ABBV-744 20nM group, p&amp;lt;0.001, Fig.1A). ABBV-744 reduced viable cell numbers in the majority of AML cases (31.7 ± 5.2%) and the cell growth suppression was more profound in the combination group (77.2 ± 6.3 %, p&amp;lt;0.001, Fig.1B). In two AML primary samples tested, combination treatment of ABBV-744 and ABT-199 induced apoptosis with caspase-3 activation and PARP cleavage through regulation of the key proteins regulating survival and proliferation pathways (e.g. (BCL-2, BCL-XL, MCL-1, c-Myc)). To identify biomarkers of response to therapy, we performed the baseline transcriptome analysis of AML cells used for in vitro response assessment (n=25) by RNA-sequencing (RNA-seq), and correlated baseline gene expression levels with in vitro response to therapy. Based on response to venetoclax or combination, samples were divided into 3 groups: sensitive to venetoclax (n=10), samples with no response to single agent or combination ("low apoptosis", n=7) and samples resistant to venetoclax as a single agent but responsive to venetoclax/ ABBV-744 combination ("synergy", n=4). AML samples sensitive to venetoclax and venetoclax/ABBV-744 combination were characterized by high level of BCL2 and lower levels of MCL1 and BCL2L1 transcripts, consistent with known inability of venetoclax to inhibit MCL-1 and BCL2L1 (Fig.1C).The resistant samples additionally expressed higher levels of anti-apoptotic genes such as GADD45, BCL2L10, PMAIP1. AML cells that showed synergy between venetoclax/ABBV-744 expressed low levels of AR, IL1R1 genes and had high CCND1 expression. The gene expression analysis indicated that the genes differentially expressed in the synergy vs. low apoptosis samples overlap with the genes inhibited by dual BCL-2/BCL-XL inhibitor ABT-737. To test the efficacy of this regimen in vivo, we established a patient-derived xenograft (PDX) from an AML patient with FLT3-ITD, DNMT3A, EGFR, IDH1, NPM1, TET2 mutations in NSG mice. Upon engraftment, mice were randomized to receive vehicle; single agent venetoclax at 50 mg/kg; ABBV-744 at 9.4 mg/kg; or venetoclax plus ABBV-744 for 21 days. After 21 days of therapy, flow cytometry data demonstrated significantly reduced leukemia burden in venetoclax treated group (9.5% ± 1.7%) but not in ABBV-744 group (22.3% ± 5.8%) compared to controls (30.8% ± 3.9%), with lowest tumor burden in the combination group (5.0% ± 0.8%, p&amp;lt;0.01) (Fig. 1E). Combination of ABBV-744 and venetoclax treatment delayed AML progression and extended the survival compared to the untreated mice (median survival, 193 days vs 99 days, p&amp;lt;0.001) (Fig. 1D). No significant impact on mice' weight was noted, and no clinical signs of toxicity recorded over the course of therapy. In summary, combinatorial blockade of BDII bromodomain and of BCL-2 anti-apoptotic pathway facilitates apoptotic cell death, suppresses proliferation in the majority of primary AML cells and produces anti-AML activity in AML PDX models in vivo at tolerable doses of both agents. This combination is currently undergoing testing in a Phase I clinical trial in AML (NCT03360006). Disclosures Kuruvilla: The University of Texas M.D.Anderson Cancer Center: Employment. Lin:AbbVie: Employment. Uziel:AbbVie: Employment, Other: stock or other options. Lu:AbbVie: Employment. Zhang:AbbVie: Employment. Huang:AbbVie: Employment. Zhang:The University of Texas M.D.Anderson Cancer Center: Employment. Shen:AbbVie: Employment. Konopleva:Astra Zeneca: Research Funding; Ablynx: Research Funding; Eli Lilly: Research Funding; Kisoji: Consultancy, Honoraria; Ascentage: Research Funding; Agios: Research Funding; Reata Pharmaceuticals: Equity Ownership, Patents &amp; Royalties; Amgen: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria, Research Funding; Cellectis: Research Funding; Genentech: Honoraria, Research Funding; Stemline Therapeutics: Consultancy, Honoraria, Research Funding; Calithera: Research Funding; F. Hoffman La-Roche: Consultancy, Honoraria, Research Funding; Forty-Seven: Consultancy, Honoraria.

Abstract 3831: Selective targeting BET family BDII bromodomain with ABBV-744 and BCL-2 with venetoclax (ABT-199) is synergistic in primary acute myeloid leukemia models

Abstract Despite advances in understanding of the biology of acute myeloid leukemia (AML), cure remains elusive for the majority of patients. ABT-199 (Venetoclax) is a small-molecule BH3 mimetic that selectively inhibits BCL-2 causing cell death. ABBV-744 is a highly selective inhibitor for the BDII of BET family proteins, exhibiting greater than 300-fold more potent binding affinity to the BDII bromodomain of BRD4 relative to BDI (Warren Kati AACR 2018; Xiaoyu Lin AACR 2018). In this study, we evaluated the anti-leukemia efficacy of the concomitant BCL-2 blockade by venetoclax and of BDII inhibition with ABBV-744 in primary AML samples. First, anti-leukemia activity of venetoclax and ABBV-744 was examined in 12 primary AML samples with diverse genomic alterations. The combination significantly enhanced cell death (61.4% ± 8.7%), as compared to the single agent treatment (51.4% ± 9.3% in venetoclax 10 nM group and 22.2% ± 3.4% in ABBV-744 50 nM group, p&amp;lt;0.001). ABBV-744 inhibited cell proliferation in the majority of AML cases (31.7% ± 8.2 %), and the cell growth suppression was more profound in the combination group (82.9% ± 6.9%, p&amp;lt;0.001). Most importantly, three of 12 patients were resistant to venetoclax, but two of these were sensitive to ABBV-744 or ABBV-744/venetoclax combination. We next performed the whole genome transcriptome analysis of pre-treatment AML cells by RNA-sequencing (RNA-seq). The samples which were sensitive to venetoclax and to the combination with ABBV-744 were characterized by high levels of BCL2 and mid-low level of MCL1 expression. In addition, low mRNA expression of AR, IL1R1 expression and high CCND1 expression correlated with response of primary AML cells to the combination of venetoclax and ABBV-744. To test the efficacy of this regimen in vivo, we established a patient-derived xenograft (PDX) from an AML patient in NSG mice. After 21 days of therapy, flow cytometry data demonstrated significantly reduced leukemia burden in venetoclax treated group (9.5% ± 1.7%) but not in ABBV-744 group (22.3% ± 5.8%) compared to controls (30.8% ± 3.9%), with lowest tumor burden in the combination group (5.0% ± 0.8%, p&amp;lt;0.01). No significant impact on mice’ weight was noted, and no clinical signs of toxicity recorded over the course of therapy. The experiment is ongoing, and the survival analysis will be reported. Next, the anti-leukemia efficacy of ABBV-744 was tested in 7 additional AML PDX models. In all of the 7 models, Combination of ABBV-744 and venetoclax treatment delayed AML progression compared to untreated mice (survival days: 141 vs 105, 275 vs 153, 62 vs 46, 136 vs 119, 138 vs 77, 129 vs 116, 94 vs 86). In summary, combinatorial blockade of BDII bromodomain and of BCL-2 anti-apoptotic pathway facilitates apoptotic cell death and suppresses proliferation in the majority of primary AML cells and produces anti-AML activity in AML PDX models in vivo. Citation Format: Tianyu Cai, Vinitha Kuruvilla, Xiaoyu Lin, Tamar Uziel, Xin Lu, Lu Zhang, Qi Zhang, Lina Han, Antonio Cavazos, Yu Shen, Marina Konopleva. Selective targeting BET family BDII bromodomain with ABBV-744 and BCL-2 with venetoclax (ABT-199) is synergistic in primary acute myeloid leukemia models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3831.

Utility of Ki67 labeling index, cyclin D1 expression, and ER-activity level in postmenopausal ER-positive and HER2-negative breast cancer with neoadjuvant chemo-endocrine therapy.

In this study, we investigated the relationships of pathological response after neoadjuvant chemo-endocrine therapy with alterations in the Ki67 labeling index (LI), expression of cyclin D1 (CCND1) and progesterone receptor (PgR), and estrogen receptor (ER) activity in breast cancer. A total of 43 Japanese post-menopausal ER-positive and human epidermal growth factor receptor 2-negative invasive breast cancer patients with tumors >2 cm or positive lymph nodes were enrolled. Exemestane alone was administered for 2 months. Neoadjuvant chemo-endocrine therapy included four cycles of doxorubicin plus paclitaxel followed by weekly paclitaxel. The immunohistochemical expression of Ki67 LI, CCND1, and PgR, and ER activity were evaluated using core needle biopsy samples at the pretreatment and post-exemestane alone stages. ER activity was evaluated through transfection of an adenovirus vector carrying an estrogen-response element-green fluorescent protein gene. In current study, marked pathological responses (including 4.7% with pathological complete response) were obtained in 34.9% of patients. Ki67 LI and PgR expression were significantly decreased after treatment. High Ki67 LI at pretreatment was a significant predictive factor of marked pathological response. At the stage of post-exemestane alone, Ki67 LI was not significantly associated with pathological response; however, high CCND1 expression was significantly correlated with high Ki67 LI. Moreover, low-level ER activity at the post-exemestane alone stage was significantly associated with marked pathological response. In conclusions, pretreatment Ki67 LI was a predictor of response to neoadjuvant chemo-endocrine therapy. CCND1 expression and ER activity at the post-endocrine therapy alone stage may be useful in determining further treatments.

Abstract LB-261: Targeting BET Family Bromodomain with ABBV-075 and BCL-2 with venetoclax (ABT-199) is synergistic in primary acute myeloid leukemia models

Abstract Despite advances in understanding of the biology of acute myeloid leukemia (AML), cure remains elusive for the majority of patients. ABBV-075 is a potent and selective BET family bromodomain inhibitor that entered phase I clinical trials. ABT-199 (Venetoclax) is a small-molecule BH3 mimetic that selectively inhibits BCL-2 causing cell death. ABBV-075 and Venetoclax were recently shown to be synergistic in AML cell lines (Bui MH, Cancer Res 2017). In this study, we evaluated the anti-leukemia effects of concomitant BCL-2 blockade by venetoclax in combination with BET inhibitor ABBV-075 in primary AML samples. First, anti-leukemia activity of venetoclax and ABBV-075 was examined in 12 primary AML samples with diverse genetic alterations. The combination significantly enhanced cell death (71.2 ± 29.3 %), as compared to the single agent treatment (51.4 ± 32.2 % in venetoclax 10 nM group and 32.8 ± 21.7% in ABBV-075 20 nM group). ABBV-075 inhibited cell proliferation in the majority of AML cases (51.3 ± 30.9 %) and the cell growth suppression was more profound in the combination group (87.9 ± 18.8 %, p&amp;lt;0.01). Most importantly, three of 12 patients were resistant to venetoclax, but two of 3 were sensitive to ABBV-075 or ABBV-075/venetoclax combination. We next performed the whole genome transcriptome analysis of pre-treatment AML cells by RNA-sequencing (RNA-seq). The samples which were sensitive to venetoclax and the combination with ABBV-075 were characterized by high level of BCL2 and mid-low level of MCL1 expression. In samples with low level of AR, IL1R1expression and high CCND1 expression, the combination of venetoclax and ABBV-075 was synergistic in inducing AML cell death. To test the efficacy in vivo, we established a patient-derived xenograft (PDX) from an AML patient with FLT3-ITD, DNMT3A, IDH1 and NPM1 mutation in NSG mice. Upon engraftment, mice were randomized to receive vehicle, single agent venetoclax at 50 mg/kg for 21 days, ABBV-075 at 0.5 mg/kg for 21 days; or venetoclax at 50 mg/kg plus ABBV-075 at 0.5 mg/kg for 21 days. After 21 days treatment, flow cytometry data demonstrated significantly reduced leukemia burden in treated groups (7.9 ± 5.9 % in venetoclax group and 12.2 ± 5.1 % in ABBV-075 group) compared to controls (33.0 ± 12.2 %), more prominently in the combination group (1.9 ± 0.8 %). In summary, combinatorial blockade of BET family bromodomain and BCL-2 pathways promotes apoptotic cell death and suppresses proliferation in the majority of primary AML cells. Ongoing studies will evaluate efficacy of this combination therapy in 10 additional primary human AML xenografts and elucidate mechanisms of synergy. Citation Format: Tianyu Cai, Vinitha Mary Kuruvilla, Tamar Uziel, Qi Zhang, Lina Han, Antonio Cavazos, Yu Shen, Marina Konopleva. Targeting BET Family Bromodomain with ABBV-075 and BCL-2 with venetoclax (ABT-199) is synergistic in primary acute myeloid leukemia models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-261.

CD8+ T cells promote proliferation of benign prostatic hyperplasia epithelial cells under low androgen level via modulation of CCL5/STAT5/CCND1 signaling pathway

Previous studies by our group have shown that low intra-prostatic dihydrotestosterone (DHT) induced BPH epithelial cells (BECs) to recruit CD8+ T cells. However, the influence of the recruited CD8+ T cells on BECs under a low androgen level is still unknown. Here, we found CD8+ T cells have the capacity to promote proliferation of BECs in low androgen condition. Mechanism dissection revealed that interaction between CD8+ T cells and BECs through secretion of CCL5 might promote the phosphorylation of STAT5 and a higher expression of CCND1 in BECs. Suppressed CCL5/STAT5 signals via CCL5 neutralizing antibody or STAT5 inhibitor Pimozide led to reverse CD8+ T cell-enhanced BECs proliferation. IHC analysis from Finasteride treated patients showed PCNA expression in BECs was highly correlated to the level of CD8+ T cell infiltration and the expression of CCL5. Consequently, our data indicated infiltrating CD8+ T cells could promote the proliferation of BECs in low androgen condition via modulation of CCL5/STAT5/CCND1 signaling. The increased secretion of CCL5 from the CD8+ T cells/BECs interaction might help BECs survive in a low DHT environment. Targeting these signals may provide a new potential therapeutic approach to better treat BPH patients who failed the therapy of 5α-reductase inhibitors.

Hypoxia-inducible factor-2 alpha promotes the proliferation of human placenta-derived mesenchymal stem cells through the MAPK/ERK signaling pathway.

Human placenta-derived mesenchymal stem cells (hPMSCs) reside in a physiologically low-oxygen microenvironment. Hypoxia influences a variety of stem cell cellular activities, frequently involving hypoxia-inducible factor-2 alpha (HIF-2α). This research showed that hPMSCs cultured in hypoxic conditions (5% O2) exhibited a more naïve morphology and had a higher proliferative capability and higher HIF-2α expression than hPMSCs cultured in normoxic conditions (21% O2). Similar to the hypoxic cultures, hPMSCs over-expressing HIF-2α showed higher proliferative potential and higher expression of CCND1 (CyclinD1), MYC (c-Myc), POU5F1 (Oct4) and the components of the MAPK/ERK pathway. In contrast, these genes were down-regulated in the HIF-2α-silenced hPMSCs. After adding the MAPK/ERK inhibitor PD0325901, cell growth and the expression of CCND1 and MYC were inhibited. Furthermore, the chromatin immunoprecipitation (ChIP) assay and electrophoretic mobility shift assay (EMSA) showed that HIF-2α bound to the MAPK3 (ERK1) promoter, indicative of its direct regulation of MAPK/ERK components at the transcriptional level during hPMSC expansion. Taken together, our results suggest that HIF-2α facilitated the preservation of hPMSC stemness and promoted their proliferation by regulating CCND1 and MYC through the MAPK/ERK signaling pathway.

Prognostic value of differential CCND1 expression in patients with resected gastric adenocarcinoma.

Cyclin D1 (CCND1) plays essential roles in cancer progression. In this study, CCND1 expression patterns in 211 cases of resected gastric adenocarcinoma (RGA) tissue were determined by immunohistochemistry, and the association between CCND1 expression levels and RGA prognosis was analyzed. RGA tissues displayed differential CCND1 expression (high expression, 52.1%; n=110, and low expression, 47.9%; n=101). CCND1 expression levels were related with median overall survival time (MST). MST in patients with high CCND1 expression was 43months, whereas with low CCND1 expression it was 62months (P=0.013). When data were stratified by postoperative treatments and CCND1 expression levels, the MST for patients treated with fluoropyrimidine plus platinum (n=140) was significantly longer than for those treated with fluoropyrimidine only (n=71) in both high and low CCND1 expression groups (65.0 vs. 29.0months, P=0.041; and 74.5 vs. 33.0months, P=0.003, respectively). Cox multivariate analyses further confirmed that high CCND1 expression was related with poor prognosis in both treatment groups [hazard ratio (HR) 1.91, 95% confidence interval (CI) 1.12-3.23; P=0.017, and HR 2.14, 95% CI 1.08-4.25; P=0.029] and that fluoropyrimidine plus platinum was more effective than fluoropyrimidine only in high CCND1 (HR 0.47, 95% CI 0.28-0.78; P=0.004) and low CCND1 (HR 0.44; 95% CI 0.23-0.82; P=0.01) expression patients. Therefore, CCND1 may be used as a prognostic biomarker for patients with RGA.

Abstract 5205: CCND1 is a miR-193b target in prostate cancer

Abstract Prostate cancer is the most frequent cancer of men in Western countries and causes second most of the cancer-related deaths. In addition to genetic changes also epigenetic alterations and micro-RNAs (miRNAs) are important in prostate cancer development. Our group has previously shown that miR-193b is hypermethylated in 22Rv1 prostate cancer cell line and that it is putative tumor suppressor in prostate cancer. In melanoma and hepatocellular carcinoma, it has been shown that CCND1, encoding cyclin D1 protein, is among the target genes of miR-193b. The aim of this project was to study if CCND1 is a target of miR-193b in prostate cancer. According to qRT-PCR and microarray analyses, the expression of CCND1 and miR-193b were inversely correlated in prostate cancer cell lines (PC-3, VCaP, 22Rv1, LAPC4, DU145, LNCaP) and in LuCaP xenografts. Using a luciferase reporter gene assay in 22Rv1 cells, we showed that miR-193b targets CCND1 3′UTR. In 22Rv1 cells with low endogenous miR-193b expression, transient pre-miR-193b transfection reduced CCND1 mRNA levels and cyclin D1 and phospho-RB protein levels. By rescue experiment we confirmed that the downregulation of cyclin D1 and phospho-RB proteins is due to miR-193b. When prostate cancer cell lines were treated with cdk4/6 inhibitor, 22Rv1 and VCaP cells with low miR-193b and high CCND1 expression showed significant growth retardation. In contrast, the inhibitor did not have any effect on the growth of PC-3 and DU145 cells with high miR-193b and low CCND1 expression. Furthermore, immunohistochemical analysis of 337 specimens showed that cyclin D1 is expressed significantly higher in castration resistant compared to hormone-naïve prostate cancers. Therefore, we conclude that CCND1 is one of the miR-193b targets in prostate cancer. Citation Format: Kirsi M. Tuppurainen, Hanna E. Rauhala, Mauro Scaravilli, Robert L. Vessella, Tapio Visakorpi. CCND1 is a miR-193b target in prostate cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5205. doi:10.1158/1538-7445.AM2014-5205

Abstract 3107: The role of LIN28 in atypical teratoid rhabdoid tumor (ATRT) pathogenesis

Abstract Atypical teratoid rhabdoid tumor (ATRT) is a highly malignant brain tumor developing almost exclusively in children. It belongs to the embryonal brain tumor group which consists of primitive tumors recapitulating early embryogenesis of nervous system. It is known that loss of INI protein expression is the hallmark of ATRT pathogenesis. LIN28 is a key gene in embryonic development and maintenance of pluripotency in stem cells. Considering the primitive nature and young age onset of ATRT, LIN28 may be an important co-player in ATRT pathogenesis. We explored the expression and functional role of LIN28 in ATRT. In tumor tissues, LIN28 is highly expressed in ATRT compared with medulloblastomas, another embroynal tumor, whereas primary let-7 microRNA is down-regulated in ATRT. ATRT also showed higher expression of CCND1 and MYC and lower expression of CDKN1C. Knockdown of LIN28 with siRNA in ATRT cell lines resulted decreased cell viability, proliferation, and migration capability. Suppression of CCND1 and MYC expression and enhanced expression of CDKN1C were also observed. Knockdown of LIN28 lead to decreased expression of other pulripotency genes (OCT4 and NANOG) and signature of mesenchymal-epithelial transition was observed after suppression of LIN28. We then introduced wild-type INI1 into ATRT cells by transfection. Restoration of INI in ATRT cell lines lead to decreased expression of LIN28 and CCND1. These results showed that LIN28 is regulated by INI1 and that loss of INI1 protein in ATRT results in unopposed expression of LIN28 and related oncogenes such as CCND1, leading to tumorigenesis. Therefore, the strategic role of LIN28 in ATRT may be utilized as an important therapeutic target. Citation Format: Ji Hoon Phi, Seung Ah Choi, Yong Hwy Kim, Young-Hoon Kim, Chul-Kee Park, Kyu-Chang Wang, Seung-Ki Kim. The role of LIN28 in atypical teratoid rhabdoid tumor (ATRT) pathogenesis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3107. doi:10.1158/1538-7445.AM2014-3107

SOX11 over-Expression Is a Specific Marker for Mantle Cell Lymphoma and Correlates with t(11;14) Translocation, CCND1 Expression, and an Adverse Prognosis.

Abstract 2662 Introduction:Diagnosis of mantle cell lymphoma (MCL) is based on cytomorphology/histology and/or immunophenotyping as well as demonstration of cyclin D1 (CCDN1) over-expression and/or presence of a t(11;14)(q13;q32)/IGH-CCND1 rearrangement. However, some cases that are considered to belong to the MCL entity lack the CCND1 over-expression and the t(11;14) translocation and thus are difficult to distinguish from other small B-cell lymphomas. This distinction is clinically very relevant, since true MCLs show an aggressive behavior while many other B-cell lymphomas do not. Recently, the over-expression of SOX11 has been demonstrated to be specific for MCLs independent of CCND1 positivity, suggesting a diagnostic role of SOX11 expression in t(11;14) negative MCLs. The prognostic role of SOX11 has been controversially discussed. Aim:To evaluate the applicability and usefulness of SOX11 expression as a diagnostic marker for differentiation of B-cell lymphomas and to determine its impact on outcome. Patients and Methods:In this study we analyzed 159 patients with B-cell lymphomas for SOX11 and CCND1 expression levels by quantitative real time PCR. Patients were diagnosed by cytomorphology, immunophenotyping, cytogenetics and FISH and based on these methods were categorized into t(11;14) positive MCL (n=55), t(11;14) negative mature B-cell neoplasms with an MCL-typical immunophenotype (n=37), CLL (n=29), and CLL/PL (n=38). The gene expression levels were quantified and are given relative to ABL1 gene expression. Based on a negative control cohort (n=40) comprising 20 peripheral blood and 20 bone marrow samples without evidence for malignancy the cut-off for rating the expression ratio positive was calculated by the mean value plus the threefold standard deviation and resulted in 0.29 for SOX11 and 2.9 for CCND1. Results:In the total cohort SOX11 expression was present in 53/159 cases (33.3%) and was strongly associated with a t(11;14) translocation (45/55, 81.8% in t(11;14) positive cases vs. 8/104, 7.7% in t(11;14) negative cases, p<0.001). Correspondingly, SOX11 expression correlated with CCND1 expression regarding positivity (45/59 (76.3%) in CCND1 positive cases vs. 8/100 (8.0 %) in CCND1 negative cases, p<0.001). Also the absolute expression levels of both genes showed a high correlation (Spearman, correlation coefficient: 0.631, p<0.001). SOX11 positive patients were younger (63.8 vs. 68.8 years; p=0.011), showed a slightly lower hemoglobin level (12.13 vs. 12.96 g/dL; p=0.04) and a lower platelet count (145,024 vs. 202,914/μl; p<0.001). A detailed analysis within the respective diagnostic subgroups revealed that SOX11 was expressed in 45/55 t(11;14) positive MCL cases (81.8%) with an overall high expression level (median: 58.9; range: 0.3–1363.8). As expected in this entity all cases were CCND1 positive. In the group of 37 t(11;14) negative B-cell neoplasms with an MCL-typical immunophenotype one case was rated positive for CCND1 expression while 6 other cases (16.2%) showed a SOX11 expression (median: 2.0; range: 0.5–322.7), suggesting that these 6 cases might be CCND1 negative MCLs. Two of the 38 CLL/PL cases were SOX11 positive but lacked CCND1 expression. In contrast, SOX11 was never rated positive in CLL cases, while 1 case showed high CCND1 expression. For a total of 107 patients the time to treatment (TTT) was available for correlation analysis. Cases with SOX11 expression had a shorter time to treatment as compared to those without (median TTT: 37 vs. 56 months, p=0.011), what was also true for CCDN1 positive (median TTT: 37 vs. 58 months, p=0.07) and t(11;14) positive cases (median TTT: 6 vs. 56 months, p=0.003). Conclusion:SOX11 expression may be used in addition to CCDN1 as a marker for identification of t(11;14) positive MCLs. However, some rare B-cell neoplasms are considered to belong to the MCL but lack the t(11;14) and CCND1 over-expression. The differential diagnosis of this entity from other small B-cell lymphomas is difficult. SOX11 expression may be considered as a useful marker in addition to CCND1 expression in identification of t(11;14) negative MCL. Patients with SOX11 expression showed a shorter time to treatment, but further analyses are warranted to proof the diagnostic role of SOX11 expression as well as its prognostic impact. Disclosures:Meggendorfer:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Equity Ownership.

CCND1 as a Predictive Biomarker of Neoadjuvant Chemotherapy in Patients with Locally Advanced Head and Neck Squamous Cell Carcinoma

BackgroundCyclin D1 (CCND1) has been associated with chemotherapy resistance and poor prognosis. In this study, we tested the hypothesis that CCND1 expression determines response and clinical outcomes in locally advanced head and neck squamous cell carcinoma (HNSCC) patients treated with neoadjuvant chemotherapy followed by surgery and radiotherapy.Methodology and Findings224 patients with HNSCC were treated with either cisplatin-based chemotherapy followed by surgery and radiotherapy (neoadjuvant group, n = 100) or surgery and radiotherapy (non-neoadjuvant group, n = 124). CCND1 expression was assessed by immunohistochemistry. CCND1 levels were analyzed with chemotherapy response, disease-free survival (DFS) and overall survival (OS). There was no significant difference between the neoadjuvant group and non-neoadjuvant group in DFS and OS (p = 0.929 and p = 0.760) when patients treated with the indiscriminate administration of cisplatin-based chemotherapy. However, in the neoadjuvant group, patients whose tumors showed a low CCND1 expression more likely respond to chemotherapy (p<0.001) and had a significantly better OS and DFS than those whose tumors showed a high CCND1 expression (73% vs 8%, p<0.001; 63% vs 6%, p<0.001). Importantly, patients with a low CCND1 expression in neoadjuvant group received more survival benefits than those in non-neoadjuvant group (p = 0.016), however patients with a high CCND1 expression and treated with neoadjuvant chemotherapy had a significantly poor OS compared to those treated with surgery and radiotherapy (p = 0.032). A multivariate survival analysis also showed CCND1 expression was an independent predictive factor (p<0.001).ConclusionsThis study suggests that some but not all patients with HNSCC may benefit from neoadjuvant chemotherapy with cisplatin-based regimen and CCND1 expression may serve as a predictive biomarker in selecting patients undergo less than two cycles of neoadjuvant chemotherapy.

Transcriptional activities of histone H3, cyclin D1 and claudin 7 encoding genes in laryngeal cancer

Uncontrolled proliferation and a decrease in cell-cell adhesion are one of the most important characteristics of malignancy. Determination of replication-dependent histone H3 can be applied as a proliferative marker. Cyclin D1 (CCND1) regulates the cell cycle by participating in the control of the G1/S phase transition. Claudins (CLDN) are components of tight junctions and may play an essential role in the loss of tissue cohesion. The aim of the study was to assess the mRNA expression of histone H3, cyclin D1, and claudin 7 genes in laryngeal squamous cell carcinoma (LSCC) and adjacent nonneoplastic tissues. The study group consisted of 32 patients with LSCC. Adjacent nonneoplastic tissues of incision lines were used as controls. Quantification of H3, CCND1 and CLDN7 mRNAs was performed by the use of real-time QRT-PCR assay. Molecular analysis showed a significantly higher expression of CCND1 (P = 0.0001; Wilcoxon test) and H3 (P = 0.0141) genes in tumor tissues than in surrounding nonneoplastic tissues. On the contrary, transcriptional activity of claudin 7 gene was higher in histologically normal tissues; however, this difference was not statistically significant (P = 0.1499). The data obtained indicate that laryngeal cancer is characterized by high proliferative potential mediated by increase in cyclin D1 and H3 mRNAs expression.

Gene Expression Profiles as Prognostic Factors in Patients with Multiple Myeloma Treated with Conventional Versus Novel Agents in Correlation with Outcome.

Background: Bortezomib (Bor), a proteasome inhibitor, and Thalidomide (Thal), an anti-angiogenic and immunomodulatory drug, have a remarkable effect in patients with relapsed or refractory MM with 30–40% response rates. In newly diagnosed patients the response rates vary from 70–85%. However, 15–30% of newly diagnosed patients do not respond to Bor or Thal. Secondly, 30% of the patients treated with these novel agents have to stop prematurely because of intolerable side effects, such as polyneuropathy, thrombocytopenia, thrombosis and gastro-intestinal symptoms.Aim: To gain new insights regarding the mechanisms of drug response and toxicity associated with these agents, we have embarked on a prospective study to analyze gene expression profiles (GEP) of myeloma specific genes in plasma cells purified from bone marrow from myeloma patients at diagnosis who have been treated with these novel agents in order to learn which genes govern outcome upon treatment with Bor and Thal.Methods: GEP of CD138 magnetic cell selected (MACS) myeloma plasma cells was performed using Affymetrix GeneChip Human Genome U133 plus 2.0 arrays. This program has been initiated in a large multicenter, prospective, randomized phase III trial, comparing Bor in combination with Adriamycin, Dex (PAD, arm A) followed by high dose therapy with stem cell rescue and maintenance therapy with Bor vs. Vincristine, Adriamycin and Dex (VAD, arm B) followed by high dose therapy with stem cell rescue and maintenance therapy with Thalidomide (HOVON65/GMMG-HD4). This cooperative trial in the Netherlands and Germany has started in April 2005 and has included 600 patients. Gene arrays were analyzed using correlation analysis in Omniviz. Differentially expressed genes in the different groups and in Bor responders were determined using a t-test with adjusted p-value (p<0.0001) and a 1000 permutation analysis (BRB tool).Results: Based on correlated GEPs we divided myeloma patients into 10 clusters. We determined differentially expressed genes in the different clusters and correlated the clusters with chromosomal aberrations like 13q loss, 9 and 11 gain and translocations. Three clusters showed high expression of MMSET/FGFR3, MAF downstream targets and CCND1, respectively.Conclusion: unsupervised cluster analysis led to the subdivision of myeloma patients into clusters, each with a specific gene expression signature. We determined differentially expressed genes in the different clusters and could confirm 3 subgroups (MMSET/FGFR3, MAF/MAFB, and a group with high CCND1 expression) as have been published before.