bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 594: DNAJB6a as tumor suppressor in esophageal squamous cell carcinoma
Authors: YU Valentine Zhuoyou , WONG Victor Chun Lam , CHAN Kwok Wah , LUNG Maria Li
Published: May 2015

Esophageal squamous cell carcinoma (ESCC) has an especially high incidence in China, with five-year survival rates in Hong Kong being of ∼14%. Functional impact and molecular mechanisms of DnaJ (Hsp40) homolog, subfamily B, member 6 (DNAJB6) in ESCC were examined. Tissue microarray (TMA) analysis was performed to investigate the clinical relevance of DNAJB6 in ESCC. In vivo and in vitro functional and molecular studies were performed to verify the TMA results and elucidate the mechanisms for DNAJB6 function. Nuclear but not cytoplasmic DNAJB6 staining was found to correlate significantly with survival in metastasis-free patients by Kaplan-Meier analysis. Expression of nuclear DNAJB6 was down-regulated in tumors compared to normal tissues. In ESCC cell lines, over-expression of the nuclear isoform (DNAJB6a) suppresses in vivo tumor growth in a localization-dependent manner. DNAJB6a over-expression suppresses G1/S transition through inhibiting the key oncogenic Akt signaling pathway and impairs proliferation, by recruiting protein phosphatase 2A through the N-terminal HPD motif. DNAJB6a silencing results in the opposite effect in both cancer cells and immortalized normal esophageal epithelial (NE) cells. DNAJB6a silencing in NE cells also results in senescence-like alterations. DNAJB6a also regulates Akt phosphorylation in other cancers. In summary, nuclear DNAJB6 staining is associated with survival in ESCC patients. DNAJB6a regulates AKT1 phosphorylation in ESCC to suppress tumorigenicity. DNAJB6a may be a useful biomarker for detecting early ESCC tumor initiation and its role in Akt signaling may yet provide opportunities for therapeutic intervention.

Note: This abstract was not presented at the meeting.

Citation Format: Valentine Zhuoyou YU, Victor Chun Lam WONG, Kwok Wah CHAN, Maria Li LUNG. DNAJB6a as tumor suppressor in esophageal squamous cell carcinoma. [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 594. doi:10.1158/1538-7445.AM2014-594
Abstract 595: Tumor suppressor SCUBE2 inhibits breast-cancer cell migration and invasion through the reversal of epithelial-mesenchymal transition
Authors: Lin Yuh-Charn , Lee Yi-Ching , Li Ling-Hui , Cheng Chien-Jui , Yang Ruey-Bing
Published: May 2015

Signal peptide-CUB-EGF domain-containing protein 2 (SCUBE2) belongs to a secreted and membrane-associated multi-domain SCUBE protein family. We previously demonstrated that SCUBE2 is a novel breast-tumor suppressor and could be a useful prognostic marker. However, the role of SCUBE2 in breast-cancer cell migration and invasion and how it is regulated during the epithelial-mesenchymal transition (EMT) remain undefined. In this study, we showed that ectopic SCUBE2 overexpression could enhance the formation of E-cadherin-containing adherens junctions by β-catenin-SOX-mediated induction of forkhead box A1 (a positive regulator of E-cadherin) and upregulation of E-cadherin, which in turn led to epithelial transition and inhibited migration and invasion of aggressive MDA-MB-231 breast-carcinoma cells. SCUBE2 expression was repressed together with that of E-cadherin in TGF-β-induced EMT; direct expression of SCUBE2 alone was sufficient to inhibit the TGF-β-induced EMT. Furthermore, quantitative DNA methylation, methylation-specific PCR, and chromatin immunoprecipitation analyses revealed that SCUBE2 expression was inactivated by DNA hypermethylation at the CpG islands by recruiting and binding DNA methyltransferase 1 during TGF-β-induced EMT. Together, our results suggest that SCUBE2 plays a key role in suppressing breast-carcinoma cell mobility and invasiveness by increasing the formation of the epithelial E-cadherin-containing adherens junctions to promote epithelial differentiation and drive the reversal of EMT.

Citation Format: Yuh-Charn Lin, Yi-Ching Lee, Ling-Hui Li, Chien-Jui Cheng, Ruey-Bing Yang. Tumor suppressor SCUBE2 inhibits breast-cancer cell migration and invasion through the reversal of epithelial-mesenchymal transition. [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 595. doi:10.1158/1538-7445.AM2014-595
Abstract 598: Forkhead transcription factor FOXF1 is a novel p53 target gene and regulates cancer cell migration and invasion
Authors: Tokino Takashi , Tamura Miyuki , Idogawa Masashi , Sasaki Yasushi
Published: May 2015

p53 is an established tumor suppressor that can activate the transcription of multiple target genes. Recent evidence suggests that p53 may contribute to the regulation of cell invasion and migration. In this study, we show that the forkhead box transcription factor FOXF1 is a novel target of p53 because FOXF1 is upregulated by p53. We show that FOXF1 is induced upon DNA damage in a p53-dependent manner. Furthermore, we identified a response element located within the FOXF1 gene that is responsive to wild-type p53, TAp73 as well as TAp63. The ectopic expression of FOXF1 inhibited cancer cell invasion and migration, whereas the inactivation of FOXF1 stimulated cell invasion and migration. We also show that FOXF1 regulates the transcriptional activity of E-cadherin (CDH1) by acting on its FOXF1 consensus binding site located upstream of the E-cadherin gene. Collectively, our results show that FOXF1 is a p53 family target gene, and our data suggest that FOXF1 and p53 form a portion of a regulatory transcriptional network that appears to play an important role in cancer cell invasion and migration.

Citation Format: Takashi Tokino, Miyuki Tamura, Masashi Idogawa, Yasushi Sasaki. Forkhead transcription factor FOXF1 is a novel p53 target gene and regulates cancer cell migration and invasion. [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 598. doi:10.1158/1538-7445.AM2014-598
Abstract 599: Id4 acts as a tumor suppressor by inducing apoptosis and senescence in p53-dependent manner
Authors: Morton Derrick Jerone
Published: May 2015

The physiological mechanisms that can restore biological activity of mutant p53 is an area of high interest given that mutant p53 expression is observed in one third of prostate cancer and more than 50% of all cancers. Here we demonstrate that Id4 (inhibitor of differentiation-4) a dominant negative regulator of bHLH transcription factors can restore the mutant p53 transcriptional activity in prostate cancer cells. Id4 is highly expressed in the normal prostate and decreased in prostate cancer due to promoter hypermethylation. Prostate cancer cell lines: DU145 harbors mutant p53 also lack Id4 expression, and LNCaP cells with wild type p53 express Id4, whereas PC3 cells are null for p53 and express low levels of Id4. The p53 mutants (P223L and V274F) in DU145 cells are within the DNA binding domain and abrogate p53 transcriptional activity due structural de-stabilization and/or DNA interactions. Ectopic expression of Id4 in DU145 cells resulted in increased apoptosis and expression of BAX, PUMA and p21, the transcriptional targets of p53. DNA binding, p53 luciferase reporter studies and ChIP analysis demonstrated that mutant p53 gains Id4 dependent DNA binding and transcriptional activity in part due to CBP/p300 dependent acetylation of p53 at lysine 373. Loss of Id4 in LNCaP cells also abrogated wild type p53 DNA binding and transcriptional activity with concomitant loss of CBP/p300 requirement and decreased acetylation of p53. To further elucidate Id4 dependent restoration of biological activity of p53 we stably transfected p53-null cell line PC3, which has endogenous Id4 with mutant p53 mimicking DU145 cells. mRNA, protein levels and apoptosis assays were used to determine the effects on cell death and to determine if mutant p53 had the ability to transactivate upstream/downstream targets (MDM2, PUMA, and p21). Our results indicate that mutant p53 in PC3 cells were able to induce expression of downstream targets as well as effect biological processes such as apoptosis and cell cycle arrest. Also, post-translational modifications on residues lys320 and lys373 indicated activation of mutant p53. Collectively, based on our studies we demonstrate that Id4 promotes post-translational modifications of p53, which in turn regulates p53 transcriptional activity. NIH grant #R01CA128914

Citation Format: Derrick Jerone Morton. Id4 acts as a tumor suppressor by inducing apoptosis and senescence in p53-dependent manner. [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 599. doi:10.1158/1538-7445.AM2014-599
Abstract 600: Maternally expressed gene 3 (MEG3) as a tumor suppressor in nasopharyngeal carcinoma
Authors: Chak Wing P. , Tong Joanna Hung-Man , Lung Raymond Wai-Ming , Chan Yat Yee , Lo Kwok Wai , To Ka Fai
Published: May 2015

Nasopharyngeal carcinoma (NPC) is a distinctive type of head and neck cancer arising from the nasopharynx. Although rarely occurring worldwide, the incidence and mortality rates of this tumor are remarkably high among Southern China including Hong Kong. The distinct geographical distribution highlights the significance of several etiologic factors in NPC tumorigenesis, including Epstein-Barr virus (EBV) infection, genetic predisposition and intake of preserved food. However, the understanding of NPC development is still unclear. In our previous genome-wide analysis, chromosome 14q32 has been suggested as a tumor suppressor region where loss of heterozygosity (LOH) was detected in >80% of NPCs. Hence, some novel candidate genes involving in NPC development may be located within this region.

By microarray study in NPC samples, consistent down-regulation of a putative tumor suppressor gene, maternally expressed gene 3 (MEG3) within the 14q32 deletion region was identified. MEG3 is an imprinted gene with preferential maternal expression and encodes for a long non-coding RNA. The silenced expression of MEG3 gene in NPC cell lines and xenografts was further confirmed by quantitative RT-PCR. Subsequent DNA methylation analysis in a panel of NPC samples demonstrated that densely methylation at a CpG island on the MEG3 promoter region was observed in all tested NPC cell lines, xenografts and 50% of primary NPCs, while normal imprinting pattern was found in non-cancerous nasopharyngeal epithelium. Interestingly, the presence of demethylation agent in culture medium could successfully rescue MEG3 expression in available NPC cell lines, indicating that promoter hypermethylation is one of the mechanisms to control MEG3 expression. On the other hand, fluorescent in situ hybridization (FISH) also showed the loss of DNA copy number at chromosome 14q32 in 60% of primary NPCs, suggesting an alternative MEG3 regulation mechanism in NPC. In our functional studies, ectopic MEG3 expression in NPC cell lines could significantly inhibit cell propagation, colony and foci formation. A series of reporter assays further suggested that MEG3 might contribute to regulate both p53 and TGF-β signaling pathways.

In conclusion, we have found that MEG3 is a novel tumor suppressor gene in NPC. Further investigation the function of MEG3 in NPC pathogenesis may have potential implications to improve NPC treatment in the future.

Citation Format: Wing Po Chak, Joanna Hung-Man Tong, Raymond Wai-Ming Lung, Yat Yee Chan, Kwok Wai Lo, Ka Fai To. Maternally expressed gene 3 (MEG3) as a tumor suppressor in nasopharyngeal carcinoma. [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 600. doi:10.1158/1538-7445.AM2014-600
Abstract 603: TNFR1 is indispensable in the development of spontaneous lung squamous cell carcinomas
Authors: Xiao Zuoxiang , Willette-Brown Jami , Hu Yinling , Xi Sichuan
Published: May 2015

We recently reported that kinase-dead IKKα knockin mice, in which an ATP binding site of IKKα, Lys 44 was replaced by alanine (IkkαKA/KA), develop spontaneous lung SCCs that were closely associated with lung inflammatory microenvironment. This mouse model resembles human lung SCCs, sheds light on the mechanisms underlying lung malignancy development, and identifies targets for therapy of lung SCCs (Xiao et al. Cancer Cell 23(4):527-40, 2013). To further elucidate what and how the inflammatory stimuli contribute to the tumorigenesis, we generated bitransgenic mice harboring IkkαKA/KA and tnfrsf1-/- (KALTFR). It was identified that the KALTFR mice display lung tumor-free even the lung inflammations are still severe, while almost all of the littermate IkkαKA/KA mice developed lung SCCs. Interestingly, we found a noticeably increased TNFR1 expression in the lung SCCs of IkkαKA/KA mice compared to normal lung tissues. It has also been detected that the TNFR1 expressions are significantly higher in both transcript and protein levels in human lung SCCs with the compare to their adjacent tissues. These findings indicate that TNFR1 may play a crucial role in the development of lung SCC in either IkkαKA/KA mice or humans. Further investigation demonstrated that TNFR1 is a critical factor to regulate nuclear transcriptional factors and oncogenes including C-Myc, through one bromodomain (BRD) proteins- containing complex. These findings broaden our understanding about how inflammatory microenvironment contributes to tumorigenesis through TNFα-TNFRs engagements.

Citation Format: Zuoxiang Xiao, Jami Willette-Brown, Yinling Hu, Sichuan Xi. TNFR1 is indispensable in the development of spontaneous lung squamous cell carcinomas. [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 603. doi:10.1158/1538-7445.AM2014-603
Abstract 611: Co-targeting the PI3K and androgen receptor signal pathways in castration resistant prostate cancer
Authors: Velasco Marco A. De , Kura Yurie , Ando Naomi , Fukushima Emiko , Hatanaka Yuji , Yamamoto Yutaka , Shimizu Nobutaka , Yoshimura Kazuhiro , Nozawa Masahiro , Yoshikawa Kazuhiro , Nishio Kazuto , Uemura Hirotsugu
Published: May 2015

PTEN-deficient castration-naïve prostate tumors respond well to PI3K pathway inhibition, however, treatment response is largely ineffective in castration-resistant prostate cancer (CRPC). Reciprocal feedback regulation between the PI3K and androgen receptor (AR) signaling pathways helps maintain survival. We hypothesized that combined PI3K and AR pathway inhibition would improve the therapeutic outcome. Using an in vivo model of prostate cancer, we assessed the rational therapeutic combination of the mTOR inhibitor, everolimus, and the anti-androgen, chlormadinone acetate (CA), in PTEN deficient tumors. Treatment efficacy was examined in conditional PTEN-mutant mice that developed CRPC after surgical castration. Drug efficacy was evaluated by comparing differences in tumor burden, inhibition of tumor cell proliferation and induction of apoptosis. Treatment-specific responses were measured by downstream PI3K signal molecules, nuclear translocation of AR and activation of compensatory signaling pathways. Mice treated with everolimus or CA alone did not show a significant reduction in tumor burden compared to controls, however, the combination of everolimus and CA significantly inhibited tumor burden. Combination therapy also significantly inhibited cellular proliferation and increased apoptosis. PI3K pathway activation was effectively suppressed by everolimus as measured the levels of phosphorylated S6. No significant differences were noted in the nuclear translocation of the AR between control and treatment groups. Mice treated with the combination therapy demonstrated suppression of MAPK pathway activation. Interestingly, all treatments resulted in an increase of STAT3 activation. In summary, our findings show that AR re-challenge can sensitize CRPC to PI3K inhibition. This study provides a rationale for the developing combination therapies for CRPC by co-targeting the AR and PI3K signal transduction pathways.

Citation Format: Marco A. De Velasco, Yurie Kura, Naomi Ando, Emiko Fukushima, Yuji Hatanaka, Yutaka Yamamoto, Nobutaka Shimizu, Kazuhiro Yoshimura, Masahiro Nozawa, Kazuhiro Yoshikawa, Kazuto Nishio, Hirotsugu Uemura. Co-targeting the PI3K and androgen receptor signal pathways in castration resistant 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 611. doi:10.1158/1538-7445.AM2014-611
Abstract 612: Endostatin is a novel inhibitor of androgen receptor function in prostate cancer
Authors: Lee Joo Hyoung , Isayeva Tatyana , Larson Matthew , Chanda Diptiman , Chesnokov Igor , Ponnazhagan Selvarangan
Published: May 2015

Abnormalities in androgen-androgen receptor (AR) axis have long been recognized for their roles in promoting prostate tumor growth and metastasis. Conventional therapies targeting androgen ablation show transient tumor regression, however, when the tumor recurs during castration-refractory metastatic stage, the current treatment options are very limited. Although the precise mechanism by which prostate cancer (PCa) cells circumvent the androgen deprivation is unclear, recurring tumors have been shown to retain AR activity and upregulation of AR-target gene expression such as prostate specific antigen (PSA), suggesting that targeting AR remains a key component of developing novel therapeutic strategies. Here, we show a profound effect of endostatin, an endogenous angiogenesis inhibitor, on proliferation and invasion of AR-positive PCa cells by targeting AR function. The present study identified that intracellular trafficking of endostatin and direct interaction with AR disrupts AR nuclear translocation and the consequent transcriptional activation of PSA gene. In addition, our structural modeling and site-directed mutagenesis studies suggest that the binding mechanism may include the interaction of bulky side chains (F31 and F34) present in endostatin with the co-activator binding interface (AF-2) in AR ligand-binding domain (LBD). Regarding drug resistance to the second-generation androgen antagonists in patients with metastatic PCa, recent studies revealed that a missense mutation in AR LBD (F876L) and the consequential antagonist-to-agonist switch confers continued AR activity. In this context, our study suggests that endostatin can be recognized as an endogenous AR inhibitor where its molecular interaction with AR may prevent from agonistic switch of AR function by fully masking the AF-2 subdomain. Overall, the current finding provides new insights into endostatin whose anti-cancer activity is not only limited to inhibiting angiogenesis, but can also be expanded to suppressing AR-mediated PCa progression by disrupting AR transactivation.

Citation Format: Joo Hyoung Lee, Tatyana Isayeva, Matthew Larson, Diptiman Chanda, Igor Chesnokov, Selvarangan Ponnazhagan. Endostatin is a novel inhibitor of androgen receptor function 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 612. doi:10.1158/1538-7445.AM2014-612
Abstract 615: TRPM8 channel as a novel molecular target in androgen-regulated prostate cancer cells
Authors: Asuthkar Swapna , Velpula Kiran , Zakharian Eleonora
Published: May 2015

TRPM8 channels are highly expressed in prostate cancer cells but the physiological and pathological functions of TRPM8 in these cells are not known. In addition to prostate cancer (PC) cells, TRPM8 is expressed in sensory neurons where it acts as a sensor of cold. Recently, trpm8 has been identified as both an important gene regulated by androgens and a critical regulator of Ca2+ homeostasis. The androgen and its receptor (AR) are pivotal in the biology of sex hormone-regulated malignancies, with PC the most affected tumor. From the chromatin-immunoprecipitation (ChIP) analysis, we observed that an androgen response element (ARE) mediates androgen regulation of TRPM8. Further, using immunoprecipitation and calcium-imaging experiments, we identified that TRPM8 Ca+2 channels are functionally associated with androgens (testosterone and 5α-dihydrotestosterone) in the prostate. We also identified that unlike prostate cancer cell lines (LNCAP, PC3 and RWPE2) and normal prostate tissues, prostate tumor tissues (grade 1-4) showed high levels of TRPM8 which was predominantly localized as the plasma membrane protein at the tumor periphery. Of note, the decreased stabilization of TRPM8 on the plasma membrane was associated with increased expression of AR protein. These results suggest an important role of androgen signaling in trpm8 gene transcription corroborating to cancer progression and clinical lethality in prostate cancer.

Citation Format: Swapna Asuthkar, Kiran Velpula, Eleonora Zakharian. TRPM8 channel as a novel molecular target in androgen-regulated prostate cancer cells. [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 615. doi:10.1158/1538-7445.AM2014-615
Abstract 617: Repression of cell proliferation and androgen receptor activity by 2’-hydroxyflavanone in prostate cancer cells
Authors: Mizokami Atsushi , Ofude Mitsuo , Kumaki Misako , Izumi Kouji , Konaka Hiroyuki , Kadono Yoshifumi , Shin Minkyoung , Zhang Jian , Keller Evan T. , Namiki Mikio
Published: May 2015

Background: Since androgen-androgen receptor (AR) axis is the main stream for development and progression of prostate cancer, androgen-deprivation therapy (ADT) is conducted as a first line hormonal therapy using medical castration, such as LH-RH agonists, LH-RH antagonist, and antiandrogen for advanced PCa. After an initial response to ADT, however, PCa eventually loses responsiveness to ADT and progresses into castration-resistant prostate cancer (CRPC). Prevention of the development of castration-resistant from hormone-naïve prostate cancer is an important issue in maintaining the quality of life of the patients. We explored the effect of 2’-hydroxyflavanone on proliferation and androgen responsiveness using prostate cancer cell lines.

Materials and Methods: To investigate the effect of 2’ -hydroxyflavanone on proliferation, prostate cancer cells were treated with 2’-hydroxyflavanone. Androgen-responsiveness in LNCaP cells was confirmed by luciferase assay after transfection of luciferase reporter driven by prostate specific antigen promoter. To detect androgen receptor (AR) expression, reverse transcriptase polymerase chain reaction and western blot analysis were conducted.

Results: 2’ -Hydroxyflavanone inhibited the proliferation of PC-3 and DU145 cells by induction of apoptosis. 2’ -Hydroxyflavanone inhibited the proliferation of LNCaP cells stimulated by androgens and attenuated androgen-responsiveness through down-regulation of AR protein.

Conclusion: 2’ -Hydroxyflavanone not only inhibited proliferation of prostate cancer cells, but also repressed androgen-responsiveness, suggesting that it might be an useful agent in preventing recurrence of prostate cancer.

Citation Format: Atsushi Mizokami, Mitsuo Ofude, Misako Kumaki, Kouji Izumi, Hiroyuki Konaka, Yoshifumi Kadono, Minkyoung Shin, Jian Zhang, Evan T. Keller, Mikio Namiki. Repression of cell proliferation and androgen receptor activity by 2’-hydroxyflavanone in prostate cancer cells. [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 617. doi:10.1158/1538-7445.AM2014-617
Abstract 620: Evidence for combination tamoxifen and betulinic acid to treat hormone responsive- and unresponsive breast cancer by attenuation of Pygopus expression
Authors: Tzenov Youlian , Andrews Philip , Popadiuk Cathy , Kao Kenneth R.
Published: May 2015

The presence of the estrogen and progesterone hormone receptors (ER and PR) in advanced breast cancer tumour nuclei is predictive for response to hormone disruptive therapy. However, the accuracy of ER and PR testing can be variable and although patients receive significant benefit from hormone therapy, they ultimately become refractory to treatment. The chromatin remodeling protein: human Pygopus2 (hPygo2), is important for oncogenic growth and cell cycle progression and may serve as a diagnostic or targetable biomarker for breast cancer. Here we demonstrate that ER-alpha (Erα)−Sp1 transcription factor (Sp1) complexes associate with chromatin at adjacent motifs within the hPygo2 promoter in both hormone receptor positive and negative breast cancer cells. Erα agonists induced, while antagonists inhibited hPygo2 mRNA and protein expression. Erα and Sp1 occupancy at the hPygo2 promoter required intact promoter binding sites and functional DNA binding domains. Estrogen (E2) increased, while Fulvestrant decreased, Erα binding to the hPygo2 promoter with no effect on Sp1. Furthermore, Sp1-directed siRNA reduced Sp1 occupancy at the hPygo2 promoter and decreased hPygo2 mRNA and protein expression in both Erα-expressing (+) and Erα-nonexpressing (-) cells. Betulinic acid, an Sp1 inhibitor, caused cell cycle arrest, reduced Sp1 binding to the hPygo2 promoter and reduced hPygo2 expression in Erα- cell lines. The activation of hPygo2 expression either cooperatively or independently by Erα and Sp1 suggests their potential diagnostic and therapeutic benefit for both hormone responsive and unresponsive breast cancer.

Citation Format: Youlian Tzenov, Philip Andrews, Cathy Popadiuk, Kenneth R. Kao. Evidence for combination tamoxifen and betulinic acid to treat hormone responsive- and unresponsive breast cancer by attenuation of Pygopus expression. [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 620. doi:10.1158/1538-7445.AM2014-620
Abstract 622: S-equol, an estrogen receptor β agonist, inhibits tumor growth and progression of breast cancer
Authors: Samayoa Cathy , Krishnegowda Naveen K. , Krishnan Samaya R. , Vadlamudi Ratna K. , Tekmal Rajeshwar R.
Published: May 2015

Breast cancer is the primary cause of cancer-associated mortality in women worldwide. Estrogen and the Estrogen Receptors (ER) play a significant role in breast cancer, with over two-thirds of breast cancers expressing ERα. Current endocrine therapy, such as aromatase inhibitors, target estrogen biosynthesis and anti-estrogens target ERα. However, therapeutic resistance frequently arises. In addition to the importance of ERα, ERβ has also been shown to play a critical, but opposing role in breast cancer. ERβ has been shown to inhibit the growth of ERα-positive breast cancer cells. The ratio of ERα to ERβ, in addition to the cross talk between ER's and growth factor signaling, has been implicated in the development of therapeutic resistance. Recently, several plant-derived compounds that exhibit ERβ agonist activity have been identified. S-equol is a potent ERβ agonist and a metabolite from the soy isoflavone daidzein, and has been previously shown to alleviate menopausal symptoms in a clinical trial. Activation of ERβ, or its over expression, shifts the balance of ER's from the oncogenic action of ERα to the tumor suppressor activity of ERβ, and therefore may be a valuable therapeutic approach in the treatment of breast cancer. In this study we sought out to determine the efficacy of the ERβ agonist, S-equol, in inhibiting the growth and progression of breast tumors using a syngeneic mouse model. We used mouse mammary tumor cells expressing endogenous ERβ, and to determine the contribution of ERβ, cells with knockdown of ERβ were generated using shRNA. ERβ mRNA and protein expression was analyzed using qRT-PCR and western blot respectively. Syngeneic tumors were established and mice were treated with either a vehicle control or S-equol. Treatment with S-equol reduces tumor volume and inhibits the progression of mouse mammary tumor cells in tumors expressing ERβ. Our mechanistic studies show that S-equol reduces the expression of ERα, and increases the expression of p53 and p27 in an ERβ dependent manner. Additionally, S-equol modulates the expression of inflammatory molecules involved in aromatase expression and promotes the differentiation of cancer stem cells. In conclusion, this study suggests that targeting ERβ may be a valuable strategy in treatment of breast cancer.

Citation Format: Cathy Samayoa, Naveen K. Krishnegowda, Samaya R. Krishnan, Ratna K. Vadlamudi, Rajeshwar R. Tekmal. S-equol, an estrogen receptor β agonist, inhibits tumor growth and progression of breast 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 622. doi:10.1158/1538-7445.AM2014-622
Abstract 628: Pan-Bcl-2 inhibitor, GX15-070 (Obatoclax), decreases human T regulatory lymphocytes while preserving effector T Lymphocytes: A rationale for its use in combination immunotherapy
Authors: Kim Peter S. , Jochems Caroline , Grenga Italia , Donahue Renee N. , Tsang Kwong Y. , Gulley James L. , Schlom Jeffrey , Farsaci Benedetto
Published: May 2015

Bcl-2 inhibitors are currently being evaluated in clinical studies for treatment of patients with solid tumors and hematopoietic malignancies. In this study we explored the potential for combining the pan-Bcl-2 inhibitor GX15-070 (GX15; obatoclax) with immunotherapeutic modalities. We evaluated the in vitro effects of GX15 on human T-cell subsets obtained from PBMCs in terms of activation, memory, and suppressive function. Our results indicated that in healthy-donor PBMCs, mature-activated T cells were more resistant to GX15 than early-activated T cells, and that GX15 preserved memory but not non-memory T-cell populations. Furthermore, GX15 increased the apoptosis of regulatory T cells (Tregs), profoundly down-regulated FOXP3 and CTLA-4 in a dose-dependent manner, and decreased their suppressive function. Treating PBMCs obtained from ovarian cancer patients with GX15 also resulted in increased CD8+:Treg and CD4+:Treg ratios. These results support preclinical studies in which mice vaccinated before treatment with GX15 showed the greatest reduction in metastatic lung tumors as a result of increased apoptotic resistance of mature CD8+ T cells and decreased Treg function brought about by GX15. Taken together, these findings suggest that when a Bcl-2 inhibitor is combined with active immunotherapy in humans, such as the use of a vaccine or immune checkpoint inhibitor, immunotherapy should precede administration of the Bcl-2 inhibitor to allow T cells to become mature, and thus resistant to the cytotoxic effects of the Bcl-2 inhibitor.

Citation Format: Peter S. Kim, Caroline Jochems, Italia Grenga, Renee N. Donahue, Kwong Y. Tsang, James L. Gulley, Jeffrey Schlom, Benedetto Farsaci. Pan-Bcl-2 inhibitor, GX15-070 (Obatoclax), decreases human T regulatory lymphocytes while preserving effector T Lymphocytes: A rationale for its use in combination immunotherapy. [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 628. doi:10.1158/1538-7445.AM2014-628
Abstract 632: Radiation-induced immunogenic modulation of tumor enhances antigen processing and calreticulin exposure, resulting in enhanced T-cell killing
Authors: Gameiro Sofia R. , Jammeh Momodou L. , Wattenberg Max M. , Tsang Kwong Y. , Ferrone Soldano , Hodge James W.
Published: May 2015

Radiation therapy (RT) is used for local tumor control through direct killing of tumor cells. Radiation-induced cell death can trigger tumor antigen-specific immune responses, but these are often noncurative. Radiation has been demonstrated to induce immunogenic modulation (IM) in various tumor types by altering the biology of surviving cells to render them more susceptible to T cell-mediated killing. Little is known about the mechanism(s) underlying IM elicited by sub-lethal radiation dosing. We have examined the molecular and immunogenic consequences of radiation exposure in breast, lung, and prostate human carcinoma cells. Radiation induced secretion of ATP and HMGB1 in both dying and surviving tumor cells. In vitro and in vivo tumor irradiation induced significant upregulation of multiple components of the antigen-processing machinery and calreticulin cell-surface expression. Augmented CTL lysis specific for several tumor-associated antigens was largely dictated by the presence of calreticulin on the surface of tumor cells and constituted an adaptive response to endoplasmic reticulum stress, mediated by activation of the unfolded protein response.

This study provides evidence that radiation induces a continuum of immunogenic alterations in tumor biology, from immunogenic modulation to immunogenic cell death. We also expand the concept of immunogenic modulation, where surviving tumor cells recovering from radiation-induced endoplasmic reticulum stress become more sensitive to CTL killing. These observations offer a rationale for the combined use of radiation with immunotherapy, including for patients failing RT alone.

Citation Format: Sofia R. Gameiro, Momodou L. Jammeh, Max M. Wattenberg, Kwong Y. Tsang, Soldano Ferrone, James W. Hodge. Radiation-induced immunogenic modulation of tumor enhances antigen processing and calreticulin exposure, resulting in enhanced T-cell killing. [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 632. doi:10.1158/1538-7445.AM2014-632
Abstract 634: Combination chemotherapy significantly reduces indoleamine 2,3-dioxygenase activity in NSCLC patients
Authors: Schafer Cara C. , Wang Yong , Sawant Anandi , Jin Tong Huan , Ponnazhagan Selvarangan , Grant Stefan C. , Deshane Jessy S.
Published: May 2015

Heterogeneity and immunosuppression in the tumor microenvironment (TME) has dampened the efficacy of current frontline combination chemotherapies for lung cancer which has provided limited improvement in patient survival. Modified amino acid metabolism in the TME contributes to lung cancer progression and suppression of anti-tumor immunity. Enhanced tryptophan metabolism in primary lung cancer patients is indicative of advanced disease stage. One of the key regulators of tryptophan metabolism is an intracellular, heme-containing enzyme indoleamine 2,3-dioxygenase (IDO). IDO catabolizes the breakdown of tryptophan into a toxic, immunosuppressive metabolite, L-Kynurenine (L-Kyn). Induction of IDO and accumulation of its metabolite correlates with poor prognosis and overall patient survival.

We hypothesized that IDO enzymatic activity would be elevated in lung cancer patients compared to normal healthy controls. We predicted that combination chemotherapy strategies would modulate serum levels of L-Kyn and IDO activity in the immune compartment of lung cancer patients. Using an IDO enzymatic activity assay to measure the production of L-Kyn, we demonstrate that prior to receiving combination chemotherapies, Non-Small Cell Lung Cancer (NSCLC) patients (n=11) display significantly elevated levels of this immunosuppressive serum metabolite compared to healthy relatives (n=8) (Student's t-test p=0.000087; Paired Means p=0.0001). Following the second cycle of treatment, serum L-Kyn was reduced significantly (Student's t-test p=0.023; Paired Means p=0.043) in a subset of patients (n=3 of 5) suggesting response to chemotherapy, but these post-treatment levels remained elevated overall compared to healthy relatives. Additionally, greater levels of IDO enzymatic activity were observed in circulating immunosuppressive myeloid-derived suppressor cells from NSCLC patients and peripheral blood mononuclear cells prior to receiving combination chemotherapy compared to those from healthy relatives.

These preliminary studies suggest that the IDO enzymatic pathway and tryptophan metabolism may serve as important therapeutic targets and diagnostic markers in predicting clinical outcomes of combination chemotherapies in the treatment of NSCLC in humans.

Citation Format: Cara C. Schafer, Yong Wang, Anandi Sawant, Tong Huan Jin, Selvarangan Ponnazhagan, Stefan C. Grant, Jessy S. Deshane. Combination chemotherapy significantly reduces indoleamine 2,3-dioxygenase activity in NSCLC patients. [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 634. doi:10.1158/1538-7445.AM2014-634
Abstract 637: Chemotherapy enhances recombinant lipoimmunogen-based antitumor immunity against cervical cancer
Authors: Liu Shih-Jen , Chang Li-Sheng , Yeh Yi-Chen , Leng Chih-Hsiang
Published: May 2015

Although CTLs play a major role in eradicating cancer cells during immunotherapy, the development of immune escape often limits the success of such therapy. Therefore, the simultaneous induction of CTLs against cancer and administration of chemotherapy would be feasible to overcome the limitation. The recombinant lipoprotein consisting of inactive E7 (E7m) biologically linked to a bacterial lipid moiety (rlipo-E7m) induces the maturation of mouse bone marrow-derived dendritic cells through toll-like receptor 2 (TLR2), E7-specific cytotoxic T lymphocytes (CTLs) responses and inhibits tumor growth. The large tumor (> 10 mm diameter) was regressed when treated with rlipo-E7m and TLR9 agonist (oligodeoxynucleotide, CpG ODN). In addition, the combinations inhibit local immunosuppressive cells number (MDSCs, TAM and Tregs) and increase CTLs number in tumor infiltration. However, the tumor grew again at ∼ 50 days after tumor implantation. To improve the therapeutic effects of rlipo-E7m/CpG, cisplatin or gemcitabine was combined to treat large tumor. We found that the combinations increase the survival time of the tumor-bearing mice. The promising approach may be applied to other tumor antigen for other cancer immunotherapy.

Citation Format: Shih-Jen Liu, Li-Sheng Chang, Yi-Chen Yeh, Chih-Hsiang Leng. Chemotherapy enhances recombinant lipoimmunogen-based antitumor immunity against cervical 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 637. doi:10.1158/1538-7445.AM2014-637
Abstract 639: Antibody-mediated blockade of phosphatidylserine combined with radiation improves survival and tumor eradication in a rat model of non-small cell lung cancer
Authors: Belzile Olivier , Zhang Zhang , Huang Xianming , Saha Debabrata , Brekken Rolf A.
Published: May 2015

Stereotactic body radiation therapy (SBRT) uses image guidance to deliver very high doses of radiation, in no more than 5 fractions, to tumors. SBRT has shown remarkable benefit in patients with peripheral lung tumors but can cause severe toxicity to central organs when the tumor is proximally located if full potency doses are used (18 Gy x 3 fractions, for example). Bavituximab, a therapeutic monoclonal antibody that is headed to Phase III clinical trials, recognizes the immunosuppressive lipid, phosphatidylserine (PS), which becomes exposed on both tumor blood vessels and tumor cells subjected to SBRT. We hypothesized that treatment with the antibody 2aG4 (a murine version of bavituximab) would synergize with SBRT and allow tolerable doses of radiation to be used to treat centrally-located NSCLC without sacrificing efficacy. Nude rats bearing established orthotopic A549-luc NSCLC were treated with 3x12 Gy of radiation alone or combined with 2aG4 (4mg/kg) injected twice per week. Tumor growth was followed by bioluminescence. Rats treated with radiation and 2aG4 had a 100% survival rate 106 days after implantation and tumors were completely eradicated in 44% of these animals. In contrast, rats treated with radiation had a survival rate of 17% while only 11% of untreated rats survived. A toxicity study was conducted in which 3x12 Gy of radiation was delivered to central organs of tumor-free rats. This treatment does not appear to cause severe toxicity. These results suggest that bavituximab with radiation may result in improved clinical outcome in patients with centrally-located Non-Small Cell Lung Cancer (NSCLC).

Citation Format: Olivier Belzile, Zhang Zhang, Xianming Huang, Debabrata Saha, Rolf A. Brekken. Antibody-mediated blockade of phosphatidylserine combined with radiation improves survival and tumor eradication in a rat model of non-small cell lung 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 639. doi:10.1158/1538-7445.AM2014-639
Abstract 64: A p53 activating mutation drives metastatic high-grade serous ovarian cancer arising from the ovary in mice
Authors: Kim Jaeyeon , Coffey Donna M. , Ma Lang , Matzuk Martin M.
Published: May 2015

The tumor suppressor p53 is the most frequently mutated gene in high-grade serous ovarian cancer, or high-grade serous carcinoma (HGSC) − the most common and deadliest ovarian cancer. Yet it remains unknown whether p53 mutation can drive HGSC. To address this, we have incorporated a p53 activating mutation, p53R172H, into conditional Dicer-Pten double-knockout (DKO) mice, generating triple-mutant (TKO) mice. Like DKO mice, these TKO mice develop fallopian-tube-originated HGSCs, aggressively metastasizing inside the abdominal cavity, inducing ascites, and killing the mice. Then, unlike DKO mice, when both fallopian tubes are surgically removed from TKO mice, TKO ovaries alone are capable of developing HGSCs, which also metastasize to the abdominal cavity, including the omentum, diaphragm, and mesentery. This finding indicates that p53 mutation can drive HGSC in the ovary. To further examine the ability of p53 mutation to drive ovarian HGSC, we have generated p53R172H-Pten double-mutant mice (DMu), a control line for TKO mice. Intriguingly, 30% of the DMu mice (12/40 mice) develop metastatic HGSCs in the ovary, which spread to the abdominal cavity before killing the mice, just like TKO mice with fallopian tubes removed. Unlike TKO and DKO mice, however, DMu mice show no sign of tumor in the fallopian tube. Our new mouse models of ovarian cancer therefore show that p53 mutation indeed can initiate and drive metastatic HGSCs arising from the ovary. Clinically, our study suggests that besides the fallopian tube, the ovary can serve as a source of high-grade serous ovarian cancer. (Grant support: Ovarian Cancer Research Fund (OCRF); Marsha Rivkin Ovarian Cancer Challenge Grant; F32 NCI National Research Service Award (NRSA); K99/R00 NCI Pathway to Independence Award)

Citation Format: Jaeyeon Kim, Donna M. Coffey, Lang Ma, Martin M. Matzuk. A p53 activating mutation drives metastatic high-grade serous ovarian cancer arising from the ovary in mice. [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 64. doi:10.1158/1538-7445.AM2014-64
Abstract 647: CD20-specific Engineered Toxin Body demonstrates direct cell kill of multiple B-cell Non-Hodgkin's lymphoma types
Authors: Rajagopalan Sangeetha , Wirth Rachel , Erdman Jennifer , Grant Kari , Brieschke Brigitte , Flores Rodney , Null William , Liu Jen-Sing , Willert Erin K. , Higgins Jack P.
Published: May 2015

The B-cell membrane protein CD20 is expressed by both benign and neoplastic lymphocytes. It has been a superb target for immunotherapies targeting B-cell derived diseases. There are several anti-CD20 antibodies which have been developed for B-cell lymphoma therapy. The most successful example of this targeted approach is rituximab, which is approved for front and second line therapy of Non-Hodgkin lymphoma (NHL). However, rituximab has limited direct cell-kill effects, acting primarily through indirect immune responses induced by antibody-dependent cell-mediated cytotoxicity (ADCC) and/or complement dependent cytotoxicity (CDC). While CD20-targeted radiotherapeutics have shown promise, their administrative challenges have curtailed their clinical utility. Thus, a therapeutic approach with direct cell-kill activity against CD20-expressing cells would be clinically useful. We have developed an engineered toxin body (ETB) comprising a CD20 scFv and a modified Shiga-like Toxin A (MT-3724) capable of specifically recognizing and killing CD20 expressing cells. MT-3724 is selectively cytotoxic against B-cell NHL cell lines and against NHL cells obtained from patients. It demonstrated potent cytotoxicity against Burkitt's lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, and mantle cell lymphoma with IC (50) values ranging from 1.4 nM to 12 nM. Through immunofluorescent tracking, MT-3724 internalizes within one hour after binding to CD20 (+) B cells. To our knowledge, this is the first example of efficient internalization of an immunotoxin via CD20. Consistent with the in vitro activity, MT-3724 demonstrated anti-tumor efficacy in mice-bearing NHL cell line xenografts. These results show that MT-3724 is a promising targeted therapeutic agent against CD20 positive B-cell lymphomas and is currently under further development.

Citation Format: Sangeetha Rajagopalan, Rachel Wirth, Jennifer Erdman, Kari Grant, Brigitte Brieschke, Rodney Flores, William Null, Jen-Sing Liu, Erin K. Willert, Jack P. Higgins. CD20-specific Engineered Toxin Body demonstrates direct cell kill of multiple B-cell Non-Hodgkin's lymphoma types. [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 647. doi:10.1158/1538-7445.AM2014-647