bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 1906: PFKFB4, much more than just a glycolytic gene
Authors: Phillips Emma L. , Bethke Frederic , Balss Jörg , Pusch Stefan , Christen Stefan , Schnölzer Martina , Habel Antje , Capper David , Deimling Andreas Von , Fendt Sarah-Maria , Lichter Peter , Goidts Violaine
Published: August 2018

Glioblastoma is one of the most aggressive primary brain tumors in adults, with a dismal median overall survival of only 14 months after diagnosis. Glioblastoma stem-like cells (GSCs) are particularly resistant to current therapies, capable of self-renewal and tumour initiation and are hence thought to be major contributors to patient relapse. A kinome and phosphatome wide screen revealed glycolysis gene 6-Phosphofructo-2-Kinase/Fructose-2,6-Biphosphatase 4 (PFKFB4) as an important candidate gene for GSC survival. Notably, silencing PFKFB4 in an orthotopic xenograft mouse model of glioblastoma completely rid the mice of the tumor. Here we show a brand new function of PFKFB4, independent of its role in glycolysis - namely in the regulation of HIF1α, which is upregulated in GSCs. Gene expression profiling revealed a downregulation in HIF1α target genes in PFKFB4-silenced GSCs, and HIF1α protein levels are also dramatically reduced upon silencing. Mass spectrometric analysis of immunoprecipitated PFKFB4 protein revealed a novel interaction partner, F-box only protein 28 (FBXO28), an E3 ubiqutin ligase. Co-immunoprecipation assays show that FBXO28 forms an SCF multi-protein ubiquitin ligase complex with CUL1 and SKP1. Ubiquitylation studies of HIF1α show that PFKFB4 stabilizes this crucial protein in GSCs by preventing its targeting by FBXO28 for proteasomal degradation. These new findings, coupled with its cancer specific expression in a variety of tumor entities, makes PFKFB4 a compelling target.

Citation Format: Emma L. Phillips, Frederic Bethke, Jörg Balss, Stefan Pusch, Stefan Christen, Martina Schnölzer, Antje Habel, David Capper, Andreas Von Deimling, Sarah-Maria Fendt, Peter Lichter, Violaine Goidts. PFKFB4, much more than just a glycolytic gene [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 1906.
Abstract 1913: Investigation of targeting SKP2 to improve the treatment response in glioma
Authors: Wang Jing , Su Hong-Kai , Chen Zhong-Ping , Qu Yue , Cai Hai-Ping
Published: August 2018

Glioma is the most frequent tumor in central nervous system (CNS), constituting about 30% of CNS tumors and 80% of malignant brain tumors. The current standard strategy for glioma treatment is surgery plus adjuvant chemotherapy and concurrent radiotherapy. However, the prognosis of glioma is still poor. It has been proved that the existence of glioma stem cells and the timely repair of DNA damages are the main causes of treatment failure. Skp2 (S-phase kinase associated protein-2), a member of F-box proteins, forms the Skp2 SCF complex with Skp1, Cullin-1, and Rbx1, serves as one of the important E3-ligases in the process of ubiquitination, and is responsible for substrate recognition. Skp2 is overexpressed in a variety of human cancers and promotes cancer progression by inducing p27 degradation. Importantly, Skp2 deficiency profoundly restricts cancer progression in multiple genetic mouse tumor models. Our group has demonstrated that Skp2 is involved in the regulation of pool size and self-renewal ability of hematopoietic stem cells, as well as breast cancer stem cells and nasopharyngeal carcinoma stem cell-like cells. However, the exact role of Skp2 in the regulation of glioma stem cells and DNA damage repair in glioma has not been elucidated yet. We proposed that targeting Skp2 could promote radio- and chemotherapy sensitivity through suppressing function of glioma stem cells. We first detected the expression of SKP2 in glioma tumor tissues and found that SKP2 was highly expressed in glioma and related to the prognosis of glioma patients. We also adopted gene knockdown methods and inhibitor pretreatment with small molecules in order to suppress the level of Skp2, and then checked the sphere formation and self-renewal ability of glioma stem cell upon radio/chemo treatments. In this way we evaluated the role of Skp2 in repressing the glioma cell proliferation and subsequently triggering apoptosis. Our project will clarify the novel mechanisms about how Skp2 is involved in the glioma radio- and chemotherapy resistance, which will provide a potential target to improve the radio/chemo therapeutic effect through targeting one single target.

Citation Format: Jing Wang, Hong-Kai Su, Zhong-Ping Chen, Yue Qu, Hai-Ping Cai. Investigation of targeting SKP2 to improve the treatment response in glioma [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 1913.
Abstract 194: Prognostic implication of stromal hyaluronic acid protein expression in resected oropharynx and oral cavity cancer
Authors: Ko Yoon Ho , Won Hye Sung , Sun Der Sheng , Hong Soon Auck , Hong Ji Hyung
Published: August 2018

Background: Hyaluronic acid (HA) is a well-characterized component of extracellular matrix (ECM), which plays a critical role in a variety of cellular processes and regulates cell adhesion, migration, and proliferation associated with prognosis of various tumor types. However, the clinical prognostic significance in resected head and neck squamous cell carcinoma (HNSCC) is not well identified.

Methods: The resected tissues from oropharynx or oral cavity cancer patients undergoing surgery were made for tissue microarray (TMA) divided into stroma and cancer panels. Including HA protein expression, HA synthases (HAS) and hyaluronidases (HYAL1) was also assessed by immunohistochemistry.

Results: A total of 160 resected oropharynx or oral cavity cancer tissues between 1994 and 2012 were analyzed in this study. Stromal HA expression, positive in 31 (19.4%) of patients, showed a trend favoring for HPV negative tumor. Other clinicopathological characteristics did not differ according to stromal HA protein expression. However, the HA expression in stroma were statistically associated with clinical outcomes. In the multivariate analysis for clinical outcomes, stromal HA expression was found to be an independent indicator for disease recurrence. (HR, 2.716; 95 % CI, 1.323 - 5.575, P = 0.007), and the combined analysis of tumoral and stromal expression of HA showed that patients with a cancer and stroma-positive HA expression showed a significantly worse outcome than those with other combinations (RFS, P = 0.023, OS, P = 0.082). In addition, when using stratification of HPV stastus, the stromal HA expression remained an independent indicator for disease recurrence and survival(HR, 2.712; 95% CI, 1.323 - 5.56; P = 0.006).

Conclusions: The HA expression in stroma could be related with poor prognosis independently in surgically resected HNSCC. Thus, the finding suggested that HA in stroma may serve not only as a prognostic marker but also as a supposed target for HNSCC treatment.

Citation Format: Yoon Ho Ko, Hye Sung Won, Der Sheng Sun, Soon Auck Hong, Ji Hyung Hong. Prognostic implication of stromal hyaluronic acid protein expression in resected oropharynx and oral cavity cancer [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 194.
Abstract 1940: Thymine DNA glycosylase (TDG) as a novel target for melanoma
Authors: Tricarico Rossella , Mancuso Pietro , Bhattacharjee Vikram , Cosentino Laura , Nicolas Emmanuelle , Einarson Margret , Beeharry Neil , Devarajan Karthik , Katz Rich , Dorjsuren Dorjbal G. , Simeonov Anton , Kadariya Yuwaraj , Davidson Guillaume , Testa Joseph R. , Davidson Irwin , Larue Lionel , Sobol Robert W. , Yen Timothy , Bellacosa Alfonso
Published: August 2018

Melanoma is an aggressive neoplasm with increasing incidence that bears the infamous distinction of being a recalcitrant cancer, i.e. a cancer with poor prognosis, lacking progress in diagnosis and treatment. In addition to conventional therapy, melanoma treatment is currently based on targeting the BRAF/MEK/ERK signal transduction pathway and immune checkpoints; however, advanced therapeutic approaches based on novel targets are urgently needed. We reasoned that the base excision repair enzyme Thymine DNA Glycosylase (TDG) could be such a target for its dual role in safeguarding genome stability and in effecting active DNA demethylation downstream the Ten-Eleven Translocation (TET) dioxygenases. TDG knockdown in melanoma cell lines causes cell cycle arrest, senescence and death by mitotic alterations, and impairs xenograft formation. Importantly, untransformed melanocytes are not affected by TDG knockdown, and adult mice with conditional knockout of TDG are viable. Candidate TDG inhibitors, identified through a high-throughput screen, reduced viability and clonogenic capacity of melanoma cell lines. Candidate TDG inhibitors increased cellular levels of 5-carboxylcytosine, the last intermediate in DNA demethylation, which is specifically removed by TDG, indicating successful targeting. These findings suggest that TDG may provide critical functions in cancer cells, but not in normal cells, that make it a highly suitable anti-melanoma drug target. By potentially disrupting both DNA repair and the epigenetic state, targeting TDG may represent a completely new approach to melanoma therapy.

Citation Format: Rossella Tricarico, Pietro Mancuso, Vikram Bhattacharjee, Laura Cosentino, Emmanuelle Nicolas, Margret Einarson, Neil Beeharry, Karthik Devarajan, Rich Katz, Dorjbal G. Dorjsuren, Anton Simeonov, Yuwaraj Kadariya, Guillaume Davidson, Joseph R. Testa, Irwin Davidson, Lionel Larue, Robert W. Sobol, Timothy Yen, Alfonso Bellacosa. Thymine DNA glycosylase (TDG) as a novel target for melanoma [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 1940.
Abstract 1942: Synthetic lethal interaction between SETD2 loss and inhibition of PI3Kβ in clear cell renal cell carcinoma (ccRCC)
Authors: Terzo Esteban , Chiang Yun C. , Chytil Anna , Jansen Valerie M. , Rathmell Kimryn
Published: August 2018

Renal cell carcinoma (RCC) is a high-risk and high-mortality cancer for which several new targeted therapies exist. However, responses of primary clear cell RCC (ccRCC) tumors and metastases to these targeted agents are variable, and complete responses are relatively uncommon underscoring the need to identify reliable biomarkers to predict tumor behavior. Bi-allelic inactivation of the tumor suppressor gene SETD2, encoding a lysine methyltransferase, occur in approximately 20% of ccRCC tumors while the SETD2 protein deficiency rate has been reported to be as high as 34%. The phosphatidylinositide 3-kinases (PI3Ks) have emerged as important therapeutic targets for the treatment of solid and hematological malignancies, including ccRCC. Here, we show that genetic and pharmacologic inhibition of the PI3Kβ isoform results in inhibition of SETD2 deficient (-/-) ccRCC-derived 786-O and A498 cells, which exhibit increased sensitivity to PI3Kβ-specific inhibitors TGX221 (IC50 = 2.3 - 4.5 µM, respectively) and AZD8186 (IC50 = 1.79 - 0.24 µM, respectively) as compared to SETD2 proficient (+/+) 786-O cells (IC50 = >10 µM). Inhibition of the p110δ isoform alone had a modest effect on ccRCC cell growth and migration. Importantly, genetic (with siRNA) and pharmacologic (with BYL719) inhibition of p110α had no effect on the ccRCC cell lines. Inhibition of AKT with the small molecule inhibitor MK2206 recapitulated the effects seen with AZD8186 in ccRCC cell lines with SETD2 loss, suggesting PI3Kβ mediates the synthetic lethality via AKT signaling in the context of SETD2 loss in ccRCC. Our work strongly suggests a synthetic lethal-type of interaction between PI3Kβ inhibition and SETD2 loss and offers a rationale for further translational and clinical investigation of small molecule inhibitors of PI3Kβ in RCC. Xenograft studies are ongoing to investigate the in vivo efficacy of AZD8186.

Citation Format: Esteban Terzo, Yun C. Chiang, Anna Chytil, Valerie M. Jansen, Kimryn Rathmell. Synthetic lethal interaction between SETD2 loss and inhibition of PI3Kβ in clear cell renal cell carcinoma (ccRCC) [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 1942.
Abstract 195: Electrochemical treatment produces pH changes in the tumor microenvironment that are toxic to cancer cells
Authors: Perkons Nicholas , Stein Elliot , Nwaezeapu Chike , Wildenberg Joseph , Ackerman Daniel , Nadolski Gregory , Hunt Stephen , Gade Terence
Published: August 2018

Background: Locoregional therapy is playing an increasingly central role in the management of hepatocellular carcinoma (HCC), where ablation is distinguished among these techniques by its capacity to effect cure. Electrochemical treatment (EChT), a technique that facilitates necrosis via the application of direct current at low voltages shows promise; however, widespread use has been limited as studies of the underlying biology and the mechanism of action have been limited.

Methods: A novel assay for the study of EChT mechanism was designed, wherein HCC cells (2.5e5 cells/mL) were embedded in low-melting temperature agarose (1.5%). EChT was performed on these assays between nitinol cathodes (d = 0.5 mm) and platinum anodes (d = 0.5 mm) in a variety of geometries. Buffering capacity of the encapsulation assay was varied via the addition of HEPES buffer at final concentrations of 10mM, 50mM, or 200mM. Either during or immediately following EChT, the following measurements were recorded: cell viability, pH, ROS burden, transmembrane potential, and temperature.

Results: Following EChT, acidic pH was appreciated surrounding the anodes and basic pH surrounding the cathodes in regions where cell death was observed by fluorescence microscopy. Upon increasing the buffering capacity of the assay, the total area of cell death decreased (p < 10-6). There was not sufficient heat generation nor transmembrane potential to account for the observed cell death. A pH-threshold was identified, where a pH > 10.8 or < 4.4 was found to cause cell necrosis in the three different buffer conditions studied. By varying the number of electrodes, their spacing, and length of ablation, it was possible to shape the zone of cell death observed to an arbitrary geometry.

Conclusions: The mechanism of EChT-induced cancer cell death is through the spread of acidic and basic species generated by hydrolysis of water upon electron transfer at the electrodes. The diffusion of these species leads to cell death in a predictable, pH dependent manner. These results suggest that an electrochemical ablation device could treat complex HCC tumor geometries with appropriate electrode placement and charge delivery.

Citation Format: Nicholas Perkons, Elliot Stein, Chike Nwaezeapu, Joseph Wildenberg, Daniel Ackerman, Gregory Nadolski, Stephen Hunt, Terence Gade. Electrochemical treatment produces pH changes in the tumor microenvironment that are toxic to cancer cells [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 195.
Abstract 1965: Biologic evaluation of chalcone derivatives as ABCG2- and ABCB1-mediated drug resistance reversal agents
Authors: Cai Chaoyun
Published: August 2018

The overexpression of ATP-binding (ABC) transporters, including ABCG2 and ABCB1, has been shown to be related to multidrug resistance (MDR). In this work, we investigated a series of chalcones and bis-chalcones as ABCG2- and ABCB1-mediated resistance reversal agents. The results revealed that methoxyl chalcones have better reversal activities than hydroxyl chalcones toward ABCG2-mediated resistance. One methoxyl chalcone has good reversal activities in both ABCG2 and ABCB1 overexpressing cell lines. The compound can increase the accumulation of anticancer drugs in ABCG2 or ABCB1 overexpressing cell lines while combined with anticancer drugs, due to its inhibition of ABCG2 and ABCB1 (IC50 (ABCB1) =1.54 μM, IC50 (ABCG2) = 1.15 μM). In addition, this compound did not significantly alter the expression and localization of ABCG2 or ABCB1 in ABCG2 or ABCB1 overexpressing cell lines.

Citation Format: Chaoyun Cai. Biologic evaluation of chalcone derivatives as ABCG2- and ABCB1-mediated drug resistance reversal agents [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 1965.
Abstract 1980: S116836 overcomes BCR-ABL T315I mutation mediated imatinib resistance in chronic myeloid leukemia
Authors: Gupta Pranav , Zhang Guannan , Barbuti Anna Maria , Ding Ke , Pan Jingxuan , Druker Brian J. , Chen Zhe-Sheng
Published: August 2018

Chronic Myeloid Leukemia (CML) is largely caused by the Philadelphia (Ph) chromosome carrying the Break point Cluster Region-Abelson (BCR-ABL) oncogene. Imatinib is a BCR-ABL-targeted therapy and considered the standard of care in CML management. Resistance to imatinib therapy often develops because of mutations in the BCR-ABL kinase domain. In this study, we evaluated S116836, a novel BCR-ABL inhibitor, for its anti-cancer activity against BCR-ABL expressing BaF3 cells. S116836 shows potent activity against wild-type and T315I mutated BaF3 cells as compared with imatinib. S116836 inhibited the phosphorylation of BCR-ABL and its downstream signaling in BaF3/WT and BaF3/T315I cells. S116836 inhibited the mRNA expression of BCR-ABL in BaF3/WT and BaF3/T315I cells. Mechanistically, S116836 increased the cell population in the G0/G1 phase of the cell cycle, induced apoptosis and elevated ROS production in both BaF3/WT and BaF3/T315I cells. Moreover, S116836 inhibited tumor growth in BaF3/WT and BaF3/T315I tumor xenografts. Taken together, our results indicate that S116836 exhibits anti-proliferative effects and inhibits the imatinib-resistant T315I BCR-ABL mutation. S116836 may be a novel drug candidate for overcoming the resistance to imatinib in CML patients.

Keywords: S116836, Chronic myeloid leukemia, BCR-ABL, T315I, Resistance

Citation Format: Pranav Gupta, Guannan Zhang, Anna Maria Barbuti, Ke Ding, Jingxuan Pan, Brian J. Druker, Zhe-Sheng Chen. S116836 overcomes BCR-ABL T315I mutation mediated imatinib resistance in chronic myeloid leukemia [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 1980.
Abstract 1982: Obesity reversibly enhances mammary epithelial estrogen receptor alpha expression and progenitor activity
Authors: Chamberlin Tamara , D'Amato Joseph V. , Arendt Lisa M.
Published: August 2018

Obesity has been identified as an important risk factor for breast cancer in post-menopausal women and is significantly correlated with poor prognosis in breast cancer patients. Women with an increasing body mass index (BMI) are more likely to develop estrogen receptor alpha (ERα) positive tumors that are larger at the time of diagnosis and demonstrate an increased risk for metastasis. Given that obesity is becoming a global epidemic, understanding how obesity alters the biology of the normal breast is of significant clinical relevance. To examine the effects of obesity on mammary tissue, mice were fed a high fat diet to induce obesity and compared with mice fed a control diet. We also examined reduction mammoplasty tissue from women with a known BMI. In both obese female mice and women, mammary epithelial cell populations demonstrated significant decreases in basal/myoepithelial cells, when examined using either flow cytometry or cell type-specific markers (SMA and p63) in tissue. ERα expression was significantly increased in luminal cells in obese mammary tissue, compared with control mice or breast tissue from lean women. Functional assays demonstrated significantly enhanced mammary epithelial progenitor activity in obese mammary epithelial cells as well as elevated numbers of ERα positive epithelial cells that were co-labeled with markers of proliferation. Weight loss in a group of obese mice reversed increases in progenitor activity and ERα expression observed in obese mammary tissue. Obesity enhances the percentage of ERα positive epithelial cells and reduces the number of basal/myoepithelial cells within normal mammary tissue in both women and female mice. Both aspects of this phenotype have been linked to breast tumor susceptibility. These changes may contribute to lifetime risk for the development of ERα positive tumors in postmenopausal women. However, the changes in epithelial cell populations induced by obesity are reversible with weight loss, suggesting that weight loss interventions in obese women may effectively reduce breast cancer risk by decreasing the number of potential tumor progenitor cells in the breast epithelium.

Citation Format: Tamara Chamberlin, Joseph V. D'Amato, Lisa M. Arendt. Obesity reversibly enhances mammary epithelial estrogen receptor alpha expression and progenitor activity [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 1982.
Abstract 1985: Upregulation of miR-328 contributes to ovarian cancer stem cell maintenance by downregulating DDB2
Authors: Srivastava Amit K. , Cui Tiantian , Han Chunhua , Banerjee Ananya , Cai Shuri , Liu Lu , Zhang Xiaoli , Li Zaibo , Karuppalyah Selvendiran , Wani Altaf A. , Wang Qi-En
Published: August 2018

Cancer stem cells (CSCs) are considered to play a central role in the cancer progression, metastasis and the development of drug resistance. MicroRNAs (miRNAs) have important roles in regulating CSC properties and are considered to be potential therapeutic targets. Diverse aberrantly expressed miRNAs have been reported in ovarian cancer cells. However, there have been few reports about miRNAs that were associated with stemness and progression of ovarian cancer. In this study, miRNA Nanostring profiling analysis was performed to screen crucial miRNAs associated with characteristics and maintenance of CSCs in ovarian cancer. We found that miR-328-3p was remarkably upregulated in ovarian CSCs isolated from both ovarian cancer cell lines and primary ovarian tumors compared to their corresponding bulk cancer cells. We further demonstrated that enforced expression of miR-328-3p in ovarian cancer cell lines expanded the population of ALDH+ cells, enhanced their sphere formation ability, as well as increased their tumorigenicity. While inhibition of miR-328-3p limited the ALDH+ cell population, reduced their sphere formation capacity, and decreased their tumorigenicity. The orthotopic ovarian xenograft assay also demonstrated that inhibition of miR-328-3p impedes tumor growth and metastasis. The mechanistic investigation revealed that repressed ERK1/2 phosphorylation in ovarian CSCs, mainly due to reduced level of reactive oxygen species (ROS), contributes to the enhanced expression of miR-328-3p, and the maintenance of CSCs. Finally, we identified DDB2 as a direct target of miR-328-3p. Given our previous finding that DDB2 is capable of limiting the CSC population in ovarian cancers, we conclude that highly expressed miR-328-3p in ovarian CSCs, probably due to repressed ERK1/2 activity, inhibits DDB2 expression, resulting in the expansion of these CSCs. Thus, targeting miR-328 could be exploited to a novel strategy to eradicate CSCs in ovarian cancer.

Citation Format: Amit K. Srivastava, Tiantian Cui, Chunhua Han, Ananya Banerjee, Shuri Cai, Lu Liu, Xiaoli Zhang, Zaibo Li, Selvendiran Karuppalyah, Altaf A. Wani, Qi-En Wang. Upregulation of miR-328 contributes to ovarian cancer stem cell maintenance by downregulating DDB2 [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 1985.
Abstract 1988: The pivotal roles of adipocytes on regulation of cancer stemness and drug resistance in hepatocellular carcinoma
Authors: Gurung Shilpa , Lee Terence Kin-Wah
Published: August 2018

Hepatocellular carcinoma (HCC) is one of the deadliest cancers in the world with less than 10% 5-year survival rate. Liver cancer stem cells (CSCs) are sub-populations of the cancer cells that have been found to play a crucial role in tumor relapse and therapeutic resistance. Increasing reports showed the significant correlation between non-alcoholic fatty liver disease (NAFLD) and HCC development. Based on this evidence, we hypothesize adipocytes, one of the key cellular factors within the tumor microenvironment, play a crucial role in HCC development and progression via regulation of liver CSCs. To verify this, we have employed a co-culture system to dissect the potential cross-talk between fully differentiated adipocytes and HCC cells. Our pilot data showed that adipocytes enhanced the self-renewal ability of HCC cells through indirect paracrine secretion. Based on this finding, we examined the effect of the conditioned medium of adipocytes on regulation of liver CSC. Consistently, we found that HCC cells pre-incubated with conditioned medium of adipocytes showed enhanced in vivo tumorigenicity and chemo-resistance to doxorubicin and sorafenib. Furthermore, conditioned medium of adipocytes promoted the migration and invasion abilities of HCC cells. To delineate the molecular mechanism of how adipocytes, exert its CSC enhancing effect on HCC cells, we have collected the conditioned medium derived from adipocytes and analyzed their secretome profiles using Orbitrap Liquid Chromatography-Mass spectrometry. Upon analysis, we identified 209 proteins, among which we have focused on two proteins (CSF1 and FABP4) for further analysis as these are preferentially secreted from adipocytes. Respective inhibitors of the proteins are used to study the specificity of the protein in the medium and the downstream studied with the help of RNA sequencing which has been performed on conditioned medium treated HCC cells. Collectively, we demonstrated the pivotal role of adipocytes on regulation of liver CSCs through paracrine secretion. Thus, targeting adipocytes-derived signaling cascade may be a novel therapeutic strategy for treatment of fatty liver induced HCC.

Citation Format: Shilpa Gurung, Terence Kin-Wah Lee. The pivotal roles of adipocytes on regulation of cancer stemness and drug resistance in hepatocellular carcinoma [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 1988.
Abstract 1993: Rapid clonal evolution of dormant micrometastatic disease in head and neck cancer
Authors: Lagunas Angelica M. , Wu Jianchun , Crowe David L.
Published: August 2018

Head and neck cancer (HNC) is prone to locoregional recurrence which results in poor clinical outcome. Recent HNC clinical studies demonstrated that cervical lymph node dissection results in improved patient outcome even in early disease. These results suggest that dormant micrometastatic cancer occurs early in head and neck carcinogenesis. We used our established in vivo chemical carcinogenesis model of head and neck cancer to characterize dormant micrometastatic disease using genetic lineage tracing and null mutant mice. Histopathology was analyzed by hematoxylin and eosin staining. Telomere DNA damage response was determined by immunofluorescent 53BP1 localization at telomeres, and by ATM/ATR western blotting. Keratin expression and proliferation index were determined by immunohistochemistry. Apoptotic cells were determined by TUNEL analysis. Telomerase activity was determined by telomere repeat amplification protocol. Alternative lengthening of telomeres was determined by PML localization at telomeres, telomere circular DNA analysis, and chromosome orientation fluorescence in situ hybridization. Genomic instability was determined by high throughput sequencing. Cancer stem cells were sorted by flow cytometry and transplanted subcutaneously in Matrigel. Advanced HNC exhibited significant metastatic disease in the majority of lymph nodes. Early HNC and even premalignant lesions exhibited micrometastatic disease in most lymph nodes. These micrometastatic lesions contained multiple HNC clones that were dependent for proliferation on paracrine chemokine signaling from a tumorigenic keratin 15+ cancer stem cell population. In situ, the micrometastatic K15+ cancer stem cell population exhibited increased telomere DNA damage response including activated ATM and ATR which correlated with low proliferation index. Clonal evolution of K15+ micrometastatic cancer stem cells was dependent on extremely short telomeres. These cells expressed both telomerase and recombination-based alternative lengthening of telomeres (ALT) activities. K15+ micrometastatic cancer stem cells exhibited high levels of genomic instability resulting from telomere fusions and expressed metastasis-specific gene expression programs. These patterns of micrometastatic gene expression revealed targeted therapeutic vulnerabilities. These results indicate that dormant micrometastatic disease develops rapidly during head and neck carcinogenesis from normal epithelial cells with short telomeres and is sensitive to targeted therapies.

Citation Format: Angelica M. Lagunas, Jianchun Wu, David L. Crowe. Rapid clonal evolution of dormant micrometastatic disease in head and neck cancer [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 1993.
Abstract 1995: PTHrP drives tumor initiation signaling pathways in the PyMT model of breast cancer progression
Authors: Zhang Rui , Li Jiarong , Badescu Dunarel , Karaplis Andrew , Ragoussis Jiannis , Kremer Richard
Published: August 2018

Parathyroid hormone-related peptide (PTHrP) was first discovered in cancer patients as the primary cause of malignancy associated hypercalcemia (MAH) & is overexpressed in most human breast tumors. We previously showed that PTHrP ablation, in the MMTV-PyMT murine model of breast cancer progression can dramatically prolong tumor latency, slows tumor growth & metastases. Here we examined the signaling pathways under the control of PTHrP in the pre-neoplastic stage of tumor development (hyperplasia) by generating MMTV-PyMT model in which the mammary epithelium, expressing membrane-targeted GFP, is distinguished from the membrane-targeted red fluorescent backlight of stromal & nonepithelial-derived mammary tissues. We constructed Pthrpflox/flox; Cre+ mT/mG tumor mice (PTHrP conditional KO) & Pthrpwt/wt; Cre+ mT/mG tumor mice (WT control). Next, we applied FACS to enrich the GFP+ mammary epithelial cells isolated from PTHrP KO & control tissues for subsequent RNAseq analyses. We then examined differentially expressed genes (DEGs) by comparing purified cell populations from PTHrP KO & control tissues. We identified 939 DEGs (p value <0.01) & used DAVID bioinformatics resources to systematically analyze the KEGG pathways. Among the most significant pathways, extracellular matrix (ECM), focal adhesion,PI3K-Akt, RAS and WNT pathways were up-regulated in WT control cells compared to KO cells. In summary, PTHrP controls critical signaling pathways involved in breast cancer initiation at the pre-neoplastic stage & suggests that PTHrP ablation is a promising therapeutic strategy in breast cancer.

Citation Format: Rui Zhang, Jiarong Li, Dunarel Badescu, Andrew Karaplis, Jiannis Ragoussis, Richard Kremer. PTHrP drives tumor initiation signaling pathways in the PyMT model of breast cancer progression [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 1995.
Abstract 1998: Novel interplay between TANK-binding kinase (TBK1) and Yes-associated protein (YAP1) in KRAS mutant NSCLC
Authors: Saha Biswarup , Jaiswal Neha , Bora-Singhal Namrata , Chellappan Srikumar
Published: August 2018

Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related mortality in the United States. Lung adenocarcinomas are highly correlated with smoking and are characterized by mutations in KRAS, EGFR, BRAF and other oncogenes. Among them, KRAS mutations are widespread in adenocarcinomas in smokers and known to be a key player in various downstream signaling pathways contributing to genesis and progression of these tumors. More recently, the non-canonical IκB kinase, Tank Binding Kinase 1 (TBK1), has been found to facilitate tumorigenesis in KRAS mutant cancers. TBK1 has well documented functions in immune response, cell survival as well as in mitosis. While it has been suggested that TBK1-mediated regulation of Akt signaling might facilitate oncogenesis, the molecular mechanisms underlying TBK1 function downstream of KRAS is not fully elucidated. In a similar vein, Yes associated protein 1 (YAP1), the oncogenic component of the Hippo signaling cascade has been found to promote KRAS mediated oncogenesis and could substitute for the loss of Kras in mouse models of pancreatic cancer. In the present study, we have demonstrated a unique and novel molecular interplay between TBK1 and the oncogenic components of Hippo effector molecules, YAP1/TAZ. YAP1 and its ortholog, TAZ are known to be transcriptional co-activators, function to maintain organ size but often get activated in various types of cancer.

We find that TBK1 physically interacts with YAP1, and knock-down (KD) or knocking-out (KO) of TBK1 resulted in a significant elevation of YAP1/TAZ expressions at protein level. We have tested four different KRAS and EGFR mutant NSCLC cell-lines; interestingly, the upregulation of YAP1 upon depletion of TBK1 was only restricted to the KRAS mutant cell-lines. Further, depletion of TBK1 led to the enrichment of YAP1 in the nucleus; notably, there were only minimal changes in the levels of MST1/2 and LATS, raising the possibility that these changes occur independent of the classic hippo signaling pathway. Depletion of TBK1 also resulted in the induction of EMT-like features in these cells, and increased the proportion of stem-like side-population. Mechanistically, TBK1 physically interacts with YAP1 in the cultured cells, and could phosphorylate it in vitro, on T110, T114, S128 and S131 residues. The underlying molecular mechanism(s) by which TBK1 regulates YAP1 expression are under investigation, and we hypothesize that this regulatory event is exclusive to KRAS mediated oncogenesis, especially in the context of NSCLC.

Citation Format: Biswarup Saha, Neha Jaiswal, Namrata Bora-Singhal, Srikumar Chellappan. Novel interplay between TANK-binding kinase (TBK1) and Yes-associated protein (YAP1) in KRAS mutant NSCLC [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 1998.
Abstract 200: FOXD1 promotes stromal investment in clear cell renal cell carcinoma
Authors: Bond Kyle H. , Fetting Jennifer , Gupta Ashwani , Duarte Christine W. , Karolak Michele , Congdon Clare B. , Emery Ivette , hAinmhire Eoghainín Ó , Humphreys Benjamin D. , Oxburgh Leif
Published: August 2018

Forkhead transcription factors (FOX) play a role in the development of many cancers, including clear cell renal cell carcinoma (ccRCC). FOXD1, essential for angiogenesis in the developing kidney, has not previously been implicated in ccRCC. Tumor microarrays (TMAs) containing 41 patient-derived ccRCC tumors and 26 healthy cortex samples were stained for FOXD1, with 65% of tumor samples staining positively. Due to the effect of FOXD1 on angiogenesis, staining for stromal markers PECAM, αSMA, PDGFRβ and NG2 was also performed. A direct correlation was found only between FOXD1 and PDGFRβ (p<4.5x10-6), suggesting interstitial fibroblast recruitment by FOXD1+ cancer cells. Increased expression of FOXD1 was found to correlate with increased stage (p=9.1x10-5) and reduced survival (FOXD1 low= 2830 days; FOXD1 high= 1913 days) from a Kaplan-Meier analysis of RNA-seq data from The Cancer Genome Atlas (TCGA). Potential FOXD1 target binding sites were determined using the TRANSFAC FOXD1 binding site matrix. SLIT2, a factor known to inhibit migration of pericytes, was uncovered from the analysis and was further found to be downregulated in response to FOXD1 overexpression in renal proximal tubule cells (RPTECs). To test the influence of SLIT2 on interstitialfibroblast migration, scratch assays on NRK-49F (rat kidney fibroblast) and Gli-1 (cardiac fibroblast) cells were performed and showed that SLIT2 reduced PDGFBB-induced cell migration (p<0.027). A multiplex proximity ligation assay for multiple signaling pathways showed that SLIT2 treatment reduced the STAT signaling response to PGDFBB. To model the influence of SLIT2 on ccRCC stromal invasion in vitro, we devised a novel 3D invasion assay. In summary, 786-O ccRCC cancer cells were seeded into a 200um thick silk scaffold and cultured for 3 days to allow deposition of ECM. Scaffolds were then placed on top of a monolayer of interstitial fibroblasts and invasion into the cancer cell-filled scaffold was measured using confocal microscopy. The results showed a significant reduction of cell invasion into the cancer-filled matrix with the addition of recombinant SLIT2 to media (5µm p=1.34x10-4; 50µm p=1.64x10-2). In conclusion, we show that FOXD1 expression has prognostic relevance to patient survival in ccRCC, and that this may be due to modulation of SLIT2 expression.

Citation Format: Kyle H. Bond, Jennifer Fetting, Ashwani Gupta, Christine W. Duarte, Michele Karolak, Clare B. Congdon, Ivette Emery, Eoghainín Ó hAinmhire, Benjamin D. Humphreys, Leif Oxburgh. FOXD1 promotes stromal investment in clear cell renal cell carcinoma [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 200.
Abstract 2000: The regulatory role of endothelial nitric oxide synthase signaling in the growth of prostate cancer stem-like cells
Authors: Gao Weijie , Wu Dinglan , Wang Yuliang , Wang Zhu , Ma Taiyang , Chan Franky Leung
Published: August 2018

Cumulative studies indicate that there is a small subpopulation of cancer cells present in cancers or solid tumors referred as cancer stem-like cells (CSCs) as they share some common characteristics with stem cells, particularly self-renewal and multipotency, and these CSCs may contribute to the tumor initiation, treatment resistance and relapse. Recent advances also indicate that prostate cancer stem-like cells (PCSCs) may play a critical role in the development of castration-resistant prostate cancer (CRPC) and also its metastasis. Here, we demonstrated using an established CRPC xenograft model (VCaP-CRPC) that both the xenograft tumors and a xenograft-derived cell line contained more PCSC population, which showed higher expression levels of endothelial nitric oxide synthase (eNOS) and elevated intracellular NO production. PCSCs, isolated from prostaspheres derived from prostate cancer cell lines (DU145, LNCaP and VCaP) using a newly developed agar-based non-attachment 3D-culture method, exhibited up-regulation of eNOS and increased intracellular NO levels. Interruption of eNOS signaling by NOS inhibitors and shRNA-knockdown could significantly suppress the sphere formation capability of prostate cancer cells. Exogenous overexpression of eNOS could promote the tumor growth capacity of prostate cancer cells and their metastasis in vivo, while knockdown of eNOS could prevent the tumor growth and metastasis. Combined transcriptome-sequencing analysis and qPCR validation of prostaspheres upon eNOS-knockdown identified some potential targets of eNOS/NO signaling in the PCSC-enriched prostaspheres. In summary, our results suggest that eNOS/NO signaling may play a positive role in the growth regulation of PCSCs and eNOS might be a potential target for advanced prostate cancer. This study is supported by a General Research Fund from the Research Grants Council of Hong Kong (project code 14107617) and an Innovation and Technology Fund (GHP/003/16GD) from the Innovation and Technology Commission of Hong Kong.

Citation Format: Weijie Gao, Dinglan Wu, Yuliang Wang, Zhu Wang, Taiyang Ma, Franky Leung Chan. The regulatory role of endothelial nitric oxide synthase signaling in the growth of prostate cancer stem-like cells [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 2000.
Abstract 201: Multiregion joint somatic genotyping in ovarian cancer proves optimal tumor sampling for clinical sequencing
Authors: Pongor Lorinc , Munkacsy Gyongyi , Vereczkey Ildiko , Pete Imre , Gyorffy Balazs
Published: August 2018

Introduction. Genetic heterogeneity of ovarian tumors is caused by the accumulation of novel mutations through time. A result of this process is a spatially heterogeneous tumor with a fractal-like architecture, comprising of a mixture of spatially separated clones, sub-clones, and single cells. Spatial heterogeneity renders clinical sequencing a challenging task because clinically relevant mutations can go undetected. Here, we compare the genetic composition of single tumors with multiple DNA samples extracted from different regions of the tumor in diverse combinations to examine the effects of spatial separation on tumor heterogeneity.

Methods. We derived tumor samples from five patients with ovarian cancer. Three tumor tissue samples were collected from each patient, in each of these, DNA was extracted from a total of eight segments of different sizes (5x3x8 total segments). Exome sequencing was performed and we examined DNA mutations identified from a biopsy sample, from three merged tissue segments with immediate vicinity to the biopsy, and from segments from spatially distant tissue samples merged into one. We compared identified mutations and copy-number variations called from the exome-seq data.

Results. Three patients had heterozygous germline alterations affecting the BRCA pathway, paired with somatic TP53 mutations, one patient had multiple somatic mutations affecting the PI3K pathway, the last patient had the most common activating mutations in the PIK3CA and KRAS genes. When comparing the different sequencing runs, increasing the size of the tumor sample did not affect the overall quantity of mutations identified. Clonal mutations were identifiable in all samples, while sub-clonal mutations shifted. In case of a hypermutating phenotype, increasing the sequenced sample size strongly decreased the number of identified somatic mutations, most probably due to dilution of sub-clones. These observations are confirmed by an in silico model, in which we shuffled regions from publicly available multi-region sequencing data into known compositions.

Conclusion. In hypermutating tumors the number of detected mutations increases as the size of the sequenced sample decreases. Generally, increasing the tumor sample size did not affect the number of identifiable mutations. Our findings suggest that clinical sequencing using small biopsy samples can generate adequate mutation calling results.

Citation Format: Lorinc Pongor, Gyongyi Munkacsy, Ildiko Vereczkey, Imre Pete, Balazs Gyorffy. Multiregion joint somatic genotyping in ovarian cancer proves optimal tumor sampling for clinical sequencing [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 201.
Abstract 2014: CAMSAP3, a tubulin minus-end binding protein, attenuates epithelial-to-mesenchymal transition and alters microtubule dynamic in non-small cell lung cancer
Authors: Pongrakhananon Varisa , Wattanathamsan Onsurang , Takeichi Masatoshi
Published: August 2018

Cancer metastasis become the major cause of mortality in cancer patients, which the cancer migration exerts this aggressiveness. Epithelial to mesenchymal transition (EMT) plays an important role on cell movement and survival of the cancer cells during metastasizes. The establishment of front-rare polarization is involved with the alteration of cytoskeleton and cell-cell interaction, contributing to the notable morphological transformation from cobble stone-like epithelial to fibroblast-like mesenchymal feature. Microtubule dynamic has gained attention as a modulator behind these phenotypic changes and the EMT-related signal trafficking. Tubulin behavior has been shown to be governed by microtubule-binding proteins including a family of Calmodulin-regulated spectrin-associated proteins (CAMSAPs), a tubulin minus-end binding proteins. To illustrate the role of CAMSAPs on mesenchymal transition, CAMSAP2 and 3 genes were knockout using CRISPR-Cas9 system. Wound scratching and transwell migration assays demonstrate that non-small cell lung cancer becomes more motile with an elevation of actin stress fiber and focal adhesion protein paxillin following CAMSAP3 knockout, whereas CAMSAP2 depletion shows no significant effect. The mesenchymal markers including Slug, N-Cadherin and ZEB1 were substantial up-regulated, in opposite to the epithelial marker E-cadherin. Furthermore, CAMSAP3 removal causes a decrease of tubulin dynamic and consequently sustains an active state of EMT-regulatory proteins. This study reveals the negative

regulatory effect of CAMSAP3, a non-centrosomal microtubule minus-end binding protein, on mesenchymal transition and cell motility.

Citation Format: Varisa Pongrakhananon, Onsurang Wattanathamsan, Masatoshi Takeichi. CAMSAP3, a tubulin minus-end binding protein, attenuates epithelial-to-mesenchymal transition and alters microtubule dynamic in non-small cell lung cancer [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 2014.