bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 5991: Glucose metabolic reprogramming in HBV-related hepatocellular carcinoma through C-terminal truncated HBx protein suppression of TXNIP
Authors: Zhang Yu
Published: September 2020

Hepatitis B virus (HBV) infection has been proven strongly associated with the initiation and progression of hepatocellular carcinoma (HCC). However, a substantial amount of virally-induced mechanisms that cause malignant transformation in the liver remains largely unexplored, especially for HBV-induced metabolic reprogramming. In the present study, we identified and confirmed the HBV integration breakpoints clustering at the C-terminal X gene is a tumor-specific event, and the C-terminal HBx is expressed more frequently in the tumors than normal liver tissues. Then, the oncogenic effect of the truncated HBx was validated in vitro and in vivo. Transcriptome sequencing revealed that TXNIP, as a downstream target of truncated HBx, negatively regulates glucose metabolism. Screening of public database and our clinical cohort exhibited that TXNIP is significantly down-regulated in HCC patients with truncated HBx expression, compared to those with full-length HBx expression and HBV-negative patients. Lower expression of TXNIP is negatively correlated with patient survival. We also reported evidence that truncated HBx down-regulates TXNIP expression by transactivation via the transcriptional repressor NFACT2. Additionally, the re-introduction of TXNIP inhibited the metabolic reprogramming from mitochondrial respiration to aerobic glycolysis induced by truncated HBx and further resulted in HCC growth arrest in vitro and in vivo. In summary, our finding proposes a promising diagnostic and therapeutic strategy for HBV-induced HCC.

Citation Format: Yu Zhang. Glucose metabolic reprogramming in HBV-related hepatocellular carcinoma through C-terminal truncated HBx protein suppression of TXNIP [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5991.
Abstract 60: Semaphorin 7a promotes cell survival and resistance to endocrine therapy in ER+ breast cancer
Authors: Lyons Traci Renae
Published: September 2020

Estrogen receptor positive (ER+) breast cancers comprise the largest percentage, ~70%, of all breast cancers diagnosed in the Unites States. Despite the development of effective anti-estrogen based therapies, such as tamoxifen, aromatase inhibitors, and fulvestrant, 1 in 5 women with an ER+ tumor will experience recurrence within 10 years of diagnosis. Thus, the largest number of breast cancer-related deaths each year are attributed to ER+ breast cancers. Additionally, metastatic ER+ breast tumors uniformly develop resistance to anti-estrogen-based therapies, including combination targeted therapies with CDK4/6 and mTOR inhibitors, which represent some of the best therapeutic advances of the last five years and reflect our increasing understanding of cross-targeting to overcome acquired resistance. Our studies have shown that Semaphorin 7a expressing tumors are resistant to ER targeting therapies, including tamoxifen and fulvestrant. Furthermore, Semaphorin 7a expressing tumors do not respond to CDK4/6 inhibition. We have utilized luciferase reporter assays to show that Semaphorin 7a is initially regulated by ER; we then utilized overexpression studies to show that sustained upregulation of Semaphorin 7a results in downregulation of ER in xenograft models. This downregulation of ER is accompanied by upregulation of cell survival signaling mediated by pro-survival kinase Akt. In TCGA and METABRIC datasets, Semaphorin 7a expression is associated with significant decreases in overall survival. Furthermore, using KmPlot analysis, we have shown that Semaphorin 7a expression in patients treated with endocrine therapy predicts for poor survival rates. Thus, we sought to determine whether Semaphorin 7a expressing tumors would be susceptible to PI3K/Akt targeted therapies. We observe that Semaphorin 7a expressing tumors are sensitive to PI3K/Akt inhibition, as well as to inhibition of the ER target gene Bcl2. Thus, we suggest that patients with high Semaphorin 7a expression be considered as candidates for the addition of Venetoclax, an FDA approved Bcl2 targeted therapy, to their current therapeutic regimen. As Venetoclax is in Phase II trials in combination with fulvestrant for advanced CDK4/6 inhibitor refractory breast cancer, we believe our results could be rapidly translated to the clinical setting and will improve outcomes for the thousands of ER+ breast cancer patient that develop resistance to endocrine therapy.

Citation Format: Traci Renae Lyons. Semaphorin 7a promotes cell survival and resistance to endocrine therapy in ER+ breast cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 60.
Abstract 6007: Post-fasting refeeding enhances intestinal stem cell-mediated regeneration and tumorigenesis
Authors: Imada Shinya
Published: September 2020

Although short-term fasting interventions improve intestinal stem cell (ISC) function, little is known about how much of this benefit comes from fasting versus the post-fasting refeeding response. Given the central role that ISCs play in driving injury-induced repair and in initiating early intestinal tumors, it is possible that fasting by boosting ISC function not only improves regeneration but also unexpectedly elevates intestinal tumorigenesis. To understand how fasting/refeeding interventions influence the role of ISCs in regeneration and early tumor formation, we assessed the function of ISCs to repair the intestinal lining after injury and to contribute to intestinal tumors. Notably, we find that 24 hours post-fasting refeeding boosts intestinal stem and progenitor cells proliferation and increases ISC function in organoid and in fate mapping assays compared to AL and fasted cohorts. Mechanistically, elevated mammalian target of rapamycin (mTOR) activity mediates the effects of refeeding in ISCs as treatment with mTOR inhibitor blocks these effects. Surprisingly, loss of the APC tumor suppressor gene in ISCs increased intestinal tumor numbers in the refed state compared to the AL and fasted states in an mTOR dependent manner. We propose that refeeding through mTOR signaling stimulates ISC function and in a context-dependent manner this can either improve repair after injury or promote intestinal tumorigenesis.

Citation Format: Shinya Imada. Post-fasting refeeding enhances intestinal stem cell-mediated regeneration and tumorigenesis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6007.
Abstract 6030: Role of immature CD44 o-glycosylation in enrichment of cancer stem cell population for aggressive pancreatic cancer
Authors: Leon Frank , Nimmakayala Rama K. , Chugh Seema , Gupta Rohitesh , Rachagani Satyanarayana , Batra Surinder K. , Ponnusamy Moorthy P.
Published: September 2020

Pancreatic cancer (PC) is an aggressive malignancy expected to be the second leading cause of cancer death by 2030. Accumulating reports demonstrate that truncated o-glycans are largely observed in PC and correlates with poor prognosis and reduced patient survival. We have previously shown that loss of C1GALT1 (Core 1 n-acetylgalactose transferase) expression in PC results in the upregulation of genes associated with aggressive PC and metastasis. To study the involvement of immature O-glycophenotype in pancreatic cancer stem cells (CSCs), we selected the single-transmembrane CD44 glycoprotein based on our unbiased approach using proteomics and RNA-sequence analysis.This highly o-glycosylated protein with diverse cellular activity is essential in cell-cell recognition and is a widely occurring CSC marker identified in several cancers. However, the functional properties of CD44 o-glycan variation in pancreatic CSCs are not well understood. Thus, we hypothesize that truncation of o-glycans on CD44 imparts activation of downstream targets responsible for self-renewal and maintenance of CSC in aggressive PC. Using a proteomics approach of enriched truncated o-glycans, we discovered CD44 as one of the top identified proteins in CRISPR/Cas9 C1GALT1 knockout PC cells. We observed enhanced expression of markers responsible for CSC self-renewal and characteristics as measured by FACS-based side-population analysis and tumor sphere formation as a result of a loss in C1GALT1. To determine a potential mechanism by which truncated o-glycans on CD44 enhances CSC properties, we deleted C1GALT1 and CD44 in PC cells. A loss of CD44 o-glycan truncation significantly reduced expression of the self-renewal CSC marker Nanog and phosphorylated p65, a major component in the NF-κB signaling pathway. Furthermore, pancreatic tumorigenesis was significantly reduced in athymic mice injected with PC cells as a result of C1GALT1 and CD44 deletion. These results provide a novel mechanism by which truncated o-glycans on CD44 is responsible for activating downstream targets for self-renewal and maintenance of CSCs. Our findings begin to contextualize the contributions of aberrant glycosylation on glycoproteins to accelerate metastatic properties by enhanced expression and characteristics of pancreatic CSC.

Citation Format: Frank Leon, Rama K. Nimmakayala, Seema Chugh, Rohitesh Gupta, Satyanarayana Rachagani, Surinder K. Batra, Moorthy P. Ponnusamy. Role of immature CD44 o-glycosylation in enrichment of cancer stem cell population for aggressive pancreatic cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6030.
Abstract 6044: BR101801: A potent and selective inhibitor of DNA-PK, potentiates the activity of topoisomerase II inhibitor in vitro and causes tumor inhibition in cancer models
Authors: Son Mi Kwon , Wang Seungho , Lee Bo Ram , Jeon Byeongwook , Kim Soo jung , Yang Enhui , Park Min , Lee Joo han , Myung Jayhyuk
Published: September 2020

Introduction: The DNA-dependent protein kinase (DNA-PK) plays an instrumental role in the overall survival and proliferation of cells. As a member of the phosphatidylinositol 3-kinase-related kinase (PIKK) family, DNA-PK is best known as a mediator of the cellular response to DNA damage. DNA-PK plays a key role in the repair of DNA double-strand breaks (DSBs) that are probably the most deleterious form of DNA damage. Inhibition of DNA-PK has been considered as an attractive approach to decrease resistance to therapeutically induced DNA DSBs. In this context, DNA-PK has emerged as an intriguing therapeutic target in the treatment of a variety of cancers, especially when used in conjunction with genotoxic chemotherapy. We have developed a high potent and selective inhibitor of DNA-PK, BR101801, which inhibits topoisomerase II inhibitor-induced γ-H2AX with an IC50. In this study, BR101801, a first-in class inhibitor of DNA-PK, will be introduced as a novel candidate of human cancer therapy.

Methods: The enzyme selectivity of BR101801 was determined by Eurofins kinase profiling services. To assess the effect of BR101801 on DNA-PK in LoVo cell, cells were pre-incubated with etoposide for 2 hours and then exchanged into new media containing BR101801 or other compounds without etoposide. Activity of BR101801 was evaluated based on prolongation of γ-H2AX using western blot. In vitro GI50 was tested by WST-based viability assay in various cancer cell lines. Synergy of the combination with cytotoxic agents was analyzed using CT-26 mouse model, respectively.

Results: In the enzymatic analysis, BR101801 showed selectivity on DNA-PK. (IC50 DNA-PK = 6 nM, respectively). The combined inhibitory effect of DNA-PK was comparable to the effect of BR101801. The several cytotoxic agents that are known as a booster of γ-H2AX, including doxorubicin and etoposide were selected and all combination treatments of BR101801 with the cytotoxic agents showed significant result by having less than a combination index value of 0.5. The in vivo efficacy of the combination with doxorubicin and oxaliplatin was assessed in a CT-26 colon cancer model

Conclusions: These data confirm that DNA-PK inhibition of BR101801 enhanced the efficacy of rage of DSB inducing agent in vitro and in vivo, providing a clear rationale for its clinical use.

Citation Format: Mi Kwon Son, Seungho Wang, Bo Ram Lee, Byeongwook Jeon, Soo jung Kim, Enhui Yang, Min Park, Joo han Lee, Jayhyuk Myung. BR101801: A potent and selective inhibitor of DNA-PK, potentiates the activity of topoisomerase II inhibitor in vitro and causes tumor inhibition in cancer models [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6044.
Abstract 6071: Role of nitric oxide in the invasive pancreatic cancer cells
Authors: Fujita Mayumi , Somasundaram Veena , Basudhar Debashree Basudhar , Cheng Robert Y.s. , Ridnour Lisa A. , Imadome Kaori , No Jae Hong , Bharadwaj Gaurav , Wink David A.
Published: September 2020

Pancreatic cancer is a metastatic tumor with an extremely low 5-year survival rate. Metastasis is a main cause of patient mortality as it is extremely difficult to treat. Thus, understanding the characteristics of cancer cell populations exhibiting the invasive phenotype is fundamental for developing novel strategies to counter metastasis and improve therapeutic efficacy. To characterize these invasive phenotypes, we compared the human PANC-1 parent cell line with PANC-1 cells (INV) that invaded through transwell inserts. INV cells exhibited increased invasiveness and higher resistance to Carbon-ion radiation compared to whole cultured, control PANC-1 cells (WCC). PANC-1 invasion was reduced by nitric oxide synthase (NOS) inhibitors, suggesting that nitric oxide (NO) plays a cardinal role in PANC-1 invasion. Cell invasion through extracellular membrane (ECM) involves complex regulation of cell adhesion and de-adhesion to ECM proteins. Suspended INV cells, as well as INV cells undergoing de-adhesion, showed enhanced NO production as well as induction of several pro-metastatic, and stemness-related genes. NOS inhibitor, L-NAME, reduced the expression of these pro-metastatic or stemness-related genes, and hampered spheroid formation ability, suggesting that NO can potentially influence PANC-1 aggressiveness. In addition, in vitro studies indicated that a MEK-ERK-dependent, JAK-independent mechanism promoted NOS/NO modulation of PANC-1 invasion. Furthermore, xenograft studies with INV and WCC in an NSG mouse model revealed a higher ability of INV in liver metastasis than WCC, and L-NAME diminished the metastasis observed in INV injected mice. In summary, these results implicate NO as a key mediator of therapeutic resistance to Carbon-ion radiation and metastasis of PANC-1 cells; inhibition of NOS demonstrates therapeutic potential as observed in the animal model by specifically targeting the metastatic cells.

Citation Format: Mayumi Fujita, Veena Somasundaram, Debashree Basudhar Basudhar, Robert Y.s. Cheng, Lisa A. Ridnour, Kaori Imadome, Jae Hong No, Gaurav Bharadwaj, David A. Wink. Role of nitric oxide in the invasive pancreatic cancer cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6071.
Abstract 6077: The role of STAT3 and Snai2 in ovarian cancer metastasis
Authors: Walker David F. , Lacroix Sarah J. , Makar Yuri , Walker Sarah R.
Published: September 2020

Ovarian cancer remains a deadly disease due in part to ovarian cancer metastasis. Recent work has demonstrated that ovarian cancer forms clusters of cells (spheroids) that attach to the peritoneum by clearing the mesothelial cell layer and then invade the underlying matrix to metastasize. Understanding the key pathways and downstream transcription factors that are driving ovarian cancer metastasis may allow us to develop new therapies to treat metastatic ovarian cancer. Importantly, we have found that the STAT3 transcription factor is necessary for mesothelial clearance by ovarian spheroids. To identify key downstream STAT3 target genes involved in ovarian cancer metastasis, we analyzed publicly available STAT3 chromatin immunoprecipitation (ChIP)-seq datasets for genes associated with epithelial-to-mesenchymal transition. One of the genes that we identified was the transcriptional modulator Snai2 (Slug). To verify that Snai2 was downstream of STAT3, we transfected ovarian cancer cells with siRNA to STAT3 and analyzed Snai2 gene expression. We found reduced expression of Snai2 with siRNA to STAT3 suggesting that STAT3 upregulates Snai2 expression. To determine the effects of Snai2 on mesothelial clearance by ovarian spheroids, we transfected ovarian cancer cells with siRNA to Snai2 or control, generated spheroids, and performed a mesothelial clearance assay. We found that reduced expression of Snai2 decreased the ability of the ovarian cancer cells to clear the mesothelial cell layer suggesting that Snai2 plays an important role in the early stages of ovarian cancer metastasis. Recognizing that Snai2 is a transcriptional modulator, we were interested in determining if Snai2 affected STAT3 activity. Analysis of available Snai2 ChIP-seq data identified three putative Snai2 binding sites associated with the STAT3 gene, suggesting that Snai2 may also regulate STAT3 gene expression. To determine if Snai2 affects STAT3 gene expression, ovarian cancer cells were transfected with siRNA to Snai2 and STAT3 mRNA expression was analyzed. From this, we found that reducing Snai2 expression also reduced STAT3 expression. Taken together, our data suggests that STAT3 and Snai2 form a feedforward loop promoting upregulation of each other and together they promote the early stages of ovarian cancer metastasis. Therefore, developing an inhibitor to one of these transcription factors would affect both proteins and may be beneficial to prevent or treat metastatic ovarian cancer.

Citation Format: David F. Walker, Sarah J. Lacroix, Yuri Makar, Sarah R. Walker. The role of STAT3 and Snai2 in ovarian cancer metastasis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6077.
Abstract 6078: Suppression of tumor-intrinsic IFNγ signaling in ARF6-mediated metastatic progression of melanoma
Authors: Rich Coulson P. , Rogers Aaron , Yoo Jae Hyuk , Wolff Roger , Odelberg Shannon J. , Holmen Sheri L. , Grossmann Allie H.
Published: September 2020

ARF6 is a small GTPase that controls the subcellular location of proteins to regulate invasion, proliferation and tumor microvesicle shedding. ARF6 is aberrantly activated in cutaneous melanoma and stimulates invasion in BRAFV600E-melanoma by controlling localization and activation of beta-catenin and PI3K/AKT. In uveal melanoma, ARF6 facilitates GNAQQ209L signaling to drive tumor growth by trafficking GNAQ to cytoplasmic vesicles. Although distinct diseases, pharmacologic inhibition of ARF6 reduces disease progression in both uveal and cutaneous melanoma models. These data underscore the importance of intracellular trafficking mechanisms in cancer and highlight the potential clinical utility of targeting this machinery by inhibiting ARF6. In order to genetically dissect the role of ARF6 in melanomagenesis, we utilized RCAS-mediated delivery of Cre recombinase, with or without constitutively activated ARF6 (Arf6Q67L), specifically to melanocytic cells to induce constitutive activation of BRAFV600E (BrafCA), deletion of Cdkn2a (Cdkn2af/f) and (Arf6f/f). Activation of ARF6 accelerates spontaneous metastasis while loss of Arf6 delays tumor onset and reduces tumor incidence and growth, dramatically improving survival of BrafCA;Cdkn2af/f mice, including in the presence of Pten loss. Unexpectedly, tumors expressing ARF6Q67L show significantly reduced expression of interferon-gamma (IFNγ) response genes, reduced CD8 (T cell) transcripts and an anti-apoptotic proteomics profile. Knockdown of Arf6 sensitizes BRAFV600E melanoma cells to IFNγ treatment reducing cell viability in vitro. Together these data demonstrate that ARF6 modulates BRAFV600E-driven tumor progression and suggest that ARF6 activation suppresses tumor intrinsic IFNγ signaling to evade the anti-tumor immune response during metastatic progression. Our in vivo models provide a new paradigm for understanding vulnerabilities in BRAF-mediated oncogenesis and implicate intracellular trafficking mechanisms as potentially actionable machinery in melanoma development, immune evasion and disease progression.

Citation Format: Coulson P. Rich, Aaron Rogers, Jae Hyuk Yoo, Roger Wolff, Shannon J. Odelberg, Sheri L. Holmen, Allie H. Grossmann. Suppression of tumor-intrinsic IFNγ signaling in ARF6-mediated metastatic progression of melanoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6078.
Abstract 6080: IL1RAP augments Cysteine metabolism and drives oxidative stress adaptation and lung metastasis in Ewing sarcoma
Authors: Zhang Hai-Feng , El-Naggar Amal M. , Cheng Hongwei , Prudova Anna , Delaidelli Alberto , He Jian-Zhong , Negri Gian Luca , Lizardo Michael , Yang Tianqing , Morin Gregg , Li Wei , Dimitrov Dimiter S. , Sorensen Poul H.
Published: September 2020

The high oxidative stress cancer cells experience during the metastatic cascade is an important determinant of metastasis. How cancer cells adapt to those stressful conditions remains elusive. Here our global translatome and proteome analyses have uncovered novel signatures exploited by oncogene-transformed cells to adapt and survive oxidative stress. We revealed that various oncoproteins promote the expression of the IL1RAP to alleviate oxidative stress and facilitate stress adaptation. Mechanistically, IL1RAP controls Cysteine metabolism, a key substrate and determinant of antioxidant glutathione synthesis. CTH, a crucial enzyme for de novo Cysteine synthesis and redox regulation, was identified as a key functional mediator of IL1RAP. Moreover, global interactome analysis uncovered IL1RAP as a novel component and enhancer of the System Xc− transporter (SLC7A11), which is involved in Cystine uptake. Thus, IL1RAP enhances Cysteine supply via both uptake and biogenesis. IL1RAP depletion rendered Ewing sarcoma cells susceptible to oxidative stress and ferroptosis in vitro, and dramatically mitigated local invasion and lung metastasis in mice. In patients with Ewing sarcoma, high-expression of IL1RAP in the tumors correlated with poor event-free survival. Therefore, we have defined a novel pro-metastatic mechanism driven by IL1RAP-mediated Cysteine metabolism and redox regulation.

Citation Format: Hai-Feng Zhang, Amal M. El-Naggar, Hongwei Cheng, Anna Prudova, Alberto Delaidelli, Jian-Zhong He, Gian Luca Negri, Michael Lizardo, Tianqing Yang, Gregg Morin, Wei Li, Dimiter S. Dimitrov, Poul H. Sorensen. IL1RAP augments Cysteine metabolism and drives oxidative stress adaptation and lung metastasis in Ewing sarcoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6080.
Abstract 6114: Host and neoplastic cells utilize Axl for breast cancer bone metastasis
Authors: Tanaka Mai , Siemann Dietmar W.
Published: September 2020

Breast cancer is the most commonly diagnosed malignancy and the second leading cause of cancer-related death for women in the United States. Approximately 90% of cancer-related death results from metastasis, and the bone is the most common site of metastasis for breast cancer. Upregulation of signaling pathways can promote the metastatic phenotypes of neoplastic cells, including cell migration, invasion, proliferation and angiogenesis. In addition, when breast cancer cells disseminate to the bone, neoplastic cells upregulate signaling pathways to activate osteoclast-induced bone resorption. A key pathway involved in metastatic spread of cancer cells is the receptor tyrosine kinase Axl. Genetic and pharmacologic Axl inhibition in both human and rodent breast cancer cell lines (MDA-MB-231 and 4T1) impaired tumor cell migration and invasion. Intracardiac inoculation of Axl knockdown (KD) MDA-MB-231 cells significantly decreased the number of bone metastases compared to the scrambled control MDA-MB-231 cells. Another mechanism by which Axl inhibition impaired bone metastasis is through the modulation of osteoclast maturation and activity. Co-culture of Axl KD breast cancer cells with murine osteoclast precursor cells (Raw264.7) significantly reduced the number of tumor cell-induced osteoclast resorption pits on bone slices. In addition, genetic and pharmacologic inhibition of Axl in Raw264.7 cells as well as bone marrow derived osteoclast precursor cells, significantly reduced osteoclast formation and activity. Scrambled control and Axl KD Raw264.7 cells were cultured in serum free medium for 24 hours, and their conditioned media were collected. The conditioned media of Axl KD Raw264.7 cells revealed significantly decreased levels of monocyte chemoattractant protein 1 (MCP-1), which known to promote osteoclastogenesis, compared to the scrambled control Raw264.7 cells. In summary, we have demonstrated that inhibition of Axl decreases breast cancer metastasis to the bone through modulation of the host and neoplastic cell functions. Collectively, these data indicate that Axl signaling may be a promising therapeutic target to impair tumor progression and metastasis.

Citation Format: Mai Tanaka, Dietmar W. Siemann. Host and neoplastic cells utilize Axl for breast cancer bone metastasis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6114.
Abstract 6119: RNAi rat models for drug discovery
Authors: Wang Chia-Lin , Yang Yu-ting , Vasileva Ana , Maurice Allison , Dow Lukas , Zuber Johannes , Lowe Scott , Premsrirut Prem K.
Published: September 2020

The cost of drug development has skyrocketed to an estimated $2.6B for every FDA approved drug primarily due to failures from lack of efficacy or safety. This suggests that our current preclinical validation process has been insufficient in predicting therapeutic potential and toxicity in humans. While genetically engineered mice have become the gold standard for dissecting cancer mechanisms and evaluating novel drug targets in vivo, the rat has historically been the major model species in many biomedical fields, notably toxicology and carcinogenicity testing; and for many scientists, the rat still remains the preferred rodent due to their larger size for surgical manipulation, repeat blood sampling, and their cognitive and physiological characteristics that more closely resemble humans than their mouse counterparts. Moreover, many hormone-dependent tumors cannot be engineered in mouse models and are better modeled in rats. Here, we take advantage of our two-step engineering approach and exploit the efficiency of CRISPR-based targeting to develop RNAi rat models that enable inducible and reversible gene silencing to simulate therapeutic regimes. We demonstrate that our approach allows us to rapidly generate RNAi rat models and mimic the function of the targeted small molecule inhibitors, such as BET inhibitors targeting Brd4. These results demonstrate that our high-throughput system currently used to generate RNAi mice is also applicable to the rat system and, by extension, other mammalian models. Inducible RNAi rat models will undoubtedly be powerful tools that can be used to model human cancers, to mimic the action of putative drugs, and to assess the potential of therapeutic targeting strategies in vivo prior to the costly drug development, ultimately guiding the development of safer and more effective drugs.

Citation Format: Chia-Lin Wang, Yu-ting Yang, Ana Vasileva, Allison Maurice, Lukas Dow, Johannes Zuber, Scott Lowe, Prem K. Premsrirut. RNAi rat models for drug discovery [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6119.
Abstract 6129: A scalable, non-biased screen for isolating functionally metastatic cell populations
Authors: Xiao Jerry , Lekan Alex , Stanton Kelly , Glasgow Eric , Agarwal Seema
Published: September 2020

Up to 90% of cancer-associated deaths are due to metastasis. Nonetheless, the mechanisms of metastasis are poorly understood, largely because of a lack of technologies to routinely and robustly isolate, propagate, and culture metastatic cells. Current cellular models for metastasis are hindered by a lack of scalability, a high cost per experiment, and the length of experiments. In this proof-of-principle study, we injected an invasive triple-negative breast cancer cell line, MDA-MB-231, into zebrafish embryos. Cells were monitored for invasion from the yolk sac into the zebrafish circulatory system, representing a cell population analogous to metastatic, circulating tumor cells. Within 5 days, cells had migrated from the primary injection site to the tail. Tails showing migration of cells were excised, digested, and the labeled human-cancer cells were propagated. Re-injection of propagated cells resulted in serial generations (F1 and F2). The time required for migration decreased with each succeeding generation; with the latest (F2) generation migrating to the tail within 24 hours. In addition to being more invasive, cells that migrated to the tail displayed a more mesenchymal phenotype compared to parental cells. Consistent with this finding, RNA-seq analysis showed an upregulation of genes involved in an epithelial to mesenchymal transition. Moreover, the highest differentially expressed genes have been independently associated with metastasis. Finally, in silico analyses demonstrated that the upregulated genes were clinically significant. Together these data provide proof-of-principle that we have established a scalable, rapid, and robust method that can provide routine access to invasive metastatic cells. The platform combines in vivo and in vitro components that can be used to isolate, enrich, and propagate metastatic cells from individual patients, and has the potential to advance basic and translational research in tumor metastasis. This cellular model relies solely on cell behavior and is a non-biased method that can be widely applied to all tumor types as it does not require specific biomarkers. In addition to providing a tool for advancing our understanding of metastases, the technology has the potential to be used in screening for drugs that specifically target metastatic cells; a critical unmet need in the treatment of human cancers.

Citation Format: Jerry Xiao, Alex Lekan, Kelly Stanton, Eric Glasgow, Seema Agarwal. A scalable, non-biased screen for isolating functionally metastatic cell populations [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6129.
Abstract 6163: Integrated stress response contributes to triple negative breast cancer radioresistance by regulating glutathione biosynthesis
Authors: Bai Xupeng , Ni Jie , Li Yong , Beretov Julia
Published: September 2020

Triple negative breast cancer (TNBC) is one of the most malignant gynecological cancer worldwide. Radiotherapy (RT) is an effective option for TNBC local control. However, TNBC tends to develop a radioresistant phenotype which results in a poor response of TNBC to following therapy. In this study, we identified integrated stress response (ISR) as the most activated signaling in radioresistant MDA-MB-231, MDA-MB-436, and MDA-MB-468 cells. We found that constitutive phosphorylation of eIF2α in radioresistant TNBC cells promoted the activation of activating transcription factor 4 (ATF4) via increasing its translational efficiency, which elicited the transcription of a set of genes implicated in GSH biosynthesis, including glutamate-cysteine ligase catalytic subunit (GCLC), solute carrier family 7 member 11 (SLC7A11), and cystathionine-γ-lyase (CTH), leading to a higher intracellular level of reduced glutathione (GSH) and an efficient reactive oxygen species (ROS) scavenging after irradiation (IR). Treating radiosensitive TNBC cells with salubrinal, a selective inhibitor of eIF2α dephosphorylation, can mimic the stress-resistant phenotypes of their radioresistant counterparts. In contrast, eIF2α dephosphorylation or ATF4 knockdown accelerated IR-induced ROS accumulation and increased IR-induced cell apoptosis in radioresistant TNBC cells in vitro. In combination with eIF2α dephosphorylation, IR significantly reduced the size and weight of radioresistant TNBC xenograft tumors in vivo. These data reveal ISR as an important mechanism underlying TNBC radioresistance and provide a rationale for exploring eIF2 signaling as a novel therapeutic target for TNBC treatment.

Citation Format: Xupeng Bai, Jie Ni, Yong Li, Julia Beretov. Integrated stress response contributes to triple negative breast cancer radioresistance by regulating glutathione biosynthesis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6163.
Abstract 62: Destabilization of NOXA mRNA as a common resistance mechanism to targeted therapies
Authors: Montero Joan , Gstalder Cecile , Kim Daniel J. , Sadowicz Dorota , Miles Wayne , Manos Michael , Cidado Justin R. , Secrist J. Paul , Tron Adriana E. , Flaherty Keith , Hodi F. Stephen , Yoon Charles H. , Letai Anthony , Fisher David E. , Haq Rizwan
Published: September 2020

Most targeted cancer therapies fail to achieve complete tumor regressions or attain durable remissions. To understand why these treatments fail to induce robust cytotoxic responses despite appropriately targeting oncogenic drivers, here we systematically interrogated the dependence of cancer cells on the BCL-2 family of apoptotic proteins after drug treatment. We observe that multiple targeted therapies, including BRAF or EGFR inhibitors, rapidly deplete the pro-apoptotic factor NOXA, thus creating a dependence on the anti-apoptotic protein MCL-1. This adaptation requires a pathway leading to the destabilization of the NOXA mRNA transcript. We find that interruption of this mechanism of anti-apoptotic adaptive resistance dramatically increases cytotoxic responses in cell lines and a murine melanoma model. Durable control of disease in vivo can be achieved with combinations of targeted therapy with an MCL-1 inhibitor. However, consistent with an adaptive resistance mechanism we have identified, the timing of drug treatment strongly influences the efficacy of the combination therapy. Collectively, our results identify NOXA mRNA destabilization/MCL-1 adaptation as a non-genomic mechanism that limits apoptotic responses. Our data indicate that optimal sequencing of MCL-1 inhibitors with targeted therapies could overcome widespread and clinically important therapeutic resistance, and achieve prolonged tumor responses in solid tumors.

Citation Format: Joan Montero, Cecile Gstalder, Daniel J. Kim, Dorota Sadowicz, Wayne Miles, Michael Manos, Justin R. Cidado, J. Paul Secrist, Adriana E. Tron, Keith Flaherty, F. Stephen Hodi, Charles H. Yoon, Anthony Letai, David E. Fisher, Rizwan Haq. Destabilization of NOXA mRNA as a common resistance mechanism to targeted therapies [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 62.
Abstract 6224: Novel antitumor semi-synthetic heliomycin derivatives direct targeting tNOX-NAD+-SIRT1 axis to activate apoptosis/autophagy, as determined by the cellular thermal shift assay (CETSA)
Authors: Chueh Pin Ju , Islam Atikul , Chen Xiao Qi , Chiu Shi Han , Tikhomirov Alexander S. , Shchekotikhin Andrey E.
Published: September 2020

The antibiotic heliomycin (resistomycin) has been studied since the 1950s, however, its low solubility in aqueous media has restricted its systemic bioavailability and therapeutic potential. We recently synthesized a series of novel aminomethyl derivatives that are readily soluble in aqueous solution. In this study, we examined their antiproliferative properties and underlying molecular mechanisms in p53 wild-type and p53-mutated cancer cells. We continuously monitored the cellular impact of these compounds using the xCELLigence System, a label-free, real-time cell monitoring platform that measures electrical impedance, displaying results as cell index values. The results from the system showed that heliomycin and its derivatives exhibited different biological effects on tumor cells. To further our understanding of the interactions between the novel derivatives and cellular targets, we utilized the cellular thermal shift assay (CETSA). Our CETSA results revealed that some of the derivatives that were directly engaged with tNOX enhanced its thermal stability, which in turn resulted in a marked attenuation of the intracellular NAD+/NADH ratio. This attenuated ratio signifies a decrease in the NAD+-dependent activity of Sirtuin 1 (SIRT1) deacetylase and triggers apoptosis/autophagy, with varying amounts of decrease depending on the cellular environment. We have successfully transformed heliomycin into derivatives that are much more soluble, allowing the more efficient modulating of biological activities - a significant step closer towards finding a novel anti-cancer candidate.

Citation Format: Pin Ju Chueh, Atikul Islam, Xiao Qi Chen, Shi Han Chiu, Alexander S. Tikhomirov, Andrey E. Shchekotikhin. Novel antitumor semi-synthetic heliomycin derivatives direct targeting tNOX-NAD+-SIRT1 axis to activate apoptosis/autophagy, as determined by the cellular thermal shift assay (CETSA) [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6224.
Abstract 6236: Mil protein-shelled gold nanoparticle to treat glioblastoma multiform
Authors: Kim Hyungshik , Park In-Kyu , Huh Kang-Moo , Lee Dong-Yun
Published: September 2020

Gold nanoparticle (AuNP) as a photothermal therapy (PTT) agent has been reported in various application due to its physical properties. If gold nanoparticle can be orally absorbed, its application may be extended. Unfortunately, it has low absorption efficiency from the gastrointestinal (GI) tract to blood stream. To overcome its limitation orally, here we newly synthesized lactoferrin (Lf; a milk protein)-conjugated gold nanoparticles (Lf-AuNP). Interestingly, this Lf-AuNP could be orally absorbed and targeted to the brain cancer cells due to its interaction with lactoferrin receptor (Lf-R) on the intestinal cells, Blood-Brain Barrier (BBB) and cancer cells. Based on these findings, the Lf-AuNP was orally absorbed with higher bioavailability and targeted to the brain cancer cells (U87MG cells) that was orthotopic animal model. Fortunately, when specified wavelength of laser was noninvasively irradiated to the head of orthotopic animals, the Lf-AuNP made the higher temperature to the head, thereby killing the brain cancer cells. Collectively this milk protein-based oral delivery of AuNP could be a promising strategy as a platform technology.

Citation Format: Hyungshik Kim, In-Kyu Park, Kang-Moo Huh, Dong-Yun Lee. Mil protein-shelled gold nanoparticle to treat glioblastoma multiform [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6236.
Abstract 6259: Exosomal formulation escalates cellular uptake of honokiol leading to enhanced antitumor efficacy in pancreatic cancer
Authors: Khan Mohammad Aslam , Kanchanapally Rajashekhar , Deshmukh Sachin Kumar , Srivastava Sanjeev K. , Singh Seema , Singh Ajay P.
Published: September 2020

Pancreatic cancer (PC) is the third leading cause of cancer-related death in the United States. It is challenging to treat PC due to the existence of intrinsic and extrinsic chemoresistance mechanisms. We previously reported that honokiol, a phytochemical isolated from Magnolia plant, targets important signaling pathways involved in PC associated with survival, chemoresistance and stromal remodeling, and exhibits significant anti-tumor activity both in vitro and in vivo. However, considering poor bioavailability and tumor uptake as major hurdles in clinical translation, we made efforts to develop an exosomal nanoformulation of honokiol. Mesenchymal cell-derived exosomes are non-immunogenic and express surface markers that could support tumor-targeted delivery. Maximum entrapment of honokiol was achieved when it was used in 1:4 weight ratio with exosomes and subjected to 6 cycles of sonication. Data from dynamic light scattering suggested that key features of exosomes (size, polydispersity and integrity) remained intact post-honokiol encapsulation. The comparative half maximal inhibitory concentration (IC50) data suggested 4 to 5 times greater therapeutic efficacy of exosomal honokiol as compared to that of free honokiol. Similarly, exosomal-honokiol had significantly superior effect on cell cycle progression, apoptosis and expression of cell cycle- and survival-associated proteins than that observed in PC cells treated with equivalent doses of free honokiol. We also found that the cellular uptake of honokiol was significantly greater for exosomal-honokiol as compared to its free form. Together, our work is the first demonstration of exosomal encapsulation of honokiol and its improved anti-tumor efficacy against pancreatic cancer.

Citation Format: Mohammad Aslam Khan, Rajashekhar Kanchanapally, Sachin Kumar Deshmukh, Sanjeev K. Srivastava, Seema Singh, Ajay P. Singh. Exosomal formulation escalates cellular uptake of honokiol leading to enhanced antitumor efficacy in pancreatic cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6259.
Abstract 627: Evaluating the efficacy of enzalutamide and the development of resistance in a preclinical mouse model of type-I endometrial carcinoma
Authors: Koivisto Christopher Steven , Melodie Parrish , Santosh Bonala , Ngoi Soo , Nahhas Georges J. , Liu Bei , Torres Adrian , Gallagher James , Snchez-Hodge Rebekah , Zeinner Victor , Cohn David , Backes Floor , Goodfellow Paul , Chamberlin Helen , Leone Gustavo
Published: September 2020

Androgen Receptor (AR) signaling is a critical driver of hormone-dependent prostate cancer and has also been proposed to have biological activity in female hormone-dependent cancers, including type I endometrial carcinoma (EMC). In this study, we evaluated the preclinical efficacy of a third-generation AR antagonist, enzalutamide, in a genetic mouse model of EMC, Sprr2f-Cre;Ptenfl/fl. In this model, ablation of Pten in the uterine epithelium leads to localized and distant malignant disease as observed in human EMC. We hypothesized that administering enzalutamide through the diet would temporarily decrease the incidence of invasive and metastatic carcinoma, while prolonged administration would result in development of resistance. Short-term treatment with enzalutamide reduced overall tumor burden through increased apoptosis but failed to prevent progression of invasive and metastatic disease suggesting that AR signaling may have biphasic, oncogenic and tumor suppressive roles in EMC. Enzalutamide treatment increased Progesterone Receptor (PR) expression within both stromal and tumor cell compartments. Prolonged administration of enzalutamide decreased apoptosis, increased tumor burden and resulted in the clonal expansion of tumor cells expressing high levels of p53 protein, suggestive of acquired Trp53 mutations. In conclusion, we show that enzalutamide induces apoptosis in EMC but has limited efficacy overall as a single agent. Induction of PR, a negative regulator of endometrial proliferation, suggests that adding progestin therapy to enzalutamide administration may further decrease tumor burden and result in a prolonged response.

Citation Format: Christopher Steven Koivisto, Parrish Melodie, Bonala Santosh, Soo Ngoi, Georges J. Nahhas, Bei Liu, Adrian Torres, James Gallagher, Rebekah Snchez-Hodge, Victor Zeinner, David Cohn, Floor Backes, Paul Goodfellow, Helen Chamberlin, Gustavo Leone. Evaluating the efficacy of enzalutamide and the development of resistance in a preclinical mouse model of type-I endometrial carcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 627.
Abstract 6274: Radiosensitizing effect of beta-Apopicropodophyllin is mediated from ER stress against colorectal cancer
Authors: Kang A-Ram , Lee Na-Kyung , Shin Hyun-Jin , Um Hong-Duck , Park Jong Kuk
Published: September 2020

β-Apopicropodophyllin (APP) is a synthesized derivative of podophyllotoxin (PPT), which is a natural product that has been used as a traditional drug to decrease immunosuppression, exert antiviral effects for the treatment of influenza, venereal warts, measles and herpes, and even to treat skin cancer. In this study, we tested whether β-apopicropodophyllin (APP) could act as a radiosensitizer in colorectal cancer. To demonstrate radiosensitizing activity of APP, several in vitro tests such as clonogenic assay, immunoblotting, Annexin V-Propidium iodide (PI) assay, detection of mitochondrial ROS/intracellular of H2O2, mitochondrial membrane potential detection were performed. We also tested in vivo radiosensitizing activity of APP as performing colorectal cancer-derived xenograft experiments with mice, and detected tumor volumes and apoptotic cell death. Clonogenic assay showed a combination of APP and γ-ionizing radiation (IR) inhibits cell growth and increases cell death in colorectal cancer cells. Several signal transduction pathways were examined for its potential involvement, and then these results indicated enhanced ER stress is main radiosensitization effect of APP as assessed by immunoblotting analyses. In addition, disrupted mitochondrial membrane potential and increased reactive oxygen species (ROS) were also observed in cells co-treated with APP and IR; these phenomena were followed by the cytosolic release of cytochrome c and consequent activation of caspase-3 and -9. Interestingly, pharmaceutical inhibition of JNK or p38, which prevents caspase activation, blocked the APP/IR-induced activations of ER stress and apoptotic cell death. Therefore, our results implied APP has a radiosensitizing function to induce apoptotic cell death via activation of ER stress, disruption of mitochondrial membrane potential, and induction of the caspase pathway via JNK or p38 induction.

Citation Format: A-Ram Kang, Na-Kyung Lee, Hyun-Jin Shin, Hong-Duck Um, Jong Kuk Park. Radiosensitizing effect of beta-Apopicropodophyllin is mediated from ER stress against colorectal cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6274.