bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 2790: Temporal immune changes of murine breast and melanoma tumor microenvironments following pulsed focused ultrasound
Authors: Cohen Gadi , Chandran Parwathy , Lorsung Rebecca M. , Tomlinson Lauren E. , Rosenblatt Robert B. , Burks Scott R. , Frank Joseph A.
Published: July 2021

Various cellular and biological immunotherapies have revolutionized the treatment of human malignancies by augmenting endogenous immune responses within the tumor microenvironment (TME). Although non-ablative pulsed-FUS (pFUS) has received less attention, it has been successfully used as a noninvasive treatment of solid malignancies by stimulating a pro-inflammatory immune response within the TME. Such an approach could be useful to treat peripheral micro-metastases in normal-appearing tissues. In the present study, we interrogated the immune-modulatory changes that occur within the TME following pFUS exposure. Murine B16 melanoma and 4T1 mammary carcinoma cells were subcutaneously implanted into both mice flanks (n=10 tumors/group/time point). The natural histories cytokine, chemokine, trophic factor (CCTF), and cell adhesion molecule (CAM) expression was evaluated within the TME of untreated flank tumors over 11 days. Ultrasound-guided pFUS at 1 MHz and peak negative pressures of 6 MPa was administered to tumors after reaching ~5 mm in diameter. Temporal immune-related changes in tumors were assessed following sonication using using proteomic, transcriptomic and histological analyses. Natural history growth characteristics showed a progressive increase in size for both tumors, and proteomic analysis revealed a shift toward an immunosuppressive TME. An anti-tumor TME was detected following pFUS treatment with increased expression of various pro-inflammatory CCTF and CAM as well as reduced tumor growth rates. Transcriptomic analyses following sonication identified inverted gene expression patterns in the two tumor types. Functional analysis of B16 tumors revealed increased intracellular signaling pathways associated with immune response regulations, while 4T1 tumors demonstrated reduced expression of proliferation genes. This study provides a macroscopic overview on the temporal dynamics of the TME and underline the profound magnitude of tumoral heterogeneity between tumor types as well as through progressive stages of similar primary tumors. Nevertheless, the induced immune-modulation changes suggest that pFUS may alter the expression of pro-inflammatory CCTF, CAM, towards an anti-tumoral TME, and supports the potential use of pFUS as a neoadjuvant treatment approach.

Citation Format: Gadi Cohen, Parwathy Chandran, Rebecca M. Lorsung, Lauren E. Tomlinson, Robert B. Rosenblatt, Scott R. Burks, Joseph A. Frank. Temporal immune changes of murine breast and melanoma tumor microenvironments following pulsed focused ultrasound [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2790.
Abstract 2826: BAG2 promotes tumorigenesis and metastasis by regulating the Cathepsin B cleavage in triple-negative breast cancer cells
Authors: Yang Kyung-Min , Pang Kyoungwha , Ooshima Akira , Lee Jihee , Kim Seong-jin
Published: July 2021

Breast cancer is classified into clinical subtypes based upon receptor expression (ER, PR, Her2). Among the breast cancer subtypes, triple-negative breast cancer (TNBC) refers to a form of breast cancer which lacks expression of ER, PR and HER2. Most of breast cancer treatments can be targeted for cancers that express hormonal receptors or HER2, whereas traditional chemotherapy does not work in TNBCs. Therefore, TNBC remains a clinical challenge for discovery novel specific target biomarker because of no targeted therapies for TNBC. In this study, we show that BAG2 promotes cancer progression by regulating the dual function of Cathepsin B in TNBC cells. We found that BAG2 is highly expressed in basal-like breast cancer cells, where it was associated with recurrence or distant metastasis-free survival in patients. In loss-of-function experiment, BAG2 knockdown strongly decreased the ability of tumor formation and lung metastasis in vivo in TNBC cells. Mechanistically, BAG2 induces metastasis through regulation of secretion of pro-form Cathepsin B and prevents lysosome-mediated cell death through decrease of intracellular single-chain form of Cathepsin B into cytoplasm. Thus, our results suggest that BAG2 promotes tumorigenesis and metastasis through regulation of Cathepsin B cleavage in TNBC cells and may be possible as a novel specific target for treatment of TNBC. [This work was supported by National R&D Program for Cancer Control, Ministry for Health and Welfare, Republic of Korea (HA17C0037).]

Citation Format: Kyung-Min Yang, Kyoungwha Pang, Akira Ooshima, Jihee Lee, Seong-jin Kim. BAG2 promotes tumorigenesis and metastasis by regulating the Cathepsin B cleavage in triple-negative breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2826.
Abstract 2829: Identifying germline variants for metastatic risk assessment in sporadic colorectal carcinomas
Authors: Cheah PehYean , Thean Lai Fun , Wong Michelle , Lo Michelle , Tan Emile , Tang Choong Leong
Published: July 2021

Colorectal carcinoma (CRC) is the third highest incidence cancer and leading cause of cancer mortality worldwide. Metastasis to distal organ is the major cause of cancer mortality. This trend is expected to be exacerbated by an aging population in the developed world. Genetic predisposition plays a greater role in disease progression than disease etiology in sporadic CRC. However, the underlying genetic factors for metastasis are currently unclear. We identified germline single nucleotide polymorphism (SNP) variants contributing to metastasis risk from a case-case (metastasis-positive vs metastasis-negative) genome-wide association study (GWAS) comprising 3,000 sporadic Chinese CRC cases with definitive metastasis status. Metastasis-positive case is defined as one with distal-organ involvement attributable to primary CRC; metastasis-negative case is defined as metastasis-free with 5 years or more follow-up. By interrogating summarized SNP statistics from the case-case GWAS using the Functional Mapping and Annotation (FUMA) software, we identified 5 genomic risk loci and 74 disease-relevant genes based on gene set enrichment analysis and the Molecular Signatures Database. These genes are in various pathways implicated in different metastasis steps, from epithelial-mesenchymal transition (EMT) to implantation at distal organs. They are different from the risk variants identified by case-control GWAS for disease occurrence. We will expression-profile 250 metastasis-positive vs metastasis negative cases from the GWAS panel to validate the bioinformatics analysis by quantitative real-time PCR. We will focus first on the five genes implicated in glycolysis as this is a biochemical fingerprint of malignant cells that represents one of the ‘hallmarks of cancer'. Expression profile of these genes will be validated in a second independent panel. Kaplan Meier survival analysis as function of the median expression value will be performed to assess prognostic value of these genes. The ultimate goal is to improve metastasis risk assessment for secondary prevention to reduce cancer mortality and morbidity for individual patient as well as reducing public health expenditure.

Citation Format: PehYean Cheah, Lai Fun Thean, Michelle Wong, Michelle Lo, Emile Tan, Choong Leong Tang. Identifying germline variants for metastatic risk assessment in sporadic colorectal carcinomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2829.
Abstract 2838: Epigenetic regulator MLL3 mutations promote tumor metastasis by enhancing the epithelial-mesenchymal transition state
Authors: Cui Jihong , Ge Kai , Guo Wenjun
Published: July 2021

Epigenetic dysregulations have a recognized role in cancer pathogenesis. Histone methyltransferase MLL3 (mixed-lineage leukemia protein 3) (gene symbol - KMT2C) is one of the most frequently altered genes in human breast cancer. In addition to the loss-of-function mutations, the expression levels of MLL3 are downregulated significantly in the tumor, compared to normal breast samples. Consistent with these cancer genome findings, functional studies in mouse models and human cancer cells showed loss of MLL3 promotes tumor initiation and growth. However, the role of MLL3 mutation in metastasis has not been determined.

Here we showed that the MLL3 levels were further reduced in distant metastases compared to primary tumors, suggesting a potential role of MLL3 loss in metastasis. We demonstrate that MLL3 loss markedly promoted distant metastasis through specifically enhancing the step of colonization during the metastatic cascade. MLL3 loss facilitated the acquisition of an epithelial/mesenchymal (E/M) transition state by cancer cells disseminated to the lung. These cells have a hybrid phenotype of epithelial and mesenchymal features. We further showed that the E/M transitional cells induced by MLL3 loss have greatly increased tumor-initiating ability and multi-organ metastatic ability. We further demonstrated that MLL3 lose facilitated luminal breast tumor cells to acquire the E/M hybrid features and promoted endocrine therapy resistance, suggesting that loss of MLL3 also promotes the epithelial-mesenchymal plasticity and therapeutic resistance.

To identify vulnerability of MLL3-mutant cancer cells, we performed small molecule screening and discovered that MLL3-mutant cancer cells are markedly more sensitive to BET inhibitors than MLL3-WT cells. The in vivo study confirmed that the BET inhibitor repressed the MLL3-mutant tumor growth much more efficiently than MLL3-WT tumors. Furthermore, the BET inhibitor is effective in suppressing the growth of established MLL3-mutant lung metastasis. Taken together, our findings indicate that MLL3 mutations promotes metastasis and therapeutic resistance by enhancing epithelial-mesenchymal plasticity. However, these aggressive cancer cells are uniquely vulnerable to BET inhibition, suggesting a potential therapeutic strategy for MLL3-mutant cancers.

Citation Format: Jihong Cui, Kai Ge, Wenjun Guo. Epigenetic regulator MLL3 mutations promote tumor metastasis by enhancing the epithelial-mesenchymal transition state [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2838.
Abstract 2851: Trop-2 inactivates E-cadherin for metastatic diffusion in the absence of EMT
Authors: Alberti Saverio , Guerra Emanuela , Relli Valeria , Lattanzio Rossano , Ceci Martina , Boujnah Khouloud , Garbo Valeria , Moschella Antonino , Altomare donato F. , Depalo Raffaella , Trerotola Marco
Published: July 2021

Trop-2 is a type I transmembrane glycoprotein, which is overexpressed in the majority of carcinomas, where it drives tumor cell proliferation. In its mature, glycosylated/functionally-competent form, Trop-2 associates with worse prognosis. A global quest was conducted for identifying decisive drivers of cancer metastasis. Cancer cell spheroids, wound healing and cell aggregation assays were utilized to assess cell-cell adhesion capacity. Immunofluorescence confocal microscopy and flow cytometry analysis were utilized to quantify expression of target signaling proteins, IHC analysis quantified the expression of target molecules in primary tumors and metastases. Pre-clinical models of orthotopic growth of colon cancer and metastatic diffusion to the liver were utilized. Xenotransplant transcriptome profiling analyzed transcription of EMT-related determinants. This led us to identify Trop-2 as a unique upregulated gene in colon cancer metastasis. Trop-2 was here shown to bind highly-expressed E-cadherin at the cell membrane. Trop-2 binding released E-cadherin from the cytoskeleton, inhibited cell-cell adhesion functions and activated signaling through β-catenin. This occurred in the absence of transcriptional downregulation of the CDH1 gene by EMT-associated transcription factors. The Trop-2/E-cadherin/β-catenin program was shown to lead to anti-apoptotic signaling, increased cell migration and enhanced cancer cell survival. In an orthotopic model of colon cancer, Trop-2 caused metastatic diffusion to the liver. The Trop-2-led E-cadherin-inactivation metastasis program was then found to induce metastatic relapse and shorter overall survival of colon cancer patients. Corresponding impact was indicated on breast, colon, ovary, uterus, stomach cancer metastatic diffusion, for broad impact on metastatic diffusion in human cancer.

Acknowledgments:Support was provided by grants of Italian Ministry of Development (MI01_00424) and of University and Research (SCN_00558). M.T. was supported by the Programma Per Giovani Ricercatori “Rita Levi Montalcini” (Grant PGR12I7N1Z).

Citation Format: Saverio Alberti, Emanuela Guerra, Valeria Relli, Rossano Lattanzio, Martina Ceci, Khouloud Boujnah, Valeria Garbo, Antonino Moschella, donato F. Altomare, Raffaella Depalo, Marco Trerotola. Trop-2 inactivates E-cadherin for metastatic diffusion in the absence of EMT [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2851.
Abstract 286: Conception and preparation of new potent antimicrotubule agents
Authors: Gagné-Boulet Mathieu , Bouzriba Chahrazed , Alvarez Atziri Corin Chavez , Fortin Sébastien
Published: July 2021

Cancer is still to this day one of the first causes of death in industrialized countries. Despite great progress made so far, there is still no treatment completely efficient and without toxicity. Therefore, the development of new anticancer drugs is sought. To this end, we developed a novel family of potent antimicrotubule agents designated as N-phenyl 4-(2-oxoimidazolidin-1-yl)benzenesulfonates (PIB-SOs). Our previous structure-activity relationship studies were focused on the aromatic rings A and B of PIB-SOs leaving the sulfonate bridge poorly studied. In the present study, we have designed, prepared and evaluated in vitro new PIB-SO analogs by replacing the sulfonate moiety with a sulfur atom. The new PIB-SO analogs were prepared using classical organic chemistry tools. The antiproliferative activity of PIB-SO analogs was assessed on HT-1080 fibrosarcoma, HT-29 colon adenocarcinoma, M21 skin melanoma and MCF7 estrogen-dependent breast adenocarcinoma. Then, the most potent analogs were assessed for their effect on the cell cycle progression, their capacity to disrupt the microtubules and their ability to bind to the colchicine-binding site (C-BS). Lastly, their theoretical properties were calculated with SwissADME tool. The new PIB-SO analogs bearing the sulfur bridge exhibit antiproliferative activities in the low nanomolar to mid micromolar ranges. The most potent compounds block the cell cycle progression in G2/M phase, disrupt microtubules and bind to the C-BS. Finally, their theoretical pharmacokinetic profiles show that they are potentially suitable for animal experiments. In addition to show that the sulfonate moiety of PIB-SOs can be modulated with a sulfur atom, our study led to the discovery of a new family of potent antimicrotubule agents targeting the C-BS. This study will contribute in the long term to the development of new treatments for cancer patients.

Citation Format: Mathieu Gagné-Boulet, Chahrazed Bouzriba, Atziri Corin Chavez Alvarez, Sébastien Fortin. Conception and preparation of new potent antimicrotubule agents [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 286.
Abstract 2865: Repurposing potassium channel blockers to target metastasis in triple negative breast cancer
Authors: Ram Priyanka , Payne Samantha L. , Oudin Madeleine J.
Published: July 2021

Despite the identification of pro-metastatic markers and signaling pathways that drive the invasion of triple negative breast cancer (TNBC), there are currently no treatments that directly target invading cells. Recent evidence suggests that the bioelectric state of cancer cells can drive cell proliferation and invasion, creating an opportunity to investigate new therapies that target the bioelectric state of a cell. Our previous data suggests that of the most abundant ions present in the body, potassium (K+) is most important for controlling the bioelectric state of TNBC cells, and that K+ flux affects the invasive properties of TNBC cells. Therefore, we hypothesized that blocking K+ channels would disrupt the bioelectric state and reduce 2D migration and 3D invasion. Four FDA-approved K+ channel blockers (amiodarone, carvedilol, imipramine, and thioridazine) were selected from the Repurposing Drugs in Oncology database based on inhibition target channels and mechanisms of action. Cell viability in human TNBC MDA-MB-231 and MDA-MB-468 cells was investigated and IC50 dose response curves were generated to determine that 5 µM and 10µM were the optimal doses to study further. Interestingly, while we determined that all four drugs reduced cell viability, amiodarone and carvedilol significantly changed the bioelectric state and reduced proliferation of TNBC cells compared to negative controls. Experiments are ongoing to evaluate the effects of these inhibitors on 2D migration, 3D invasion, and in an in vivo mouse xenograft study. Understanding the mechanism behind K+ channel inhibition in TNBC could give rise to new pharmacologic targets. In addition, supplementing existing metastatic TNBC regimens with a repurposed K+ channel blocker could potentially reduce development costs, decrease time to market, and provide a new treatment option to patients.

Citation Format: Priyanka Ram, Samantha L. Payne, Madeleine J. Oudin. Repurposing potassium channel blockers to target metastasis in triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2865.
Abstract 2871: Hypermetastatic stem-like cells from 'cancer of unknown primary' (CUP) model multi-organ dissemination and unveil liability to MEK inhibition
Authors: Verginelli Federica , Pisacane Alberto , Gambardella Gennaro , D'Ambrosio Antonio , Candiello Ermes , Ferrio Marco , Panero Mara , Casorzo Laura , Benvenuti Silvia , Cascardi Eliano , Senetta Rebecca , Geuna Elena , Ballabio Andrea , Montemurro Filippo , Sapino Anna , Comoglio Paolo M. , Boccaccio Carla
Published: July 2021

Cancers of Unknown Primary (CUPs), featuring metastatic dissemination in the absence of a primary tumor, anatomically or histologically recognizable through a standardized work-up, are a fatal disease and still a biological enigma. Here, we propose CUPs as a distinct, yet unrecognized, pathological entity originating from stem-like cells endowed with unique properties, irrespective of their different genetic backgrounds. These cells were isolated and long-term propagated in vitro in highly stringent conditions as ‘agnospheres', and serially transplanted in vivo, displaying an extremely high tumorigenic potential and reproducing the undifferentiated histology of the original tumors. Early after subcutaneous engraftment, agnospheres recapitulated the CUP clinical presentation, as they spontaneously and quickly disseminated, establishing widespread metastases and retracing the whole metastatic cascade. Agnospheres invariably displayed cell-autonomous proliferation and self-renewal, mostly relying on unrestrained activation of the MAP kinase/MYC axis. This feature conferred sensitivity to MEK inhibitors, which induced apoptosis in vitro and impaired in vivo growth and dissemination. We generated and validated a transcriptional signature that, applied to original tumors, predicts eligibility to MEK inhibition in 75% of CUP patients. Altogether, these findings shed light on CUP biology, unveiling an opportunity for a targeted therapeutic intervention and, concomitantly, provide a novel in vivo model, suitable for assessing molecular determinants of the metastatic cascade.

Citation Format: Federica Verginelli, Alberto Pisacane, Gennaro Gambardella, Antonio D'Ambrosio, Ermes Candiello, Marco Ferrio, Mara Panero, Laura Casorzo, Silvia Benvenuti, Eliano Cascardi, Rebecca Senetta, Elena Geuna, Andrea Ballabio, Filippo Montemurro, Anna Sapino, Paolo M. Comoglio, Carla Boccaccio. Hypermetastatic stem-like cells from 'cancer of unknown primary' (CUP) model multi-organ dissemination and unveil liability to MEK inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2871.
Abstract 2888: Reversible and dose-dependent suppression of motility of human breast cancer cells with ONC201 and/or TR57 in culture
Authors: Evstratova Yana , Kobyakova Margarita , Odinokova Irina , Stolyarov Alexander , Mishukov Artyom , Graves Lee M. , Holmuhamedov Ekhson
Published: July 2021

The cytotoxicity and cytostatic effects of imipridone family drugs ONC201 and its analog TR57 (Madera, USA) in variety of cultured breast cancer cell line (estrogen dependent BT474) was manifested through induction of ISR (Integrated Stress Response), UPRER and UPRMT. The major consequences of drug treatment are inhibition of proliferation and mitochondrial structural and functional damage(s). Our preliminary data demonstrated that 24 h exposure of BT474 leads to dysregulation and suppression in mitochondrial Ca2+ uptake and Ca2+ capacity (Odinokova et al., 2021). In the present work, we extend our study to include the effect of these drugs on the rate of migration intact and treated cells in culture. Treatment of BT474 cells in culture for 24h resulted in decreased motility of cells from 17 ± 2 µm/h in the absence of the drugs to 5 ± 1 µm/h and to 3±1 µm/h in cells treated with 10µM of ONC201 or 50nM of TR57 (n=5). This suppression was fully reversible and removal of drugs for 120h resulted in time dependent restoration of the initial motility of BT474 cells, and TR57 caused larger inhibitory effect on motility as compared with ONC201. Buffering of extracellular Ca2+ with 5 mM EGTA or intracellular Ca2+ with 10 µM BAPTA, 30 min suppressed motility of cells (19 ± 2 µm/h vs 6 ± 1 µm/h). Gd3+, selective blocker of store operated Ca2+ entrance (SOCE) pathway at concentration of 10 µM suppressed the motility of BT474 cells, and rate of migration changed for from 16 ± 2 µm/h in control to 7± 2 µm/h, in Gd3+ treated cells. In line with our preliminary data, taken together these data indicate that ONC201 and/or TR57 induced dysfunction in mitochondrial Ca2+ homeostasis, which translates into the suppression of the migration of human breast cancer cells in culture. Support of Funding Information: Russian Science Foundation № 19-75-20145

Citation Format: Yana Evstratova, Margarita Kobyakova, Irina Odinokova, Alexander Stolyarov, Artyom Mishukov, Lee M. Graves, Ekhson Holmuhamedov. Reversible and dose-dependent suppression of motility of human breast cancer cells with ONC201 and/or TR57 in culture [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2888.
Abstract 2891: Protein phosphatase 5 regulation of cell motility
Authors: Li n , Huang Shile
Published: July 2021

Metastasis has been considered as one of the cancer hallmarks. Approximately 90% of cancer-related death is due to cancer metastasis. Tumor cell migration is a prerequisite for cancer metastasis. Protein phosphatase 5 (PP5) is a serine/threonine protein phosphatase that belongs to the phosphoprotein phosphatase (PPP) family including PP1 and PP2A. It is well known that PP5 regulates apoptosis, proliferation, and stress response. However, the role of PP5 in the regulation of cell motility is only beginning to be recognized, and the mechanism of PP5 regulation of cell motility is largely unknown. In this study, our tissue microarray (TMA) studies showed the expression of PP5 increased with the stages of prostate cancer, which is consistent with the previous findings that there is a positive correlation between PP5 overexpression and breast cancer metastases. Furthermore, by the single-cell motility assay, we found that overexpression of PP5 increased the motility of various tumor cells, whereas knockdown of PP5 decreased the cell motility, suggesting that PP5 positively regulates cell motility. As focal adhesion kinase (FAK) is a key regulator of cell motility, and a report has shown that PP5 forms a complex with ASK1, FAK, CXCR4 in wounded alveolar epithelial cancer cells, we examined whether PP5 activates FAK. The Western blotting results showed that overexpression of PP5 did not obviously affect the phosphorylation of FAK. The results suggest that PP5 regulates cell motility through a new mechanism. Further research is on the way to identify how PP5 positively regulates cell motility. (Supported by the Feist-Weiller Cancer Center, LSU Health Sciences Center, Shreveport, LA, USA.)

Citation Format: n Li, Shile Huang. Protein phosphatase 5 regulation of cell motility [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2891.
Abstract 2900: PRAMEF2 ubiquitylates and degrades LATS1 leading to enhanced nuclear accumulation of YAP and tumorigenesis
Authors: Ghosh Madhurima , Das Sanjeev
Published: July 2021

PRAMEF2 (PRAME Family Member 2) is a Cancer Testis Antigen belonging to the PRAME multi-gene family. All Cancer Testis Antigens are expressed in male testicular germ cells while remaining absent from other adult tissues. Conversely, they show aberrant re-expression in several human malignancies and also elicit immunogenic responses in cancer patients. This makes Cancer Testis Antigens and thereby PRAMEF2, effective and novel targets for cancer vaccine development. However, the physiological function of PRAMEF2 underlying tumorigenesis remains unspecified. Hence, we performed a proteomic screen to explore its unique interactome in the cell. Subsequent size exclusion chromatography and co-immunoprecipitation assays confirmed the role of PRAMEF2 as a BC-box containing substrate recognition subunit of a Cullin 2 based-E3 ubiquitin ligase complex. In the proteomic screen, several proteins were identified, however in-vivo assays revealed LATS1 to be a specific and novel interacting partner of PRAMEF2. LATS1 is a Ser/Thr Kinase of the Hippo Pathway, known to aid tumor suppression. We observed PRAMEF2 mediated ubiquitylation and subsequent proteasomal degradation of LATS1 through ubiquitylation assay. The site for ubiquitylation was mapped to the highly conserved Lysine 860 residue on LATS1. Upon activation of the Hippo Pathway, LATS1 results in phosphorylation of the transcriptional co-activator YAP, leading to its cytosolic translocation. Consequently, PRAMEF2 mediated LATS1 regulation resulted in decreased YAP phosphorylation and its increased accumulation in the nucleus. Furthermore, RTqPCR array and ChIP analysis showed the enhanced expression of YAP target genes implicated in proliferation and metastasis. Moreover, PRAMEF2 depletion also diminished the proliferative capacity, migration potential and invasiveness of tumor cells. Studies in mouse tumor models confirmed that PRAMEF2 abrogates the tumor suppressive function of LATS1 leading to ameliorated tumor growth and metastasis. Taken together, our findings help establish the role of PRAMEF2, an unique Cancer Testis Antigen, as a substrate recognition subunit of a Cullin 2- based E3 ubiquitin ligase complex which ubquitylates and degrades LATS1. Hence, PRAMEF2 realizes its oncogenic potential by increasing YAP nuclear accumulation and promoting tumor cell proliferation and metastasis.

Citation Format: Madhurima Ghosh, Sanjeev Das. PRAMEF2 ubiquitylates and degrades LATS1 leading to enhanced nuclear accumulation of YAP and tumorigenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2900.
Abstract 2932: Dysregulated metastatic capacity of ovarian cancer spheroids in response to physiologically relevant media conditions
Authors: Grieco Joseph , Compton Stephanie Edwards , Smyth James , Schmelz Eva
Published: July 2021

Ovarian cancer is the deadliest gynecological cancer in women, with a survival rate of less than 30% when the cancer has spread throughout the peritoneal cavity. Aggregation of cancer cells increases their viability and metastatic potential; however, there are limited studies that correlate these functional changes to specific phenotypic alterations. In this study, we investigated how changes in media composition affects the metastatic potential of ovarian cancer cells. This was conducted by using aggregated ovarian cancer cells incubated in different media conditions supplemented with different levels of glucose, glutamine and serum. Additionally, we identified the adherence capacity through examining secretions and expression of extracellular matrix proteins as well as mitochondrial localization and quality control protein content that could support the viability of these aggregates. We determined that spheroid formation requires more glutamine and limited serum and glucose to allow for successful aggregation. Additionally, this media composition supports a more proliferative phenotype that closely resembles the high glucose controls. Low glucose and hypoxia also limit the ability of spheroids to adhere due to limited expression and secretion of collagen type I and fibronectin proteins. Further, we find that mitochondrial localization is promptly more affiliated with the adherence site of spheroids incubated in high glucose, high oxygen media in comparison to low glucose, hypoxic spheroids where mitochondrial localization is heavily dispersed. Finally, we find that mitophagy and mitobiogenesis are codependent in regulating mitochondrial content to support viability in cancer spheroids in peritoneal media conditions. Altogether, these data indicate that ovarian cancer spheroids adapt to non-permissive conditions through limiting their adherence capacity and upregulating mitochondrial quality control functions to support viability during dissemination.

Citation Format: Joseph Grieco, Stephanie Edwards Compton, James Smyth, Eva Schmelz. Dysregulated metastatic capacity of ovarian cancer spheroids in response to physiologically relevant media conditions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2932.
Abstract 2941: Development and characterization of the ultra-immunodeficient NOD.CB17-Prkdcscid IL2rgtm1/Bcgen (B-NDG) mouse model
Authors: Wildt Sheryl June , Naden Jamie , Xiang Jie , Shen Yuelei , Zhang Meiling
Published: July 2021

The “B-NDG” (NOD.CB17-Prkdcscid IL2rgtm1/Bcgen) mouse was designed and generated by Biocytogen. IL2rg (common gamma) gene was deleted in NOD-scid mice resulting in a mouse that lacks mature B cells, T cells, NK cells and has a deficiency in cytokine signaling. This level of immunodeficiency makes this model ideal for engraftment with human immune cells and human tumor cells or tissues. Herein we describe the development and characterization of the B-NDG model, which includes growth curve, complete blood count, serum chemistry, flow cytometry, tumor growth and humanization. A cohort of B-NDG mice (N=50 males and 50 females) were weighed weekly from 3 to 9 weeks of age. A cohort of six male B-NDG mice were analyzed to determine baseline levels for complete blood cell count and serum chemistry. Splenocytes of C57BL/6, NOD-scid and B-NDG mice were isolated and examined for total T cells, B cells, NK cells, CD4+ T cells, CD8+ T cells and regulatory T cells using flow cytometry. Data showed complete lack of all T cells, B cells and NK cells in the B-NDG modelTwo different methods of humanization were examined in the B-NDG model. Human peripheral blood mononuclear cells (hPBMCs) were injected at 5x106 into the caudal vein of three B-NDG mice. After 24 days, the hCD45+ and mCD45+ cells were examined and mice had 30-80% hCD45+ cells. Of those hCD45+ cells, over 99% were identified as T cells. The second humanization method used was injection of 1x105 human CD34+ hematopoietic stem cells after irradiation. The percentage of human CD45+ cell in the peripheral blood was over 20% by eight weeks post injection in over 70% engrafted mice. At 16 weeks post injection, of the total number of human lymphocytes in the humanized mice, 60% were B cells, 2% were NK cells and over 10% were T cells. The percentage of human T cells increases steadily with the extended observation window. We have demonstrated that B-NDG mice have several features that translate many benefits as compared to other immunodeficient models. This model has demonstrated better tumor growth than the NOD-scid and C.B17 scid mice for cell lines as well as PDX lines. We have also shown that this model is extremely useful for studies that require humanization of an immunodeficient mouse model.

Citation Format: Sheryl June Wildt, Jamie Naden, Jie Xiang, Yuelei Shen, Meiling Zhang. Development and characterization of the ultra-immunodeficient NOD.CB17-Prkdcscid IL2rgtm1/Bcgen (B-NDG) mouse model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2941.
Abstract 2950: Humanized C5AR1 mouse model (B-hC5AR1 mice) for evaluation of anti-C5AR1 mAb in vivo efficacy
Authors: Nie Yanhui , Zhou Xiaofei , Zhang Jing , Chromikova Veronika , Shen Yuelei
Published: July 2021

C5AR1 (complement C5a receptor 1) is a classical G protein-coupled receptor that is mainly expressed on granulocytes, monocytes, dendritic cells and MDSCs (Myeloid-derived suppressor cells) in several cancer types. Myeloid-derived suppressor cells (MDSCs) are a heterogeneous group of immature cells that play a significant role in inhibiting T cell-related responses. C5a can directly bind and activate MDSCs to upregulate C5AR1 expression, and that in turn leads to an increased stimulation of MDSCs by C5a, which further increases immunosuppression. The enhancement of cancer cell invasion and growth by the C5a-C5AR1 system suggests that this system is a possible target for cancer therapy. Currently, there is a lot of focus on research and development of antibody drugs targeting C5AR1. In order to generate a suitable animal model in which to evaluate the in vivo activity of humanized anti-C5AR1 antibodies, we have developed C5AR1 humanized mice (B-hC5AR1 mice) in which the murine C5ar1 gene is replaced with the human C5AR1 gene by homologous recombination. The viability of this model is supported by previous studies showing that mouse and human C5a bind human C5AR1 with comparable affinities. Our analysis shows that human C5AR1 was detectable in dendritic cells, granulocytes and macrophages isolated from spleen and dendritic cells isolated from bone marrow in homozygous B-hC5AR1 mice but not in wild type mice. Percentage of T cells, B cells, NK cells, DCs, granulocytes, monocytes, macrophages, CD8+ T cells, CD4+ T cells and Tregs in homozygous B-hC5AR1 mice were similar to those in the wild type mice, demonstrating that introduction of human C5AR1 in place of its mouse counterpart does not change the overall development, differentiation or distribution of these cell types. Following these results, we performed an in vivo efficacy evaluation of an anti-human C5AR1 antibody using the humanized mice. Mouse pancreatic ductal adenocarcinoma Panc02 cells were subcutaneously implanted into homozygous B-hC5AR1 mice (female, 6-7 week-old, n=5). Mice were grouped when tumor volume reached approximately 100 mm3, at which time they were treated with anti-human C5AR1 antibodies. We observed that anti-human C5AR1 antibodies were efficacious in controlling tumor growth in B-hC5AR1 mice. Mouse C5AR1 and human C5AR1 was detectable on MDSCs from TILs isolated of wild type mice and B-hC5AR1 mice, respectively. In conclusion, B-hC5AR1 mice are a promising model for preclinical in vivo pharmacodynamic assessment of anti-C5AR1 antibodies.

Citation Format: Yanhui Nie, Xiaofei Zhou, Jing Zhang, Veronika Chromikova, Yuelei Shen. Humanized C5AR1 mouse model (B-hC5AR1 mice) for evaluation of anti-C5AR1 mAb in vivo efficacy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2950.
Abstract 2954: Humanized CCR8 mouse model provides a translational tool for anti-human CCR8 antibody drug development
Authors: Wang Linlin , Shang Chengzhang , Zhang Jing , Chen Yichao , Kokabee Leila , Lin Qingcong
Published: July 2021

CCR8 is reported as an important chemokine receptor expressed on intra-tumoral Tregs and plays a critical role in CCR8+Treg-mediated immunosuppression via inducing STAT3-dependent overexpression of FOXP3, CD39, IL-10, and granzyme B on Tregs with interaction of CCL1 (CCR8 ligand). CCR8 becomes a promising target in the anticancer drug development. Generation of humanized CCR8 mouse model can greatly improve translation and accelerate CCR8-related drug development. To evaluate the anti-tumor efficacy of human CCR8 blockade in tumor mouse models, Biocytogen has successfully generated a novel CCR8 humanized knock-in mouse strain to support anti-human CCR8 antibody drug development. In this mouse model, the full length of encoding sequences of mouse CCR8 gene were replaced by the full length of encoding sequences of human CCR8 (hCCR8) gene. CCR8 gene expression in WT and B-hCCR8 mice were tested by RT-PCR. Human CCR8 mRNA was detected in hCCR8 mice model but not in wild-type mice. Protein expression of human CCR8 in hCCR8 mice was evaluated by flow cytometer in a colon cancer cell line MC38 tumor bearing mouse model. The human CCR8 protein were successfully detected in tumor-infiltrating Tregs in B-hCCR8 mice bearing MC38 tumor, but not in Treg cells from spleen. An anti-tumor efficacy study showed significant tumor growth inhibition of hCCR8 antibodies in B-hCCR8 mice bearing MC38 tumors, suggesting that the B-hCCR8 mouse model is an effective tool for in vivo efficacy evaluation of therapeutic hCCR8 antibodies to support anti-human CCR8 antibody clinical development.

Citation Format: Linlin Wang, Chengzhang Shang, Jing Zhang, Yichao Chen, Leila Kokabee, Qingcong Lin. Humanized CCR8 mouse model provides a translational tool for anti-human CCR8 antibody drug development [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2954.
Abstract 2956: Humanized IL17A/hIL17F mouse model provides a preclinical tool for the evaluation of therapeutic drugs targeting to IL-17 pathway
Authors: Li Chong , Zhao Lei , Zhu Tao , Chen Yichao , Kokabee Leila , Guo Chaoshe
Published: July 2021

Interleukin 17 (IL-17) is described as a pro-inflammatory cytokine produced by activated CD4 cells. Evidence shows that IL-17 is highly up-regulated at sites of inflammatory tissues of autoimmune diseases and amplifies the inflammation through synergy with other cytokines, such as TNF (tumor necrosis factor) α. Therefore, targeting IL-17/IL-17R, IL-17-producing pathways or IL-17-mediated signaling pathways can be considered for future therapy in autoimmune diseases. To evaluate the efficacy of IL17A and IL17F antibodies, we generated a double humanized B-hIL17A/hIL17F mouse in Biocytogen. In this model, the sequences encoding the extracellular domains of the mouse IL17A gene and the IL17F gene were replaced with the corresponding human sequences. IL17A and IL17F protein expression was detected in B-hIL17A/hIL17F mice by ELISA using a species-specific IL17A ELISA kit. In efficacy study of anti-human IL17A and IL17F antibody with psoriasis model induced in B-hIL17A/hIL17F mice, mice were treated with different dose of bimekizumab produced in house. Erythema and scaling score of the skin was significantly reduced compared with non-treatment group. The results suggest B-hIL17A/hIL17F mouse model is an effective tool for in vivo efficacy evaluation, which may represent a promising model for screening IL17 related drug candidates for the treatment of psoriasis disease.

Citation Format: Chong Li, Lei Zhao, Tao Zhu, Yichao Chen, Leila Kokabee, Chaoshe Guo. Humanized IL17A/hIL17F mouse model provides a preclinical tool for the evaluation of therapeutic drugs targeting to IL-17 pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2956.
Abstract 2966: Novel ex vivo model for ERα positive breast cancer
Authors: Munne Pauliina M. , Martikainen Lahja , Räty Iiris , Bertula Kia , Nonappa Nonappa , Peura Aino , Mutka Minna , Leidenius Marjut , Meretoja Tuomo , Kovanen Panu , Mattson Johanna , Heikkila Paivi , Joensuu Heikki , Ikkala Olli , Klefstrom Juha T.
Published: July 2021

Three-dimensional ex vivo cultures from patient-derived tumor tissue offer new opportunities for drug development and personalized treatment of cancer. However, one of the main challenges in establishing a tumor type faithful ex vivo tissue culture system is in optimization of biochemical and physical microenvironment to preserve desired phenotypic features. A collaboration with the Helsinki University Hospital enables us to receive live patient-derived breast cancer tissue samples from most breast cancer surgeries performed in the Southern Finland, typically samples arriving twice a week. Together with a group of materials scientists from Aalto Technical University we have explored seven different 3D culture matrices and defined their biomechanical properties and stiffness values with rheological measurements. Four investigated matrices preserved the luminal CK8+ cell phenotype of the luminal cancers they derived from, whereas rest of the matrices that included commonly used Matrigel and collagen scaffolds promoted a switch in the cell identity from luminal CK8+ to basal CK14+ phenotype. Furthermore, the genetic expression profiles examined with NGS corresponded to observed phenotypic switch. We noticed significant species-specific differences between mouse mammary and human breast samples in their morphology, phenotypic responses and ERα+ preservation to microenvironmental matrix stiffness. Furthermore, a combination of scaffold stiffness and composition of biochemical matrix constituency is required for long-term maintenance of ERα+ luminal tumour phenotype. Therefore, both physical properties and composition of the microenvironment need to be optimized for maintenance of breast tumor type specific phenotype and genetic profiles of ex vivo tumor explant cultures.

Citation Format: Pauliina M. Munne, Lahja Martikainen, Iiris Räty, Kia Bertula, Nonappa Nonappa, Aino Peura, Minna Mutka, Marjut Leidenius, Tuomo Meretoja, Panu Kovanen, Johanna Mattson, Paivi Heikkila, Heikki Joensuu, Olli Ikkala, Juha T. Klefstrom. Novel ex vivo model for ERα positive breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2966.
Abstract 297: Innovation in delivering synthetically challenging bicyclic arginase inhibitors to enhance immunotherapy
Authors: Li Derun , Zhang Hongjun , Lyons Thomas W. , Martinot Theodore A. , Achab Abdelghani , lu Min , Nogle Lisa M. , McMinn Spencer , Mitcheltree Matthew J. , Childers Matthew , Pu Qinglin , Gathiaka Symon , Palani Anand , Chakravarthy Kalyan , DeCastro Amy D. , O'Neil Jennifer , Afshar Roshi , Walsh Nicole C. , Fan Peter W. , Cheng Mangeng , Miller Richard , Doty Amy , Palte Rachel , Kim Hai-Young , Saurí Josep , Beard Adam , Brynczka Christopher , Fischer Christian
Published: July 2021

Arginase overexpression has been associated with poor survival rates in advanced cancer patients treated with Keytruda. High levels of Arginase may lead to a depletion of arginine within the tumor microenvironment, inhibiting the immune response. Thus, arginase inhibition has the potential to greatly enhance immunotherapy treatment. Discovering novel arginase inhibitors was met with several challenges including analyzing/purifying extremely polar chemical matter without chromophores, synthetically challenging space, and poor bioavailability of compounds with ClogP less than minus two.

Cross-functional collaborations and innovations rapidly overcame these challenges. Structural chemistry and modeling guided the design of novel arginase inhibitors. Analytical and purification groups developed innovative analytical/purification methods. Novel technologies including ReatIR and Vapourtec flow system, provided key intermediates to enable chartering into synthetically challenging space. Through creative cyclization strategies and active transport strategy to improve bioavailability, the team ultimately delivered a diverse set of potent and extremely synthetically challenging arginase inhibitors to study the potential of arginase inhibition. Preliminary in vivo evaluation showed single agent efficacy in an EMT6 model. These arginase inhibitors have the potential to enhance immunotherapy.

Citation Format: Derun Li, Hongjun Zhang, Thomas W. Lyons, Theodore A. Martinot, Abdelghani Achab, Min lu, Lisa M. Nogle, Spencer McMinn, Matthew J. Mitcheltree, Matthew Childers, Qinglin Pu, Symon Gathiaka, Anand Palani, Kalyan Chakravarthy, Amy D. DeCastro, Jennifer O'Neil, Roshi Afshar, Nicole C. Walsh, Peter W. Fan, Mangeng Cheng, Richard Miller, Amy Doty, Rachel Palte, Hai-Young Kim, Josep Saurí, Adam Beard, Christopher Brynczka, Christian Fischer. Innovation in delivering synthetically challenging bicyclic arginase inhibitors to enhance immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 297.