bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 2770: A novel MIRO2/MYO9B/RhoA signaling axis controls tumor cell invasion and metastasis
Authors: Boulton Dillon P. , Hughes Connor J. , Vaira Valentina , Jindal Ajita , Raza Masoom , Purdy Stephen Connor , Sant Shilpa , Ford Heide L. , Caino M. Cecilia
Published: March 2024

Metastasis of cancer cells to vital organs remains the leading cause of cancer related deaths, emphasizing a strong need for actionable targets in advanced stage cancer. To address this, we study novel dysregulated mitochondrial signaling mechanisms that cells utilize to metastasize. Here, we focus on how outer mitochondrial membrane protein—Mitochondrial Rho GTPase 2 (MIRO2)—promotes tumor cell invasion and metastasis. Our previous work identified higher MIRO2 mRNA expression in cancer vs. normal patient samples in a multitude of cancer types, which correlated with worse patient outcomes. Furthermore, we demonstrated that MIRO2 was critical for prostate cancer cell growth and survival in vitro and in vivo. However, it remains unknown if MIRO2 only affects primary tumor growth or if this protein is important throughout tumor progression. Using siRNA mediated knockdown (KD) of MIRO2 we find MIRO2 KD ubiquitously reduces tumor cell invasion in breast, melanoma, pancreatic, and prostate cancer cells. Utilizing metastatic prostate and breast cancer models, we demonstrate that mice injected with MIRO2 shRNA cells have significantly lower metastatic burden compared to mice injected with control shRNA cells. To determine the mechanism that MIRO2 controls invasion and metastasis, we looked at which of the top protein-binding partners for MIRO2 may regulate tumor cell invasion. Of our top hits, we found that depletion of atypical myosin IXB (MYO9B) by siRNA most significantly reduced tumor cell invasion and phenocopied MIRO2 depletion. MYO9B has a well-defined role in controlling cell motility via inactivation of RhoA. Excitingly, we have found 1) MIRO2 KD results in increased active RhoA, phenocopying MYO9B KD, 2) dual ablation of MIRO2 and RhoA fully rescues tumor cell invasion, and 3) MIRO2 is required for MYO9B driven invasion. Taken together, we propose a novel signaling mechanism by which MIRO2 broadly promotes invasion and metastasis through MYO9B dependent inactivation of RhoA.

Citation Format: Dillon P. Boulton, Connor J. Hughes, Valentina Vaira, Ajita Jindal, Masoom Raza, Stephen Connor Purdy, Shilpa Sant, Heide L. Ford, M. Cecilia Caino. A novel MIRO2/MYO9B/RhoA signaling axis controls tumor cell invasion and metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2770.
Abstract 2779: The significance of TIMP-1 and hypoxia in cancer stemness: A synergistic role in chemoresistance
Authors: Thirusenthilarasan Ilamathi , Xiao Wei , Rojiani Mumtaz V.
Published: March 2024

Chemotherapeutic resistance in non-small-cell lung cancer (NSCLC) poses a formidable clinical challenge. Numerous studies have highlighted the critical role of tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) in tumorigenesis and, increasingly, in the development of chemoresistance. We have studied multiple aspects of the MMP-independent, tumor-promoting function of TIMP1 in human NSCLC. Our recent studies have found a pivotal role for TIMP1 in chemoresistance. In order to gain an enhanced comprehension of the mechanisms underpinning TIMP-1-mediated chemoresistance, we have sought to determine the contribution of CSCs and hypoxia. TIMP-1 plays a role in propagating stemness, although relatively unexplored in the context of CSCs within NSCLC. A second crucial factor in the development of chemoresistance in NSCLC is the interplay between intratumoral hypoxia and CSCs. As the tumor grows, metabolic demand for oxygen increases and hypoxic regions are seen in areas furthest from the blood vessels. We have shown TIMP1 levels to increase under experimentally-induced hypoxia. In the present study, we cultured NSCLC cell lines and their TIMP1 KD clones in an assay with serum-free media and found a significant decrease in spheroid size in TIMP1 KD clones. Postulating a reduction in CSC population in KD clones, we determined the NSCLC CSC markers CD133 and ALDH’s expression in the NT and KD TIMP1 clones. Flow cytometry results showed that CD133pos and ALDHhigh populations were significantly decreased in TIMP1 KD clones relative to NT clones. Moreover, KD clone of TIMP1 showed a decrease in the levels of CSC-specific Transcription factors Oct 4A and Nanog. Taken together, our findings confirm that reduction in TIMP1 expression negatively impacts CSC populations. Furthermore, we determined the mRNA levels of CD44, another CSC marker also expressed in lung cancer is upregulated under hypoxia (1% O2) in both the NT and KD clones of TIMP1. Thus, we postulate that TIMP1 enhances chemoresistance by promoting CSC propagation within a hypoxic tumor microenvironment.

Citation Format: Ilamathi Thirusenthilarasan, Wei Xiao, Mumtaz V. Rojiani. The significance of TIMP-1 and hypoxia in cancer stemness: A synergistic role in chemoresistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2779.
Abstract 2796: Colibactin mutation signatures are associated with younger age of onset in colorectal cancer
Authors: Gerstberger Stefanie , Lumish Melissa , Hartner Saskia , Shah Farheen , Choi Seongmin , Luthra Anisha , Jiang Qingwen , Woo Hyung Jun , Mahmoud Ahmed , Walch Henry , Asawa Simran , Donoghue Mark , Cercek Andrea , Yaeger Rona , McPherson Andrew , Sanchez-Vega Francisco , Ganesh Karuna
Published: March 2024

Escherichia coli (E. coli) strains carrying the polyketide synthase (pks) island produce the genotoxin colibactin associated with pathogenesis of colorectal cancer (CRC). Colibactin binds directly to DNA, causing alkylating damage leading to double stranded DNA breaks. Colibactin mutagenesis results in mutations in hexameric A-T rich DNA motifs that can be identified by whole genome sequencing (WGS) as mutation signatures SBS28 and SBS88 (SBS-pks). Since WGS is not routinely used clinically, we developed an approach to specifically identify SBS-pks using a clinical targeted exon capture assay, MSK-IMPACT. In an institutional pan-cancer clinical cohort of 78,905 tumors we identified 15,967 samples with > 20 mutations detected by MSK-IMPACT. We identified 150/1,884 samples with microsatellite stable CRC with >10% mutations attributable to SBS-pks. Examining clinicopathologic and genomic features of SBS-pks+ compared to SBS-pks- CRC tumors, we found that SBS-pks+ signatures are significantly enriched in younger age of onset compared to SBS-pks- CRC patients (median age 55 vs median age 59, p= 0.0028). SBS-pks+ CRC exhibit similar driver mutations and tumor mutational burden (TMB) compared to SBS-pks- CRC but are associated with increased fraction of genome altered (FGA). To mechanistically investigate colibactin-associated damage we developed a novel long-term human colon organoid-microbe co-culture model system that takes advantage of reversal of polarity in suspension culture. WGS of human CRC organoids co-cultured for three months with pks+ and delta-pks E. coli NC101 confirmed induction of pks+ specific SBS-pks signatures, validating the model. Leveraging this model and a biobank of normal colon organoids from donors aged </> 50 years, we are investigating the molecular mechanisms underlying the association of SBS-pks with younger age of onset CRC.

Citation Format: Stefanie Gerstberger, Melissa Lumish, Saskia Hartner, Farheen Shah, Seongmin Choi, Anisha Luthra, Qingwen Jiang, Hyung Jun Woo, Ahmed Mahmoud, Henry Walch, Simran Asawa, Mark Donoghue, Andrea Cercek, Rona Yaeger, Andrew McPherson, Francisco Sanchez-Vega, Karuna Ganesh. Colibactin mutation signatures are associated with younger age of onset in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2796.
Abstract 2816: Comparison of MB-49_luc bladder carcinoma cell clones in the orthotopic superficial bladder tumor model in C57BL/6 female mice
Authors: McArthur Mollie , Tran Melissa , Koznecki Aliccia , Ketteringham Helen , Silvio Corrine , Wenzel Shannan , Davis Cheryl , Nadaraia-Hoke Shorena , Holding Chris , Salem Logan , Caruso Victoria , Hoerner Ben , Spencer Madison , Josey Marcus , Tannu Alexa
Published: March 2024

In preclinical research, cell inoculation via bladder wall injection is a standard for studying invasive bladder cancer into the bladder muscle wall. However, a subset of patients presents with non-muscle invasive (superficial) disease. The standard of care for these patients is excision coupled with BCG treatment via catheterization. There is a need to further establish mouse models of cell inoculation via bladder catheterization for early drug development. While the injection places cells into the bladder wall, catheterization coats the inside of the bladder by first instilling Poly-L-Lysine. After instilling Poly-L-Lysine, instillation of luciferase-tagged cell suspension occurs.

MB49-luc cell lines were generated to determine the optimal cell lines for use in analyzing treatments for superficial bladder carcinoma. MB49-luc cells display varying degrees of luciferase activity and growth in vitro. A subset of cell lines were selected for further analysis in vivo, via bioluminescent imaging, survival analysis, flow cytometry and necropsy. The data provides a preclinical reference for the selection of a cell line for analysis of new drug candidates in C57BL/6 female mice inoculated via bladder catheterization.

Citation Format: Mollie McArthur, Melissa Tran, Aliccia Koznecki, Helen Ketteringham, Corrine Silvio, Shannan Wenzel, Cheryl Davis, Shorena Nadaraia-Hoke, Chris Holding, Logan Salem, Victoria Caruso, Ben Hoerner, Madison Spencer, Marcus Josey, Alexa Tannu. Comparison of MB-49_luc bladder carcinoma cell clones in the orthotopic superficial bladder tumor model in C57BL/6 female mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2816.
Abstract 2818: Investigating the role of TNIK in the pathogenesis of lung squamous cell carcinoma
Authors: Chang Jin Hee , Chowdhury Dipanwita Dutta , Council Danielle N. , Nguyen Triet , Jagtap Shreya , Chan Aaron , Khan Muhammad Ajmal , Connis Nick , Torres-Ayuso Pedro , Brognard John , Rezaee Moahmmad , Lafargue Audrey , Hann Christine , Tran Phuoc T.
Published: March 2024

Lung squamous cell carcinoma (LSCC) is the second most prevalent type of lung cancer with no FDA-approved targeted therapies. Platinum-based chemotherapy and immunotherapy remain the cornerstone of current treatments for advanced LSCC, and the 5-year survival rate is less than 10%. Despite abundant knowledge of the mutational landscape of LSCC, there is a paucity of effective targeted therapies. Amplification of chromosome 3q26 is the most common genomic alteration in LSCC and this leads to the overexpression of oncogenic kinases like TNIK. TNIK amplification occurs in approximately 50% of LSCC cases and is associated with tumorigenesis and therapy resistance in other cancers. Recent studies showed that TNIK maintains the survival of LSCC cells and drives LSCC progression. Furthermore, inhibiting the kinase eradicates tumor growth in TNIK-high preclinical models. These findings highlight the potential of TNIK as a therapeutic target in a subset of LSCC patients with 3q amplification. However, one major limitation of previous studies is the lack of animal models with squamous histology and patient-relevant genotypes. Therefore, we have created a novel conditional TNIK-overexpressing genetically engineered mouse model (GEMM) to better understand the roles of TNIK in tumor progression and therapy resistance in LSCC and elucidate mechanisms underlying LSCC pathogenesis. We are also producing a novel TNIK-overexpressing LSCC GEMM based on the simultaneous activation of KrasG12D and deletion of Fbxw7 (KF model). We will present validation studies such as histological and molecular profiling of our conditional TNIK GEMM and initial data on the TNIK overexpressing LSCC transgenic mouse model (KF (LSCC control) vs KF-TNIK (LSCC-TNIK)).

Citation Format: Jin Hee Chang, Dipanwita Dutta Chowdhury, Danielle N. Council, Triet Nguyen, Shreya Jagtap, Aaron Chan, Muhammad Ajmal Khan, Nick Connis, Pedro Torres-Ayuso, John Brognard, Moahmmad Rezaee, Audrey Lafargue, Christine Hann, Phuoc T. Tran. Investigating the role of TNIK in the pathogenesis of lung squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2818.
Abstract 2819: Novel drug resistant CDX models for anti-cancer drugs discovery
Authors: Dai Hai-Ting , Bing Tie-Jun , An Zhi-Da , Yang Jie , Pang Tian-Yu , Gao Jia-Wei , Bo Yun-Yi , Yu Wen-Jen
Published: March 2024

Oncogene BRAF is located on chromosome 7q34, consisting of 766 amino acids and 22 exons, encoding a serine/threonine protein kinase belonging to the RAF family and plays a critical role in the MAPK signaling pathway. The mutation of BRAF was documented in nearly 8% of all human cancers including melanoma (60%), thyroid (60%), and lung adenocarcinoma (10%). The most common mutation of BRAF is V600E (Class I), which was found in more than 70% in these cancers. Despite the clinical success of approved small molecule inhibitors of BRAF V600E (vemurafenib, dabrafenib and encorafenib), this remains an area of unmet medical need because of primary or acquired drug resistance. The construction of BRAF drug-resistant cell lines plays a pivotal role in cancer research. It serves as a crucial tool for discovery of the mechanisms of drug resistance. In this study, we have developed a systematic approach for constructing drug-resistant cell lines, inducing resistance through the gradual escalation of drug concentrations. The validation of drug resistance involves morphological recordings of parent and drug-resistant cell lines, drug resistance index in vitro through IC50 measurements and in vivo through the inhibition of tumor growth (TGI) which compare to those in the parental cell line. We successfully established Vemurafenib and Dabrafenib resistant A375 cell lines (A375 Vemurafenib R, A375 Dabrafenib R) with 21 and 31.5 folds of resistance index, respectively. The in vivo drug resistance efficacy was also tested by implanting Vemurafenib resistant cells subcutaneously into NOD-SCID mice and showed that A375 Vemurafenib R exhibited robust tolerance to 50 mg/kg Vemurafenib treatment (P.O.21 days, 50mpk, TGI=17.4%), whereas the parental cells were nearly completely inhibited (P.O.21 days, 50mpk, TGI=92.3%). In addition, single-cell sequencing of A375 Vemurafenib R revealed significant gene expression differences in the MAPK and EGFR signaling pathways, aiding in the identification of new targets, and signaling pathways associated with drug resistance, thereby providing potential targets for new drug development.

Citation Format: Hai-Ting Dai, Tie-Jun Bing, Zhi-Da An, Jie Yang, Tian-Yu Pang, Jia-Wei Gao, Yun-Yi Bo, Wen-Jen Yu. Novel drug resistant CDX models for anti-cancer drugs discovery [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2819.
Abstract 282: SFRP4+ cancer associated fibroblasts activate epithelial to mesenchymal transitions in malignant cells and dictate adverse clinical outcomes in stomach cancer
Authors: Lee Seungho , Cho Austin , Kim Hyoung-Il , Hur Hoon , Cheong Jae-Ho , Kim Tae-Min
Published: March 2024

Cancer-associated fibroblasts (CAFs), a pivotal component in the tumor microenvironment (TME) of gastric cancer (GC), play a crucial role in tumor progression and resistance to treatment. In this study, we performed analyses to build a comprehensive cellular landscape of fibroblasts in GC EMT using three single-cell RNA cohorts including ours (GSE183904, GSE134520 and GSE167297). We observed distinct fibroblast subtypes (CXCL14+/POSTN+, CXCL14+/APOE+, SFRP4+, and SFRP2+ fibroblasts). Among the subtypes, we observed that SFRP4+ fibroblasts sharing cellular origins with normal tissue-residing SFRP2+ myofibroblasts. Notably, SFRP4+ fibroblasts showed a regional prediction for tumor and deep tumor layers compared to normal and superficial tumor layers, respectively. To further understand the role of these fibroblasts, we employed spatial transcriptome data. Spatially resolving the cellular interactions within the TME, the adjacency of SFRP4+ CAFs to malignant cells undergoing epithelial-to-mesenchymal transition (EMT), was observed. Ligand-receptor analyses demonstrated SFRP4+CAFs regulate key genes of EMT in malignant cells (TGFBI, COL1A1, SPARC, COL3A1, COL1A2, and BGN). The regulatory roles of SFRP4+ CAFs in driving the epithelial-mesenchymal transition (EMT) of malignant cells were further validated in vitro. Clinical relevance was also demonstrated with the correlation between the abundance of SFRP4+ CAF and clinical outcomes, i.e., high SFRP4+CAF abundance was consistently associated with shorter survival of GC patients across cohorts. Thus, we present SFRP4+ CAFs as major fibroblast subtypes in GC TME. Spatial cellular architectures and in vitro evidence support that SFRP4+fibroblasts activate the EMT of malignant cells along with their association with patient prognosis. This study advances our understanding into the mechanistic roles of SFRP4+ fibroblasts in GC pathogenesis and progression, paving the way for potential targeted therapeutic strategies.

Citation Format: Seungho Lee, Austin Cho, Hyoung-Il Kim, Hoon Hur, Jae-Ho Cheong, Tae-Min Kim. SFRP4+ cancer associated fibroblasts activate epithelial to mesenchymal transitions in malignant cells and dictate adverse clinical outcomes in stomach cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 282.
Abstract 2864: Transcriptional repressor REST controls autophagy in Sonic Hedgehog medulloblastoma through the VHL-HIF1α axis
Authors: Singh Ashutosh , Cheng Donghang , Swaminathan Jyothishmathi , Zheng Yan , Gordon Nancy , Gopalakrishnan Vidya
Published: March 2024

Introduction: Medulloblastoma (MB) is the most common malignant brain tumor in children. It is categorized into four molecular subgroups, each with unique genetic and epigenetic alterations, molecular signatures, and differences in clinical features and treatment responses. Targeted therapy for MB has failed in the clinic, prompting new studies of mechanisms involved in MB tumorigenesis and progression.

Experimental Procedure: The transcriptomic information of the MB patients was obtained from the publicly available RNAseq datasets and was analyzed for differential expression and clustering analysis. Patient-derived orthotopic xenografts of medulloblastoma tumors with high- and low-REST expression were histologically and biochemically studied for autophagy markers. Furthermore, the SHH-MB cell lines were studied to get a mechanistic understanding of the REST-driven autophagy in this MB subgroup.

Summary: Our data showed that autophagy, a process responsible for cellular recycling, and genes associated with this pathway exhibit distinctive expression patterns in human MB subgroups. Specifically, we demonstrate that autophagy is induced in one of the MB subgroups (Sonic Hedgehog-SHH) in response to elevated expression of the RE1-silencing transcription factor (REST), a transcriptional repressor and a canonical regulator of neuronal differentiation genes. REST is also a driver of SHH-MB metastasis. Pharmacological inhibition of autophagy decreases MB cell growth, suggesting it is a pro-survival pathway in REST-driven SHH MBs. In mechanistic studies, we found REST-dependent autophagy induction to involve upregulation of the hypoxia-inducible factor 1-alpha (HIF1α) due to REST-mediated silencing of the von Hippel-Lindau (VHL) gene. The VHL gene product promotes HIF1α ubiquitination and proteasomal degradation, and its loss in SHH-MBs with elevated REST expression drives HIF1α stabilization and nuclear localization to activate target autophagy-related genes.

Conclusion: Our work is the first to link VHL to MB pathology. VHL loss of function in cancers has been mainly attributed to mutational events. Here, we report a novel mechanism of VHL loss, namely its epigenetic silencing by REST. Our findings provide the foundation for future pre-clinical investigations to examine the feasibility of autophagy blockade as a therapeutic strategy for REST-driven SHH MBs.

Citation Format: Ashutosh Singh, Donghang Cheng, Jyothishmathi Swaminathan, Yan Zheng, Nancy Gordon, Vidya Gopalakrishnan. Transcriptional repressor REST controls autophagy in Sonic Hedgehog medulloblastoma through the VHL-HIF1α axis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2864.
Abstract 2880: Radiation induces myofibroblast differentiation of pericytes
Authors: Yamazaki Tomoko , Jordan Kelley R. , Crittenden Marka R. , Gough Michael J. , Young Kristina H.
Published: March 2024

Radiation not only kills cancer cells directly through DNA damage, but also indirectly by inducing vascular changes. Endothelial cells (ECs) in small blood vessels and capillaries connect with pericytes, a type of mural cells which are important for vascular stability and structure. Effects of radiation on tumor vasculature are critical for the therapeutic efficacy, however, its impacts on pericytes remain largely unknown. In this study, we focused on the effect of radiation on pericytes and sought to elucidate the mechanism by which radiation affects pericytes. We show 20Gy for C3H10T1/2 (10T1/2) mouse embryonic mesenchymal pericyte precursor cells and 10Gy for human pericytes from placenta (hPC-PL) cells induces pericyte differentiation and activation of adhesion molecules. Radiation enhanced lymphocyte adhesion and decreased permeability in co-culture of ECs and pericytes primarily through endothelial cells, not via pericytes. Furthermore, radiation promoted transdifferentiation of pericytes into myofibroblasts. Supernatant from irradiated cells in combination with a TGFβ inhibitor showed that TGFβ signaling is involved in pericyte differentiation by radiation but not all pericyte marker genes are regulated by TGFβ. Therefore, we performed phospho-kinase array analysis and revealed Akt signaling pathway activation 10 minutes following radiation, which was verified by the phosphorylation of GSK3β and PRAS40. Along with this, generation of intracellular reactive oxygen species (ROS) occurs as early as 5 min following radiation. Inhibitors of Akt and ROS suppress the expression of myofibroblast markers increased by radiation.

To investigate whether radiation induces pericyte differentiation into myofibroblast in tumors, we employed NG2DsRedBAC (NG2DsRed) transgenic mice where DsRed is expressed under the control of NG2 promoter to trace pericytes. The endogenous expression of DsRed was detected in pericytes in the tumors implanted to NG2DsRed mice. Flow cytometry analysis of tumors harvested 7 days after radiation shows radiation increased vimentin+ cells and decreased DsRed+ cells, which indicates radiation induced pericyte differentiation into myofibroblast. In conclusion, radiation promotes pericyte maturation and transdifferentiation into myofibroblast via Akt signaling. Further study using Akt inhibitor with radiation to tumor-bearing N2DsRed mice will be warranted.

Citation Format: Tomoko Yamazaki, Kelley R. Jordan, Marka R. Crittenden, Michael J. Gough, Kristina H. Young. Radiation induces myofibroblast differentiation of pericytes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2880.
Abstract 2896: Elucidating the role of transglutaminase 2 (TGM2) in pancreatic ductal adenocarcinoma pathogenesis and its therapeutic implications
Authors: Wright Polina , Atfi Azeddine
Published: March 2024

Pancreatic cancer remains one of the deadliest malignancies, with limited therapeutic options and a poor prognosis due to delayed symptom presentation and disease detection. Pancreatic ductal adenocarcinoma (PDAC) has a 5-year survival rate of less than 8%, predominantly attributed to oncogenic KRAS mutations and loss-of-function mutations in TP53, SMAD4, and P16. Our lab is interested in studying Transglutaminase 2 (TGM2), a multifunctional enzyme involved in protein cross-linking and cellular signaling, because it is highly correlated with cancer cell survival, malignancy, metastasis, and treatment resistance. Clinically, overexpression of TGM2 is associated with poorer survival in patients with Pancreatic Ductal Adenocarcinoma so it is imperative to understand the mechanism of its action in disease progression. To elucidate the role of TGM2 in PDAC, we studied the targeted deletion of TGM2 in the context of a mouse model of PDAC and saw a significant reduction in PDAC incidence and precursor lesion formation, suggesting that TGM2 has a role in the tumor microenvironment. Using single cell sequencing, we found TGM2 levels to be elevated in certain cell populations, specifically endothelial cells and cancer cells. We discovered high TGM2 levels in TIE2-GFP mouse vessels and subsequently found high TGM2 levels in patient tumor vasculature. Further studies aim to conditionally ablate TGM2 in endothelial cells to assess its mechanistic involvement in PDAC initiation and progression. Additionally, we are studying concurrent conditional TGM2 and SMAD4 deletions in mice to investigate pancreatic cancer precursor lesion formation and are further evaluating the protective mechanisms of a TGM2 deletion in a more aggressive mouse model of PDAC. Our preliminary findings substantiate TGM2 as a critical player in PDAC pathogenesis, opening avenues for targeted therapeutics to ultimately improve patient prognosis.

Citation Format: Polina Wright, Azeddine Atfi. Elucidating the role of transglutaminase 2 (TGM2) in pancreatic ductal adenocarcinoma pathogenesis and its therapeutic implications [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2896.
Abstract 293: Distinctive roles of mammary adipomes in breast cancer progression: Insights from functional analysis and differential gene expression
Authors: Thangavel Hariprasad , Lizardo Kezia , Glazer Robert I. , Nagajyothi Jyothi F.
Published: March 2024

Breast Cancer (BC) is a heterogeneous disease that impacts treatment response. This is due in part to the tumor microenvironment (TME), which contributes significantly to BC pathogenesis, including metastasis. Women with higher breast density and reduced breast fat face an increased BC risk across multiple tumor subtypes. This is exemplified by the correlation between increased tumor progression, the appearance of fibrosis and the loss of mature adipocytes. While the role of adipokines in this process has been studied extensively, the contribution of adipocyte-derived extracellular vesicles (EV) or "adipomes" in tumorigenesis and metastasis remains poorly understood. To address this question, we developed a novel technique for isolating intact adipomes from normal mammary fat (MF) obtained from wild-type C57BL/6 mice as well as from tumor-associated MF (TAMF) obtained from mice inoculated orthotopically with EO771 mammary tumor cells. Adipome isolation involved high-speed centrifugation to separate macro- and micro-vesicles, purification using the ExoQuick EV isolation system, and immunomagnetic capture of adipomes. Characterization of adipomes by transmission electron microscopy revealed two adipome populations, large L-adipomes of ~350 nm, and small S-adipomes of ~40 nm. Notably, the quantity of L-adipomes in tumor-associated mammary fat (TAMF) was 10-fold greater than the amount of L-adipomes in the MF of non-tumor-bearing mice. To identify possible functional roles of L-adipomes in the pathogenesis of BC, lipidomic profiling was undertaken. L-Adipomes from TAMF showed significantly altered lipid species in comparison to MF-derived L-adipomes. Adipomes were further characterized by treating EO771 cells for 72 hr with TAMF- or MF-derived adipomes at a cell to adipome ratio of 1:10, followed by RNAseq analysis. Differentially upregulated genes in TAMF-derived L-adipomes included Esr1, Erbb2, Tgfb1, Bcl2, MMP2, and Notch associated with fibrosis, EMT, angiogenesis and invasion and downregulation of genes linked to tumor suppression, apoptosis, and mitochondrial metabolism. Overall, our study illustrates the importance of tumor-associated adipomes in regulating gene expression in tumor cells, which emphasizes the specificity of adipome content relating to the cell-of-origin and the host's pathologic and metabolic state and in modulating signaling pathways involved in BC progression.

Citation Format: Hariprasad Thangavel, Kezia Lizardo, Robert I. Glazer, Jyothi F. Nagajyothi. Distinctive roles of mammary adipomes in breast cancer progression: Insights from functional analysis and differential gene expression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 293.
Abstract 2935: NGS panel customization for comprehensive genomic profiling
Authors: Ruvolo Michael , Khare Akanksha , Matcha-Balaguruvappa Nishita , Wilkins Katherine , Forsmark Linus , Gupta Swati
Published: March 2024

Introduction: We have developed a custom SureSelect Cancer design pipeline for the creation of target enrichment gene panels for comprehensive profiling of single nucleotide variations (SNP/INDEL), gene-level copy number variations (CNV), and DNA translocations (TL) as well as content for measuring of microsatellite instability (MSI) and tumor mutational burden (TMB).

Methods: The performance of individual probes was measured for the 679-gene SureSelect Cancer CGP panel and the information used to generate a reference database. When creating a new design, the gene list is cross-referenced with the database and when applicable, the probe information is applied to the new design. If genes are included in the custom design that are not present in the database, a second design algorithm selects the optimal probe based on its sequence characteristics (% GC, homology, etc.). Content is selected based on the biomarker to be measured and can be defined based on gene name (SNV/indel and CNV) or genomic coordinates (SNV/indel, TL). Pre-defined content for MSI measurement can be added to any design to cover 288 microsatellite sites. The performance of these sites in determining MSI status was demonstrated by comparing to the MSI-PCR reference method. To assess the performance of the design methodology, designs of various sizes (50 to 650+ genes) and content type were generated and assayed on reference samples and FFPE with known biomarkers. Sequencing metrics were evaluated for capture specificity and coverage of the target regions.

Results: The design methodology allowed for sub-selection of content present in the 679-gene SureSelect Cancer CGP assay as well as addition of de-novo content. The design algorithm uses empirical data on probe performance to provide robust and reproducible results for the range of supported design sizes. Biomarkers were reliably detected with SNV detection down to 1% allele frequency, detection of gene amplifications and translocations concordant with FISH, and MSI and TMB determination concordant with PCR and Exome sequencing respectively. The design workspace was implemented in the SureDesign platform, providing a web-based interface for creating the designs.

Conclusions: We demonstrated full customization capability of the SureSelect Cancer target enrichment platform that pairs user-defined content with an optimized probe design methodology to enable the sensitive detection of the common types of DNA alterations found in FFPE samples.

Citation Format: Michael Ruvolo, Akanksha Khare, Nishita Matcha-Balaguruvappa, Katherine Wilkins, Linus Forsmark, Swati Gupta. NGS panel customization for comprehensive genomic profiling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2935.
Abstract 294: The HOXB7 protein promotes breast cancer cell growth through activation of the CCL5/CCR5 pathway in the crosstalk of adipocyte
Authors: Akume Ahone Gina , Jin Kideok
Published: March 2024

Homeobox genes are regulatory genes that encode nuclear proteins acting as transcription factors during normal development and differentiation. One such gene, HOXB7, plays a role in various developmental processes, including hematopoietic differentiation, lymphoid development, and mammary gland development. However, the role of HOX genes in breast cancer development remains largely unexplored. Our previous studies revealed that HOXB7 expression was significantly elevated in primary cancer and distant metastasis. HOXB7 overexpression promoted cell proliferation in SKBR3 breast cancer cells, leading to robustly vascularized xenografts in immunodeficient mice. Furthermore, HOXB7 enhanced tumor growth through TGF beta signaling and recruitment of macrophages, indicating the role of HOXB7 in the crosstalk of stromal components. We found evidence that HOXB7 overexpression in ER+ breast cancer cells promotes tumor cell growth through increased expression and signaling of CCL5/CCR5 in crosstalk with adipocytes. By screening secreted factors in adipocytes induced by TCM of MCF-7-HOXB7 cells, it was also found that CCL5 is highly expressed in HOXB7-overexpressing MCF-7 cells. The CCR5 inhibitor, maraviroc, sensitized MCF-7-HOXB7 cells compared to control cells. These findings suggest that blocking the interaction between CCL5 and CCR5 signaling significantly inhibits ER+ breast cancer with HOXB7 overexpression. We aim to further investigate if HOXB7 overexpression in ER+ breast cancer cells promotes tumor cell growth through increased expression and signaling of CCL5/CCR5 in crosstalk with adipocytes and the role of CCL5 in breast cancer cell growth and migration.

Citation Format: Ahone Gina Akume, Kideok Jin. The HOXB7 protein promotes breast cancer cell growth through activation of the CCL5/CCR5 pathway in the crosstalk of adipocyte [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 294.
Abstract 2945: Using pooled minimal genome-wide sgRNA libraries to drive oncology drug discovery
Authors: Lukasiak Sebastian , Kalinka Alex , Gupta Nikhil , Saeed Khalid , Papadopoulos Angelos , McDermott Ultan , Hannon Gregory J. , Ross-Thriepland Douglas , Walter David
Published: March 2024

The emergence of CRISPR technologies has enabled rapid and precise gene editing opportunities. The Functional Genomics Centre (FGC) - a joint venture between Cancer Research UK’s (CRUK) drug discovery engine, Cancer Research Horizons, and AstraZeneca has been launched to better utilize and improve the CRISPR technology within cancer research. The FGC leverages the strengths of both CRUK’s and AstraZeneca’s world-class oncology research and drug discovery experience, with the potential to drive scientific innovation in how new drug targets are identified and validated, and uniquely democratize access to the latest cutting-edge research tools and expertise for many thousands of scientists and clinicians funded by CRUK.

Here, we will showcase how the FGC is leading the development and refinement of functional genomics technologies. Through independent evaluation of publicly available CRISPR tools and leveraging this data we have designed optimized, minimal genome-wide CRISPR libraries. These libraries permit a reduction of the experimental scale of CRISPR screens by 50% and enable our Target Discovery platform to impact more projects, faster, at a lower cost and importantly - more sustainably.

Citation Format: Sebastian Lukasiak, Alex Kalinka, Nikhil Gupta, Khalid Saeed, Angelos Papadopoulos, Ultan McDermott, Gregory J. Hannon, Douglas Ross-Thriepland, David Walter. Using pooled minimal genome-wide sgRNA libraries to drive oncology drug discovery [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2945.
Abstract 2970: Anti-diabetic drug, semaglutide, prevents doxorubicin-induced endothelial cell toxicity
Authors: Bakhit Sandra , Amaefuna Chelsea , Ramana Kota
Published: March 2024

Doxorubicin (Dox) is part of the anthracycline drug family and is known for its broad and effective use as a chemotherapeutic agent. It is commonly used to treat solid tumors and malignancies such as leukemia, breast, lung, and ovarian cancers. Unfortunately, its use in treating advanced cancers is limited due to its cardiotoxic side effects. Therefore, various adjuvant approaches are required to increase Dox's efficacy and to reduce unwanted toxicities including cardiotoxicity. Semaglutideis a glucagon-like peptide-1 (GLP-1) receptor agonist commonly used to treat type-2 diabetes. In his study, the effect of Semaglutide in preventing Dox-induced endothelial cell toxicity was investigated. Our data indicate that Semaglutide prevents Dox-induced cell death in human umbilical vein endothelial cells (HUVEC) in a dose- and time-dependent manner. Similarly, live and dead cell staining assay also confirms Semaglutide prevents Dox-induced endothelial cell death. Semaglutide also prevented the Dox-induced apoptosis of HUVECs. It also prevented the Dox-induced reactive oxygen species and activation of caspase-3. Further, Semaglutide regulated the expression of various anti- and pro-apoptotic and inflammatory factors induced by Dox. Inconclusion, our current results suggests that the anti-diabetic drug, Semaglutide, could prevent Dox-induced endothelial cell death by regulating the expression of various pro-apoptotic, anti-apoptotic and inflammatory markers. Thus, our data indicate that Semaglutide could be used as a potential adjuvant drug to reduce the adverse effects of anthracycline chemotherapeutic drugs.

Citation Format: Sandra Bakhit, Chelsea Amaefuna, Kota Ramana. Anti-diabetic drug, semaglutide, prevents doxorubicin-induced endothelial cell toxicity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2970.
Abstract 2976: The potential use of butyrate and propionate to inhibit oncogenic phenotypes in oral squamous cell carcinoma and triple negative breast cancer
Authors: Maisonet Angel J. Amaral
Published: March 2024

Background

Oral squamous cell carcinoma (OSCC) and triple-negative breast cancer (TNBC) are aggressive cancers with low survival rates, particularly in Hispanics. Since the underpinning mechanisms of oncogenesis for both OSCC and TNBC are multifactorial, the development of novel targeted therapies is limited. The tumor microbiome has been shown to modulate the microenvironment. The microbial product short-chain fatty acids (SCFA) may represent a putative target for treatment as they can potentially inhibit cell growth, proliferation, migration, and induce morphologic changes in cancer cells. We evaluated the impact of the SCFAs butyrate, acetate, and propionate on the oncogenic phenotypes of OECM-1 and MDA-231 cell line models for OSCC and TNBC respectively.

Methods

OECM-1 and MDA-231 cell lines were cultured in RPMI-1640 medium supplemented with 10% FBS. Cytotoxicity was determined using an alamarBlue assay by treating the cells with SCFA in a series of 7 concentrations starting at 125 mM (1:2 dilution factor) for 24, 48, and 72 hours (h). Proliferation quantification was performed using trypan blue dye after treatment with SCFA for 24h, 48h and 72h. Migration and invasion were measured after treatment for 24h with SCFAs. Comparative analysis of our results was performed using GraphPad Prism v10.

Results

Higher concentrations of all SCFA decreased cell viability in OSCC and TNBC at all time points. In OECM-1 cells, the average inhibitory concentration of 50% (IC50) was 3mM for butyrate, 382mM for acetate, and 64mM for propionate while in MDA-MB-231 cells, the average IC50 was 4.3mM for butyrate, 462mM for acetate, and 20mM for propionate. Both OECM-1 and MDA-MB-231 cells treated with butyrate and propionate at 5mM resulted in a slower migration after 24h of treatment. Additionally, there was a significant reduction of cell number and proliferation rate after 48h and 72h of butyrate treatment at the concentrations 1mM (p=0.0425) and 5mM (p=0.0007) in OSCC.

Conclusions

Our results show that butyrate and propionate can reduce oncogenic phenotypes in OSCC and TNBC and may represent a potential target for the development of alternative treatment strategies for both cancers. Future work includes measuring the impact of these SCFA in epithelial-mesenchymal transition, loss of adhesion, and cell death.

Citation Format: Angel J. Amaral Maisonet. The potential use of butyrate and propionate to inhibit oncogenic phenotypes in oral squamous cell carcinoma and triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2976.
Abstract 2992: miR-200-mediated inactivation of cancer-associated fibroblasts attenuates lung cancer invasion and metastasis
Authors: Cheon Inyoung , Lee Sieun , Ahn Young-Ho
Published: March 2024

Cancer-associated fibroblasts (CAFs) play a crucial role in the tumor microenvironment and contribute to the development and progression of lung cancer. We aimed to elucidate whether miRNA-200 (miR-200), which is recognized for its ability to suppress EMT, prevents CAFs from promoting cancer progression. Inactivation of CAFs mediated by miR-200 suppressed lung cancer cell invasion, migration, tumorigenesis, and metastasis. Additionally, miR-200 attenuated the ability of CAFs to facilitate the aggressiveness of lung cancer cells, encourage M2 polarization of macrophages, and increase the angiogenic sprouting of vascular endothelial cells. NRP2, known as a co-receptor of VEGFR, was identified as a target of miR-200, mediating the functional activity of miR-200 in CAFs. NRP2-VEGFR signaling promoted the secretion of VEGFD and PTN from CAFs to activate cancer cell migration and invasion. Based on these findings, we suggest that miR-200 impedes cancer progression and metastasis by remodeling CAFs, and that miR-200 and NRP2 are potential therapeutic targets in the treatment of lung cancer.

Citation Format: Inyoung Cheon, Sieun Lee, Young-Ho Ahn. miR-200-mediated inactivation of cancer-associated fibroblasts attenuates lung cancer invasion and metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2992.
Abstract 303: Revealing molecular mechanism of G protein-coupled receptor kinase 2 (GRK2)-mediated extracellular matrix remodeling in ovarian cancer
Authors: Lei Weng I , Yu Shan , Lei Chi Teng , Zhang Zhiming , Liu Tzu Ming , Chang Wakam , Lee Leo Tsz On
Published: March 2024

G protein-independent pathway (β-arrestin dependent signaling cascade) is initiated by activated GPCRs phosphorylated by G protein-coupled receptor kinases (GRKs), triggering multiple signaling pathways. The purpose of this research is to reveal the role of GRK2 on the tumor microenvironment (TME). Through in vitro assays, we showed that GRK2 overexpression led to loss of cell-matrix and cell-cell adhesion. The cell spreading assay further indicated that the loss of focal adhesion (FA) assembly and cell size decreased in GRK2-overexpressing cells. In vivo experiments demonstrated that tumor formation and metastasis were suppressed in xenograft mouse models implanted with GRK2-overexpressing ovarian cancer cells. In addition, label-free imaging detection of the collagen pattern suggested that GRK2 overexpression caused bundle loss but the formation of thicker, twisted collagen fibers in xenograft tumor samples. Together with the transcriptome and proteomic data, we hypothesize that GRK2 suppresses cell motility and metastasis by reducing FA and extracellular matrix (ECM) remodeling in ovarian cancer. This research revised the mechanisms underlying GRK2-mediated ovarian cancer metastasis and suggested the potential to target the GRK2-meditated pathway for cancer suppression.

Citation Format: Weng I Lei, Shan Yu, Chi Teng Lei, Zhiming Zhang, Tzu Ming Liu, Wakam Chang, Leo Tsz On Lee. Revealing molecular mechanism of G protein-coupled receptor kinase 2 (GRK2)-mediated extracellular matrix remodeling in ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 303.
Abstract 3032: Generation of a FOXC2-deficient melanoma cell line using CRISPR-Cas9 gene editing
Authors: Clark Devanand M. , Conde Patrick D. , Hargadon Kristian M.
Published: March 2024

Melanoma, a cancer derived from pigment-producing melanocytes of the skin, is a highly aggressive cancer characterized by rapid growth and metastasis. FOXC2 is a forkhead box transcription factor family member that is often dysregulated in various cancers, including melanoma, where its expression correlates with metastatic progression and drug resistance. To gain insight into the oncogenic activity of FOXC2 in melanoma, we have employed CRISPR-Cas9 gene editing technology to generate a novel FOXC2-deficient B16-F10 murine melanoma cell line. This model will be a useful tool for studying the tumor-promoting functions of FOXC2.

Citation Format: Devanand M. Clark, Patrick D. Conde, Kristian M. Hargadon. Generation of a FOXC2-deficient melanoma cell line using CRISPR-Cas9 gene editing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3032.