Mouse Model Of Metastasis Research Articles

Abstract 4381: Role of Kindlin-2 in the regulation of integrins and TBR1 signaling in TNBC

Abstract Background: Kindlins are a small gene family of FERM domain-containing adaptor proteins that function as essential drivers of integrin activation. Kindlin-2 (K2) is the most widely expressed member of the Kindlin family; its homozygous deletion in mice is embryonically lethal, K2 expression is also dysregulated in several human cancers, including the breast. We established K2 as a major driver of TNBC tumors progression and metastasis through the regulation of several hallmarks of cancer. A well-established pathway whereby K2 regulates TNBC oncogenic behavior is through the regulation of integrin inside-out signaling by directly binding to the cytoplasmic tail of integrin β subunit. Interestingly, our recent studies found K2 to play a major role in activating the TGF-β-mediated regulation of the CSF1/EGF signaling through macrophage polarization to the M2 tumorigenic state and their increased tumor infiltration, as well as inhibiting tumor infiltration of cytotoxic lymphocytes, therefore, driving tumor immune evasion. Here we present novel findings involving K2 in the stabilization of the β1-Integrin:TβRI complexes by establishing a physical bridge that links β1-Integrin to TβRI. Loss of K2 results in the degradation of this protein complex and therefore, the inhibition of the oncogenic pathways downstream of these two major regulators of several hallmarks of cancer. Methods: We applied a combination of in vitro assays; CRISPR/Cas9 gene editing, cell migration, 3D tumor-sphere, solid binding, co-immunoprecipitation, cell adhesion and spreading, as well as western blot and flow cytometry analyses with MDA-MB-231 and 4T1 TNBC cell lines. We also used preclinical in vivo mouse models of TNBC tumor progression and metastasis to support or investigations. Results: We found that the K2/β1-Integrin direct interaction is mediated through the C-terminal F3 domain of K2, while the K2/TβRI direct interaction is mediated through the F2 domain of K2. Disruption of this bridge, via CRISPR/Cas9-mediated knockout of K2, leads to β1-Integrin and TβRI degradation, and inhibition of the oncogenic pathways downstream of both β1-Integrin and TβRI, and inhibited tumor growth and metastasis in levels comparable to loss of expression of either β1-Integrin and TβRI. Treatment of the K2-deficent cells with the proteasome inhibitor MG-132 restored expression of both β1-Integrin and TβRI, confirming that K2 is required for the stabilization of the β1-integrin/TβR1 complexes at the protein level. Rescue of K2 expression in the K2-KO cells restored their oncogenic activities both in vitro and in vivo. Conclusion: We identified K2 to play a novel and major in the stabilization of the β1-integrin/TβR1 complexes and the regulation of their downstream oncogenic signaling. These studies have a significant translational impact as they may identify new therapeutically targeted pathways that are desperately needed for the treatment of TNBC tumors. Citation Format: Neelum A. Yousaf Zai, Lamyae El Khalki, Wei Wang, Justin Szpendyk, Khalid Sossey-Alaoui. Role of Kindlin-2 in the regulation of integrins and TBR1 signaling in TNBC [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 4381.

Abstract 2922: Sensory innervation as a novel driver of ovarian cancer progression

Abstract One of the main challenges currently faced in ovarian cancer treatment is that the primary platinum/taxane therapy used to treat patients does not meaningfully extend survival. The overall survival for ovarian cancer patients with advanced disease is less than 30% with most patients recurring within 5 years of initial therapy. Once resistant disease develops there are few second line therapy options, which inevitably leads to disease progression. Thus, there is a critical need to identify new therapies that can overcome ovarian cancer chemoresistance and produce meaningful increases in patient survival. In recent years, new efforts have focused on targeting the tumor microenvironment (TME), the complex mixture of non-cancer cells that surround and support the cancer cells within the tumor. Different strategies have evolved to target the TME, such as immune checkpoint inhibitors or angiogenesis inhibitors. However, these TME targeting therapies have limited effect when used to treat ovarian cancer patients. Most ovarian tumors have a microenvironment that is highly immunosuppressive and do not respond or are resistant to angiogenesis inhibitors. Thus, new TME targeting therapies must be developed that can induce a therapeutic effect in ovarian tumors and can be used to treat most ovarian cancer patients. A novel TME component ripe for therapeutic targeting are the nerve fibers that infiltrate tumors in a process termed tumor innervation. Recent studies in several cancers have shown that tumor innervation can promote tumor growth/metastasis. However, in ovarian cancer the role of innervation in promoting cancer progression remains undefined. Here we show that Transient Receptor Potential cation channel subfamily V member 1 (TRPV1)+ sensory innervation plays a significant role in driving ovarian cancer growth and metastasis. Analysis of patient samples shows that sensory nerve fiber innervation is much higher in ovarian tumors vs benign reproductive tissue. In addition, ablation of TRPV1 sensory nerve fibers in vivo causes reduced tumor burden and prolongs survival in a syngeneic mouse model of ovarian cancer metastasis. Taken together, our results establish TRPV1+ sensory innervation as a novel driver of ovarian cancer growth/metastasis and a potential therapeutic target for ovarian cancer treatment. Citation Format: Matthew Knarr, Katherine Cummins, Dusan Racordon, Hunter Reavis, Timothy Lippert, Ryan Hausler, Priyanka Rawat, Suyeon Ryu, Jamie Moon, Dave Hoon, Roger Greenberg, Paola Vermeer, Ronny Drapkin. Sensory innervation as a novel driver of ovarian cancer progression [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 2922.

Abstract 6404: GRB2 stabilizes RAD51 at reversed replication forks restricting genomic instability and associated innate immune response

Abstract Growth factor receptor-bound protein 2 (GRB2) is a cytoplasmic adapter for tyrosine kinase signaling and a nuclear adaptor for homology-directed-DNA repair. Here we find nuclear GRB2 protects DNA at stalled replication forks from MRE11-mediated degradation in the BRCA2 replication fork protection axis. Mechanistically, GRB2 binds and inhibits RAD51 ATPase activity to stabilize RAD51 on stalled replication forks. In GRB2-depleted cells, PARP inhibitor (PARPi) treatment releases DNA fragments from stalled forks into the cytoplasm that activate the cGAS-STING pathway to trigger pro-inflammatory cytokine production. Moreover in a syngeneic mouse metastatic ovarian cancer model, GRB2 depletion in the context of PARPi treatment reduced tumor burden and enabled high survival consistent with immune suppression of cancer growth. Collective findings unveil GRB2 function and mechanism for fork protection in the BRCA2-RAD51-MRE11 axis and suggest GRB2 as a potential therapeutic target and an enabling predictive biomarker for patient selection for PARPi and immunotherapy combination. Citation Format: Zamal Ahmed, Zu Ye, Shengfeng Xu, Yin Shi, Xueqian Cheng, Yuan Zhang, Sunetra Roy, Sarita Namjoshi, Michael A. Longo, Todd Link, Katharina Schlacher, Guang Peng, Dihua Yu, Bin Wang, John Tainer. GRB2 stabilizes RAD51 at reversed replication forks restricting genomic instability and associated innate immune response [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 6404.

Abstract 2459: ADT-030 is a novel dual-acting RAS/β-catenin inhibitor that potentiates antitumor immunity

Abstract ADT-030 is a novel indene identified from a screening campaign and chemically optimized for drug-like properties that acts as a dual inhibitor of RAS and β-catenin signaling. Here we demonstrate that ADT-030 binds RAS with high affinity and inhibits MAPK/AKT signaling with high potency and selectivity in KRAS mutant cells to inhibit proliferation and induce apoptosis. ADT-030 also inhibits phosphodiesterase 10A (PDE10) that is overexpressed in cancer cells to selectively activate cyclic guanosine monophosphate (cGMP)/protein kinase G (PKG) signaling, which induces phosphorylation and degradation of the oncogenic pool of β-catenin. ADT-030 exhibited cytotoxicity to a variety of murine cancer cell lines in vitro, and inhibited tumor growth in syngeneic immunocompetent mice when orally administered. The antitumor activity of ADT-030 was diminished in mice that were immunodeficient or depleted of CD8+ T cells, indicating a critical role of the host immune system. In a metastatic breast tumor mouse model (4T1), ADT-030 not only delayed the growth of the primary tumor but also reduced tumor colonization in the lungs, leading to increased survival. CyTOF and scRNAseq analyses provided a plethora of information revealing the impact of ADT-030 on the immune milieu in the tumor microenvironment. Notably, ADT-030 selectively induced apoptosis of myeloid-derived suppressor cells (MDSCs) while CD8+ T cells were not affected. Consequently, ADT-030 treatment resulted in a marked reduction of MDSCs, which was accompanied by increased tumor infiltration of effector CD8+ T cells. ADT-030 treatment of cancer cells led to immunogenic cell death and enhanced dendritic cell activation. Furthermore, the ability of ADT-030 to augment the antitumor activity of immune checkpoint blockade therapy underlines its potential as a desirable immune-potentiating agent to improve the outcome of cancer immunotherapy. ADT-030 represents a development candidate ready for IND-enabling safety assessment. Citation Format: Md Yeashin Gazi, Ogacheko Okoko, Xin Wang, Caitlin Brandle, Xi Chen, Khalda Fadlalla, Adam Keeton, Yulia Maxuitenko, Catherine Hedrick, Huidong Shi, Gary Piazza, Gang Zhou. ADT-030 is a novel dual-acting RAS/β-catenin inhibitor that potentiates antitumor immunity [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 2459.

Abstract 5004: Oncolytic Jurona-driven systemic and intratumoral immunotherapy combined with immune checkpoint blockade boost immune response and survival in hepatocellular carcinoma models

Abstract The members of the vesiculovirus genus have recently become attractive immunotherapeutic agents due to their natural selectivity for tumors and lack of toxicity in humans. Here, the research shows Intratumoral (IT) or Intraperitoneal (IP) injection of JURV was found to induce dynamic tumor regression in human hepatocellular carcinoma (HCC) xenografts and syngeneic models. Furthermore, IT injections of JURV were also shown to recruit and activate cytotoxic T lymphocytes and decrease tumor-associated macrophage infiltration, resulting in the delay of tumor growth in both local and distant murine HCC tumors established in a syngeneic model. Additionally, the concurrent use of JURV and anti-PD-1 antibodies was found to synergistically modulate the tumor microenvironment (TME) via increased tumor-infiltrating CD4+ T cells and depleted CD8+ PD-1+ and NK cells. Furthermore, using RIL-175, an aggressive metastatic HCC tumor mouse model, we show that intraperitoneal (IP) (systemic) administration of JURV and anti-PD1 significantly improved long-term survival (90%) compared to the (20%) JURV or anti-PD1 alone. Through proteogenomic analysis, the activation of different effectors of the immune system was observed to cooperate to alter the TME to a favorable anti-tumor immune state. These findings further support the development of JURV as an immunovirotherapy platform for HCC, with the potential to induce abscopal effects via the activation of several tumor suppressor genes and the mechanism regulating the T helper cell responses. Citation Format: Mulu Z. Tesfay, Khandoker U. Ferdous, Aleksandra Cios, Randal S. Shelton, Camila C. Simoes, Steven R. Post, Thomas J. Kelly, Martin J. Cannon, Alexei Basnakian, Bolni M. Nagalo. Oncolytic Jurona-driven systemic and intratumoral immunotherapy combined with immune checkpoint blockade boost immune response and survival in hepatocellular carcinoma models [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 5004.

Abstract 5457: Inflammatory paracrine loops established between metastasis-initiating cells and alveolar epithelium drive interactions between tumor cell subtypes that are essential for osteosarcoma lung colonization

Abstract Introduction: The development of lung metastasis drives the poor outcomes experienced by children and young adults with osteosarcoma. The mechanisms that facilitate metastatic colonization remain poorly understood. We previously determined that IL6 and CXCL8 are two osteosarcoma derived cytokines that are required for metastasis, though roles played by these cytokines have not been identified. Methods: To explore the interactions that drive cytokine expression, the role these factors play in tumor cell trafficking, and the mechanisms that facilitate metastatic niche development, we utilized transwell and co-culture systems together with a panel of metastatic osteosarcoma mouse models that allowed tumor cell manipulation via lentiviruses, CRISPR-based gene editing, and pre-clinical therapeutic evaluation approaches. Results: While metastatic osteosarcoma cells produced small amounts of IL6 and CXCL8 at baseline, lung epithelial cells markedly upregulated IL6/CXCL8 production. Ligand-receptor analysis utilizing single-cell transcriptomic data identified IL1 as an epithelial derived ligand that drives tumor IL6/CXCL8 production. Interestingly, IL6/CXCL8 production was limited to a small subset of hypo-proliferative osteosarcoma cells that have metastasis-initiating capacity. Utilizing CRISPR-mediated ablation of IL1R1 signaling within osteosarcoma cells and pharmacological inhibition with recombinant IL1R antagonist (anakinra), we elucidated that formation of pulmonary lesions in multiple mouse models of metastatic osteosarcoma required IL1 signaling. Recent work from our lab has demonstrated that osteosarcoma metastases are defined by marked phenotypic heterogeneity, though the mechanisms by which metastatic heterogeneity occurred were unclear. We hypothesized that hypo-proliferative metastasis-initiating cells may recruit circulating cells with higher proliferative capacity to establish phenotypic heterogeneity. Transwell migration assays showed that tumor cells induced the chemotactic recruitment of other tumor cells in an IL6/CXCL8-dependent manner. To test this hypothesis in vivo, we injected mice with GFP-tagged osteosarcoma cells, then injected the same mice 14 days later with RFP-labeled tumor cells from the same culture. Lungs harvested 14 days later exhibited RFP-dominant lesions surrounding a small core of GFP-labeled cells. Conclusions: Collectively, these data suggest that cytokines expressed by a subgroup of hypo-proliferative tumor cells upon interaction with lung epithelium drive the trafficking of highly proliferative circulating tumor cells into developing metastatic lesions and that disrupting these signaling networks can prevent metastatic colonization. Citation Format: Amy C. Gross, James B. Reinecke, Amanda Saraf, John Hinckley, Maren Cam, Matthew V. Cannon, Sophia Vatelle, Ryan D. Roberts. Inflammatory paracrine loops established between metastasis-initiating cells and alveolar epithelium drive interactions between tumor cell subtypes that are essential for osteosarcoma lung colonization [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 5457.

Abstract 4133: Hyaluronidase inhibitor delphinidin inhibits cancer metastasis

Abstract Cancer remains a formidable global health challenge, with metastasis being a key contributor to its lethality. Abundant high molecular weight hyaluronic acid, a major non-protein component of extracellular matrix, protects naked mole rats from cancer and reduces cancer incidence in mice. Hyaluronidase plays a critical role in degrading hyaluronic acid and is frequently overexpressed in metastatic cancer. We investigated the potential of targeting hyaluronidases to reduce metastasis. High throughput screening identified delphinidin, a natural plant compound found in fruits and vegetables, as a potent hyaluronidase inhibitor. Delphinidin-mediated inhibition of hyaluronidase activity led to an increase in high molecular weight hyaluronic acid in cell culture and in mouse tissues, and significantly reduced proliferation, migration and invasion of breast (EO771), prostate (RM1), and melanoma (B16-F10) metastatic cell lines. Addition of exogenous hyaluronidase partially reversed all of these effects. Moreover, delphinidin treatment suppressed melanoma (B16-F10) metastasis in a spontaneous mouse model of metastasis. These results provide a proof of principle that inhibition of hyaluronidase activity suppresses cancer cell migration, invasion and metastasis. These results combined with the lack of adverse effect make delphinidin an attractive potential therapeutic option for inhibition of metastasis. We believe further investigation is warranted to fully elucidate the mechanisms and optimize the use of delphinidin in cancer therapy. Citation Format: Jeremy Jonathan McGuire, Taketo Taguchi, Greg Tombline, Victoria Paige, Michelle Janelsins, Nikesha Gilmore, Andrei Seluanov, Vera Gorbunova. Hyaluronidase inhibitor delphinidin inhibits cancer 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 4133.

Abstract 5285: DKK1 blockade inhibits progression of bone metastases via multiple mechanisms and synergize with zoledronate treatment

Abstract Background: Despite the important breakthroughs of immune checkpoint blockade (ICB) therapy in multiple primary cancers, responses of ICB therapy in bone metastases remain extremely poor, largely due to the highly immunosuppressive bone metastasis microenvironment. However, the current understanding of bone metastasis microenvironment is still incomplete, and specific treatment targets for patients with bone metastasis are still lacking. Methods: Transcriptome sequencing data of gastric and breast cancer bone metastasis samples from The TCGA and GEO databases were obtained. The correlation between DKK1 expression and prognosis, intertumoral immune infiltration of bone-metastatic patients was analyzed. Mouse bone metastasis models from gastric, breast and lung cancer were established, and in vivo treatment efficacy of DKK1 and/or zoledronic acid was evaluated by micro-computed tomography (CT), micro-magnetic resonance imaging (MRI) and immunohistochemistry (IHC) staining. The changes of immune components wthin bone-tumor microenvironment were explored via flow cytometry, CyTOF and scRNA-seq analysis. The impact of DKK1 on different immune cell types was analyzed via in vitro co-culture models. Results: Serum detection of gastric cancer patients from our center showed that compared with non-metastatic patients, DKK1 concentration in patients with bone metastasis was the significantly increased (p<0.0001); and compared with other metastatic sites of breast cancer patients, bone metastases had the highest level of DKK1 mRNA expression (p<0.05). Using multiple bone metastasis mouse models, we found that DKK1 blockade significantly controlled the tumor burden of bone metastases. Results of micro-CT and TRAP staining showed that the bone destruction and osteoclast activity within bone lesions were significantly inhibited after DKK1 blockade. In addition, results of flow cytometry and immunofluorescence staining on bone metastasis samples showed that both innate and adaptive anti-immune responses were obviously improved after DKK1 blockade, including increased infiltration of CD8+ T cells, M1 macrophages and decreased infiltration of M2 macrophages. Mechanistically, DKK1 directly induced inhibition of CD8+ T cell activation, M2 polarization of macrophages and maturation of osteoclasts, which all contributes to the immune suppression in the bone metastasis microenvironment. Finally, combined therapy of DKK1 blockade and zoledronic acid had synergistic treatment effects in bone metastases. Conclusion: Our study provides novel insights into the multiple role of DKK1 in the bone metastasis immune microenvironment. DKK1 is a potential immunotherapeutic target for bone metastases, and dual blockade of DKK1 and zoledronic acid is a promising combination immunotherapy strategy for patients with bone metastasis. Citation Format: Tao Shi, Kaijie Liang, Yipeng Zhang, Xiaoyu Zhou, Baorui Liu, Jia Wei. DKK1 blockade inhibits progression of bone metastases via multiple mechanisms and synergize with zoledronate treatment [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 5285.

Abstract 1391: Therapeutic salmonella induces long-term protective trained immunity in NK cells against metastasis

Abstract Immune memory is a trait commonly associated with adaptive immunity. Nevertheless, it has been observed that activation of the innate immune system can also result in enhanced responsiveness to subsequent triggers, a form of innate immune memory known as trained immunity. This phenomenon has been primarily explored in the context of host defense against infectious disease, where myeloid cells, including monocytes, macrophages, and their precursor cells, are conditioned by specific stimuli such as β-glucan, LPS, or the bacillus Calmette-Guιrin (BCG) to boost protection against microbial pathogens. However, the induction of trained immunity in innate lymphocytes, particularly through these known or yet-to-be-discovered stimuli and its explicit contribution to tumor immunity, remain insufficiently characterized. Metastasis accounts for 90% of cancer-related deaths, and no effective clinical therapies are currently available to halt the metastatic cascade. Based on multiple cancer metastasis mouse models and immunological analysis, we found that a single dose of therapeutic Salmonella YB1, an engineered oxygen-sensitive strain, provides extensive and persistent protection against metastasis by inducing protective trained immunity in NK cells, consistently observed irrespective of the mouse genetic background or cancer cell lines. Following inflammatory resolution, salmonella YB1-trained NK cells exhibit an enhanced capacity to release IFN-γ upon secondary stimulation and increased cytotoxicity towards cancer cells, which is associated with epigenetic changes. Further investigation using single-cell transcriptomic profiling indicates that the formation of long-term anti-metastatic trained immunity in NK cells by Salmonella YB1 is dependent on IL12 and IL18. Additionally, Salmonella YB1-trained NK cells outperform common immune checkpoint therapies like PD1 blockade and TIGIT blockade in suppressing metastasis. In summary, our results revealed a novel anti-metastatic function of trained NK cells induced by therapeutic Salmonella, suggesting the induction of trained immunity in NK cells as a potential anti-tumor strategy. Citation Format: Li Rong, Jiandong Huang. Therapeutic salmonella induces long-term protective trained immunity in NK cells against 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 1391.

Abstract 5449: Exploring signaling pathways essential for stemness and metastasis of colorectal cancer cells

Abstract The prognosis of colorectal cancer (CRC) patients with distant metastasis remains poor, requiring elucidation of molecular mechanisms and identification of therapeutic targets. While cancer stem cells have been implicated in metastasis, recurrence, and drug resistance, our understanding of the regulatory mechanisms governing the stemness of CRC stem cells remains largely incomplete. In this study, we utilized an innovative autochthonous metastatic mouse model of CRCs generated by the introduction of sporadic mutations in Ctnnb1, Kras, Trp53, and Smad4 genes (CKPS mice) and CRC cell lines derived from the mice (CKPS cells). Our investigation revealed that the cAMP/PKA/CREB and TGFβ/SMAD4 pathways play a positive and negative role, respectively, in the sphere-forming and metastasis-initiating ability of CRC cells. Treatment with the CREB inhibitor 666-15 or the genetic knockout of CREB significantly reduced the spheroid-forming and metastasis-initiating potentials of CKPS cells. Intriguingly, we observed that the irinotecan treatment induced CREB phosphorylation in liver metastases, and co-treatment of 666-15 greatly potentiated the inhibitory effect of irinotecan on liver metastasis. Furthermore, we found that the transition from 2D to 3D spheroid culture of CKPS cells led to the induction of RhoC expression. Notably, RhoC knockout in CKPS cells effectively suppressed spheroid formation in vitro and inhibited metastasis formation in an intrasplenic injection liver metastasis assay. Potential therapeutic implications of these findings will be discussed. Citation Format: Teruaki Fujishita, Makoto M. Taketo, Masahiro Aoki. Exploring signaling pathways essential for stemness and metastasis of colorectal cancer cells [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 5449.

Abstract 1489: The role of EPAS1 and TEAD1 in gastric cancer liver metastasis

Abstract Gastric cancer ranks fifth in incidence and fourth for cancer-related mortality worldwide. While the liver is widely recognized as the leading site of metastasis in gastric cancer, the intricate mechanisms underpinning its tropism toward the liver and the hematogenous metastasis process remain poorly understood and demand further investigation. Hematogenous metastasis encompasses the generation of circulating tumor cells from the primary tumor site and their subsequent attachment to the metastatic niche. This process involves the transition between adherent and suspension states. In our investigation of anchorage dependency, we identified 20 transcription factors upregulated in suspension cells compared to adherent cells and 18 transcription factors upregulated in adherent cells relative to suspension cells, respectively. To investigate factors involved in gastric cancer metastasis, RNA sequencing of paired primary and metastatic tissue samples obtained from 15 patients diagnosed with advanced gastric cancer was conducted, which disclosed the selective upregulation of EPAS1 and TEAD1 in metastatic liver tissue compared to the matched primary tumor tissue and the metastatic tissue originating from other sites. Through in vitro analysis, we observed a portion of adherent cells transitioning into suspension cells when subjected to a hyperdense environment. We also observed cell reattachment upon harvesting and replating these suspension-state transited cells. Comparing EPAS1 and TEAD1 expression in these states unveiled increased expression of EPAS1 and TEAD1 in reattached cells relative to suspension-state cells. We established a gastric cancer metastasis mouse model introducing gastric cancer cells into the mouse circulatory system via intracardiac injection to validate our findings. In vivo imaging was utilized to confirm systemic metastasis. After fluorescence-based cell sorting, EPAS1 and TEAD1 expression in cancer cells collected from the blood, liver, and ovary were assessed. We detected higher expression of EPAS1 and TEAD1 in liver metastasis compared to the circulating tumor cells. Furthermore, EPAS1 and TEAD1 were upregulated in liver metastatic cancer cells compared to ovarian metastatic cancer cells. Our findings underscore EPAS1 and TEAD1 as pivotal factors governing gastric cancer liver tropism and metastasis, offering insights that may contribute to targeted interventions in the clinical landscape. Citation Format: DONG KI Lee, Hyun Woo Park, Jae-Ho Cheong, Heon Yung Gee. The role of EPAS1 and TEAD1 in gastric cancer liver 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 1489.

Abstract 3265: Secretome core fucosylation drives clonal drug resistance expansion and hypoxic control of metastasis

Abstract Cancer secretome is a reservoir for aberrant glycosylation. How therapies alter this post- translational cancer hallmark and the consequences thereof remain elusive. Here, we show that an elevated secretome fucosylation is a pan-cancer signature of both response and resistance to multiple targeted therapies. Large-scale pharmacogenomics revealed that fucosylation genes display widespread association with resistance to these therapies. In cancer cell cultures, xenograft mouse models, and patients, targeted kinase inhibitors distinctively induced core fucosylation of secreted proteins less than 60 kDa. Label-free proteomics of N-glycoproteomes identified fucosylation of the antioxidant PON1 as a critical component of the therapy-induced secretome (TIS). N-glycosylation of TIS and target core fucosylation of PON1 are mediated by the fucose salvage-FUT8-SLC35C1 axis with PON3 directly modulating GDP-Fuc transfer on PON1 scaffolds. Core fucosylation in the Golgi impacts PON1 stability and folding prior to secretion, promoting a more degradation-resistant PON1. Global secretome and PON1-specific de-N-glycosylation both limited the expansion of resistant clones in a tumor regression model. We defined the resistance-associated transcription factors (TFs) and genes modulated by the N-glycosylated TIS via a focused and transcriptome-wide analyses. In a spontaneous lung metastasis mouse model, global secretome and PON1-specific core fucosylation functionally drive hypoxia and hypoxic selection of drug-resistant clones. Our findings demonstrate that core fucosylation is a common modification indirectly induced by targeted therapies that paradoxically promotes resistance and metastasis. Citation Format: Mark Borris D. Aldonza, Keun Hong Son, Je-Yoel Cho. Secretome core fucosylation drives clonal drug resistance expansion and hypoxic control of 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 3265.

Prevention of Metastasis by Suppression of Stemness Genes Using a Combination of microRNAs.

We propose an original strategy for metastasis prevention using a combination of three microRNAs that blocks the dedifferentiation of cancer cells in a metastatic niche owing to the downregulation of stemness genes. Transcriptome microarray analysis was applied to identify the effects of a mixture of microRNAs on the pattern of differentially expressed genes in human breast cancer cell lines. Treatment of differentiated CD44- cancer cells with the microRNA mixture inhibited their ability to form mammospheres in vitro. The combination of these three microRNAs encapsulated into lipid nanoparticles prevented lung metastasis in a mouse model of spontaneous metastasis. The mixture of three microRNAs (miR-195-5p/miR-520a/miR-630) holds promise for the development of an antimetastatic therapeutic that blocks tumor cell dedifferentiation, which occurs at secondary tumor sites and determines the transition of micrometastases to macrometastases.

Co-inhibition of TGF-β and PD-L1 pathways in a metastatic colorectal cancer mouse model triggers interferon responses, innate cells and T cells, alongside metabolic changes and tumor resistance

ABSTRACT Colorectal cancer (CRC) is the third most prevalent cancer worldwide with a high mortality rate (20–30%), especially due to metastasis to adjacent organs. Clinical responses to chemotherapy, radiation, targeted and immunotherapies are limited to a subset of patients making metastatic CRC (mCRC) difficult to treat. To understand the therapeutic modulation of immune response in mCRC, we have used a genetically engineered mouse model (GEMM), “KPN”, which resembles the human ‘CMS4’-like subtype. We show here that transforming growth factor (TGF-β1), secreted by KPN organoids, increases cancer cell proliferation, and inhibits splenocyte activation in vitro. TGF-β1 also inhibits activation of naive but not pre-activated T cells, suggesting differential effects on specific immune cells. In vivo, the inhibition of TGF-β inflames the KPN tumors, causing infiltration of T cells, monocytes and monocytic intermediates, while reducing neutrophils and epithelial cells. Co-inhibition of TGF-β and PD-L1 signaling further enhances cytotoxic CD8+T cells and upregulates innate immune response and interferon gene signatures. However, simultaneous upregulation of cancer-related metabolic genes correlated with limited control of tumor burden and/or progression despite combination treatment. Our study illustrates the importance of using GEMMs to predict better immunotherapies for mCRC.

Riboflavin protects against pancreatic cancer metastasis by targeting TGF-β receptor 1

The inhibition of transforming growth factor-β1 (TGF-β1) signaling by targeting TGF-β receptor 1 (TβR1) has been considered as an ideal approach for the prevention of pancreatic cancer metastasis. Utilizing a pharmacophore model for TβR1 inhibitors, candidate compounds with the potential TβR1 binding ability were screened from the U.S. Food and Drug Administration (FDA) database, and riboflavin (RF) with a highest fit value was chosen to investigate its binding ability to TβR1 and effect on TGF-β1 signaling in pancreatic cancer cells. Molecular docking and cellular thermal shift assay (CETSA) proved that RF at pharmacological concentrations could directly bind to TβR1. Further studies showed that pharmacological concentrations of RF in vitro could block TGF-β1 signaling, suppress the migration and invasion, and prevent epithelial-mesenchymal transition (EMT) process of pancreatic cancer cells in the absence or presence of TGF-β1 stimulation, indicating that RF presented anti-metastatic effect in pancreatic cancer cells. Knockdown of TβR1 could significantly attenuate the effects of RF on the migration and EMT process in pancreatic cancer cells, further confirming that the anti-metastatic effect of RF was achieved by blocking TGF-β1 signaling after binding to TβR1. Moreover, in a mouse model of pancreatic cancer metastasis, it was certified that RF administration could block lung and liver metastases, TGF-β1 signaling and EMT process of pancreatic cancer in vivo. In summary, our findings showed that RF could block TGF-β1 signaling by directly binding to TβR1, thereby suppressing the metastasis of pancreatic cancer cells by inhibiting EMT process both in vitro and in vivo.

Intra-bone marrow injection with engineered Lactococcus lactis for the treatment of metastatic tumors: Primary report

Bone marrow has the capacity to produce different types of immune cells, such as natural killer cells, macrophages, dendritic cells (DCs) and T cells. Improving the activation of immune cells in the bone marrow can enhance the therapy of bone metastases. Previously, we designed an engineered probiotic Lactococcus lactis, capable of expressing a fusion protein of Fms-like tyrosine kinase 3 ligand and co-stimulator OX40 ligand (FOLactis), and proved that it can induce the activation and differentiation of several immune cells. In this research, we successfully establish mouse models of bone metastasis, lung metastasis and intraperitoneal dissemination, and we are the first to directly inject the probiotics into the bone marrow to inhibit tumor growth. We observe that injecting FOLactis into the bone marrow of mice can better regulate the immune microenvironment of tumor-bearing mice, resulting in a tumor-suppressive effect. Compared to subcutaneous (s.c.) injection, intra-bone marrow (IBM) injection is more effective in increasing mature DCs and CD8+ T cells and prolonging the survival of tumor-bearing mice. Our results confirm that IBM injection of FOLactis reprograms the immune microenvironment of bone marrow and has remarkable effectiveness in various metastatic tumor models.

GRB2 stabilizes RAD51 at reversed replication forks suppressing genomic instability and innate immunity against cancer

Growth factor receptor-bound protein 2 (GRB2) is a cytoplasmic adapter for tyrosine kinase signaling and a nuclear adapter for homology-directed-DNA repair. Here we find nuclear GRB2 protects DNA at stalled replication forks from MRE11-mediated degradation in the BRCA2 replication fork protection axis. Mechanistically, GRB2 binds and inhibits RAD51 ATPase activity to stabilize RAD51 on stalled replication forks. In GRB2-depleted cells, PARP inhibitor (PARPi) treatment releases DNA fragments from stalled forks into the cytoplasm that activate the cGAS–STING pathway to trigger pro-inflammatory cytokine production. Moreover in a syngeneic mouse metastatic ovarian cancer model, GRB2 depletion in the context of PARPi treatment reduced tumor burden and enabled high survival consistent with immune suppression of cancer growth. Collective findings unveil GRB2 function and mechanism for fork protection in the BRCA2-RAD51-MRE11 axis and suggest GRB2 as a potential therapeutic target and an enabling predictive biomarker for patient selection for PARPi and immunotherapy combination.

EIF4A3-negatively driven circular RNA β-catenin (circβ-catenin) promotes colorectal cancer progression via miR-197-3p/CTNND1 regulatory axis.

Circβ-catenin, our first reported circRNA, has been reported to mediate tumorigenesis in various cancers. However, its biological functions and underlying mechanisms in colorectal cancer (CRC) remain unknown. The qRT-PCR examination was used to detect the expression of circβ-catenin, miR-197-3p, and CTNND1 in cells and human tissues. Western blot was conducted to detect the protein expression levels. The biological function of circβ-catenin was verified by MTT, colony formation, wound healing, and transwell assays. The in vivo effects of circβ-catenin were verified by nude mice xenograft and metastasis models. The regulatory network of circβ-catenin/miR-197-3p/CTNND1 was confirmed via dual-luciferase reporter and RIP assays. In the present study, circβ-catenin was found to promote CRC cell proliferation and metastasis in vitro and in vivo. Mechanistically, circβ-catenin served as miRNA decoy to directly bind to miR-197-3p, then antagonized the repression of the target gene CTNND1, and eventually promoted the malignant phenotype of CRC. More interestingly, the inverted repeated Alu pairs termed AluJb1/2 and AluY facilitated the biogenesis of circβ-catenin, which could be partially reversed by EIF4A3 binding to Alu element AluJb2. Our findings illustrated a novel mechanism of circβ-catenin in modulating CRC tumorigenesis and metastasis, which provides a potential therapeutic target for CRC patients.

Low-dose radiotherapy synergizes with iRGD-antiCD3-modified T cells by facilitating T cell infiltration

Background and purposePoor penetration of transferred T cells represents a critical factor impeding the development of adoptive cell therapy in solid tumors. We demonstrated that iRGD-antiCD3 modification promoted both T cell infiltration and activation in our previous work. Interest in low-dose radiotherapy has recently been renewed due to its immuno-stimulatory effects including T cell recruitment. This study aims to explore the synergistic effects between low-dose radiotherapy and iRGD-antiCD3-modified T cells. Materials and methodsFlow cytometry was performed to assess the expression of iRGD receptors and chemokines. T cell infiltration was evaluated by immunohistofluorescence and in vivo real-time fluorescence imaging and antitumor effects were investigated by in vivo bioluminescence imaging in the gastric cancer peritoneal metastasis mouse model. ResultsWe found that 2 Gy irradiation upregulated the expression of all three iRGD receptors and T-cell chemokines. The addition of 2 Gy low-dose irradiation boosted the accumulation and penetration of iRGD-antiCD3-modified T cells in peritoneal tumor nodules. Combining 2 Gy low-dose irradiation with iRGD-antiCD3-modified T cells significantly inhibited tumor growth and prolonged survival in the peritoneal metastasis mouse model with a favorable safety profile. ConclusionAltogether, we demonstrated that low-dose radiotherapy could improve the antitumor potency of iRGD-antiCD3-modified T cells by promoting T cell infiltration, providing a rationale for exploring low-dose radiotherapy in combination of other adoptive T cell therapies in solid tumors.

Combining TIGIT Blockade with MDSC Inhibition Hinders Breast Cancer Bone Metastasis by Activating Antitumor Immunity.

Bone is the most common site of breast cancer metastasis. Bone metastasis is incurable and is associated with severe morbidity. Utilizing an immunocompetent mouse model of spontaneous breast cancer bone metastasis, we profiled the immune transcriptome of bone metastatic lesions and peripheral bone marrow at distinct metastatic stages, revealing dynamic changes during the metastatic process. We show that cross-talk between granulocytes and T cells is central to shaping an immunosuppressive microenvironment. Specifically, we identified the PD-1 and TIGIT signaling axes and the proinflammatory cytokine IL1β as central players in the interactions between granulocytes and T cells. Targeting these pathways in vivo resulted in attenuated bone metastasis and improved survival, by reactivating antitumor immunity. Analysis of patient samples revealed that TIGIT and IL1β are prominent in human bone metastasis. Our findings suggest that cotargeting immunosuppressive granulocytes and dysfunctional T cells may be a promising novel therapeutic strategy to inhibit bone metastasis. Significance: Temporal transcriptome profiling of the immune microenvironment in breast cancer bone metastasis revealed key communication pathways between dysfunctional T cells and myeloid derived suppressor cells. Cotargeting of TIGIT and IL1β inhibited bone metastasis and improved survival. Validation in patient data implicated these targets as a novel promising approach to treat human bone metastasis.