Metastatic Pathways Research Articles

Knockdown of EIF2AK2-OAS1 axis reduces ATP production inducing AMPK phosphorylation to inhibit the malignant behavior of gastric cancer cells.

Energy metabolism has always been a hot topic in cancer progression and targeted therapy, and exploring the role of genes in energy metabolic pathways in cancer cells has become key to address this issue. Eukaryotic translation initiation factor 2α kinase 2 (EIF2AK2) plays regulatory roles in cancer and disorders of energy metabolism. Indeed, the role of EIF2AK2 in energy metabolism has been underestimated. The aim of this study is to reveal the expression specificity of EIF2AK2 in gastric cancer (GC) progression and metastasis, and to demonstrate the role of EIF2AK2 in energy metabolism, cytoskeleton, proliferation, death and metastasis pathways in GC cells. Mechanistically, EIF2AK2 overexpression promoted cytoskeleton remodeling and ATP production, mediated cell proliferation and metastasis, upregulated OAS1 expression, decreases p-AMPK expression and inhibited apoptosis in GC cells. Conversely, knockdown of EIF2AK2 resulted in the opposite effect. However, overexpression of OAS1 mediated the upregulation of mitochondrial membrane potential and promoted ATP production and NAD+/NADH ratio, but knockdown of OAS1 inhibited the above effects. In addition, knockdown of OAS1 had no effect on EIF2AK2 expression, but inhibited AMPK and upregulated p-AMPK expression. In conclusion, our study identified EIF2AK2 and OAS1 as previously undescribed regulators of energy metabolism in GC cells. We hypothesized that EIF2AK2-OAS1 axis may regulate energy metabolism and inhibit cellular malignant behavior in cancer cells by affecting ATP production to induce AMPK phosphorylation, suggesting EIF2AK2 as a potential therapeutic target for cancer cell progression.

[Translated article] Longitudinal Study of Different Progression Patterns in High-Risk Cutaneous Squamous Cell Carcinoma

Background and objectiveCutaneous squamous cell carcinoma (cSCC) is the second leading cause of skin cancer mortality in Europe. Few studies have analyzed the different pathways of this tumor progression in its natural history. The main objective of this study was to analyze the different metastatic and progression pathways and their temporal occurrence in the evolution of cSCC. Material and methodWe conducted a multicenter, retrospective, and observational study of consecutive high-risk sSCCs included in the SQUAMATA project. ResultsA total of 222 out of the 1346 patients included relapsed. The most frequent route of progression was the lymphatic one (62.6%). A total of 20.2% of the cases with lymphatic progression developed distant metastases. Only 1 case (3.1%) of distant metastasis followed local recurrence without previous lymphatic metastasis. The median time to disease-related mortality was longer in patients who developed systemic metastases than in those who died of locoregional progression. ConclusionsThe mortality of patients with cSCC is mostly due to the regional progression of their lymphatic metastases. The appearance of distant metastases is practically always (96.9%) associated with previous lymphatic metastatic progression. Therefore, in the future, new studies will be needed to assess the regional management of cSCC in both surgical and adjuvant therapies.

Comparing the HER2 Status of the Primary Tumor to That of Disseminated Tumor Cells in Early Breast Cancer.

Breast cancer remains a leading cause of cancer mortality in women globally. Despite advancements in systemic therapy, the risk of distant recurrence persists even after such treatment and may be linked to disseminated tumor cells (DTCs). Variability in molecular characteristics between primary tumors (PTs) and distant metastases underscores the need to comprehensively understand metastatic pathways. This retrospective study investigated discrepancies between HER2 expression in PTs and DTCs and their implications for survival outcomes in 201 early breast cancer (EBC) patients. We found a significant association between HER2 expression in PTs and DTCs when classifying tumors as HER2-high/low/negative. Patients whose HER2 status was discordant between PTs and DTCs exhibited worse distant disease-free survival than those with concordant status. Multivariate analysis confirmed the HER2 status of DTCs as an independent prognostic factor for distant DFS. These findings emphasize the importance of assessing HER2 expression in DTCs and its potential implications for tailored therapy strategies in EBC. Furthermore, prospective trials are needed to validate these findings and explore targeted therapies based on the molecular characteristics of DTCs.

Abdominal Parietal Metastasis from Cervical Cancer: A Review of One of the Most Uncommon Sites of Recurrence Including a Report of a New Case.

Cervical cancer is the fourth most common cancer in women, the highest mortality being found in low- and middle-income countries. Abdominal parietal metastases in cervical cancer are a very rare entity, with an incidence of 0.1-1.3%, and represent an unfavorable prognostic factor with the survival rate falling to 17%. Here, we present a review of cases of abdominal parietal metastasis in recent decades, including a new case of a 4.5 cm abdominal parietal metastasis at the site of the scar of the former drain tube 28 months after diagnosis of stage IIB cervical cancer (adenosquamous carcinoma), treated by external radiotherapy with concurrent chemotherapy and intracavitary brachytherapy and subsequent surgery (type B radical hysterectomy). The tumor was resected within oncological limits with the histopathological result of adenosquamous carcinoma. The case study highlights the importance of early detection and appropriate treatment of metastases in patients with cervical cancer. The discussion explores the potential pathways for parietal metastasis and the impact of incomplete surgical procedures on the development of metastases. The conclusion emphasizes the poor prognosis associated with this type of metastasis in cervical cancer patients and the potential benefits of surgical resection associated with systemic therapy in improving survival rates.

Enhanced angiogenesis of human umbilical vein endothelial cells via THP-1-derived M2c-like macrophages and treatment with proteasome inhibitors 'bortezomib and ixazomib'.

The leading cause of cancer-related death is lung cancer, with metastasis being the most common cause of death. To elucidate the role of macrophages in lung cancer and angiogenesis processes, we established an in vitro co-culture model of A549 or HUVEC with THP-1 cells that polarized to M2c macrophages with hydrocortisone. The proteasome inhibitors bortezomib and ixazomib were investigated for their effects on proliferation, invasion, migration, metastasis, and angiogenesis pathways. The effects of bortezomib and ixazomib on gene expression in gene panels, including crucial genes related to angiogenesis and proteasomes, were investigated after the co-culture model to determine these effects at the molecular level. In conclusion, bortezomib and ixazomib showed antiproliferative effects in both cells, as well as in M2c macrophage co-culture. M2c macrophages also increased invasion in A549 cells and both invasion and migration in HUVEC. mRNA expression upregulation, specifically in the NFKB and VEGF genes, supported the metastatic and angiogenic effects found in A549 and HUVEC with M2c macrophage co-culture. Additionally, bortezomib inhibited the VEGFB pathway in HUVEC and NFKB1 in A549 cells. The significant findings obtained as a result of this study will provide information regarding angiogenesis induced by M2 macrophages.

Regulatory effects of statins on Akt signaling for prevention of cancers

Statins, which are primarily used as lipid-lowering drugs, have been found to exhibit anti-tumor effects through modulating and interfering with various signaling pathways. In observational studies, statin use has been associated with a significant reduction in the progression of various cancers, including colon, lung, prostate, pancreas, and esophagus cancer, as well as melanoma and B and T cell lymphoma. The mevalonate pathway, which is affected by statins, plays a crucial role in activating Rho, Ras, and Rab proteins, thereby impacting the proliferation and apoptosis of tumor cells. Statins block this pathway, leading to the inhibition of isoprenoid units, which are critical for the activation of these key proteins, thereby affecting cancer cell behavior. Additionally, statins affect MAPK and Cdk2, which in turn reduce the expression of p21 and p27 cyclin-dependent kinase inhibitors. Akt signaling plays a crucial role in key cancer cell features like proliferation, invasion, and apoptosis by activating multiple effectors in downstream pathways such as FOXO, PTEN, NF-κB, GSK3β, and mTOR. The PI3K/Akt signaling is necessary for many events in the metastatic pathway and has been implicated in the resistance to cytostatic drugs. The Akt/PTEN axis is currently attracting great interest for its role in carcinogenesis. Statins have been shown to activate the purinergic receptor P2X7 and affect Akt signaling, which may have important anti-cancer effects. Hence, targeting Akt shows promise as an effective approach to cancer prevention and therapy. This review aims to provide a comprehensive discussion on the specific impact of statins through Akt signaling in different types of cancer.

Abstract PO1-11-03: Women with Metastatic Disease: Modeling a Clear and Well-Defined Announcement Pathway

Abstract Introduction: The announcement of the diagnosis of metastatic breast cancer is a key step in enabling the patient to fully understand her disease and the various treatment options, and thus to be more involved in her management and follow-up. However, according to the « Croyances et réalités 2 » survey conducted in 2021 among 269 French patients with metastatic breast cancer, 38% of them felt that they "did not fully understand" the information given by their doctor during the announcement consultation. Objective: The objective of this work is to model an optimal pathway for the announcement of metastatic breast cancer based on precise guidelines and good coordination between the different actors, in order to ensure that all patients have the necessary information and support. Method: A scientific committee composed of 4 French oncologists from 4 different centers initiated a collaborative process to propose, after discussions, precise guidelines for the announcement of metastatic breast cancer. Results: A set of 13 concrete guidelines were suggested by the expert group (Table 1). The central aspect is to build a specialized and flexible support, adapted to the needs of the patient, without ever hiding the truth about her disease. The announcement process must be adapted to the patient's history (recurrent or immediately metastatic), but also to the organization and resources available in the health care institution. The experts recommend a model of announcement process in two stages, separated by a few days. First, the oncologist announces what the entry into metastatic disease represents, followed by a repeat of the announcement by a nurse trained in the specificities of metastatic breast cancer, in order to have the patient reformulate her understanding of the disease and treatment. In a second phase, a new consultation allows to present the next therapeutic steps and ensure a coordinated intervention of the different health care professionals involved: doctors, nurses, pharmacists and supportive care specialists as needed. The offer of supportive care should be flexible and it is recommended that questionnaires should be made available to the patient to help identify her precise needs. The experts recommend that this second consultation be conducted jointly by an oncologist and a nurse. Subsequently, the town physician and pharmacist must be contacted to ensure the relay in the accompaniment of the patients in partnership with the cancer care center, and thus to ensure a good sharing of information on the patient's follow-up. To help the establishment of this dynamic announcement model, the experts gave advice on implementation: training the professionals involved in the process through theoretical training and role-playing, increasing the amount of time spent on the announcement consultation, and setting up a library of online or physical information materials to share with the patient. Finally, it is recommended that the financing of this process be prepared at the institutional level before its effective implementation in order to ensure its sustainability. Conclusion: Organizing a consultation for the announcement of metastatic breast cancer is essential to optimize the experience of the announcement and support. It must be a structured, funded and coordinated process between different health professionals specially trained for this approach. Table 1: Summary of guidelines for a metastatic breast cancer notification pathway. Citation Format: Florence Dalenc, Séverine Guiu, Marie-Ange Mouret-Reynier, Nathalie Quenel-Tueux. Women with Metastatic Disease: Modeling a Clear and Well-Defined Announcement Pathway [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-11-03.

CX3CL1 (Fractalkine)-CX3CR1 Axis in Inflammation-Induced Angiogenesis and Tumorigenesis.

The chemotactic cytokine fractalkine (FKN, chemokine CX3CL1) has unique properties resulting from the combination of chemoattractants and adhesion molecules. The soluble form (sFKN) has chemotactic properties and strongly attracts T cells and monocytes. The membrane-bound form (mFKN) facilitates diapedesis and is responsible for cell-to-cell adhesion, especially by promoting the strong adhesion of leukocytes (monocytes) to activated endothelial cells with the subsequent formation of an extracellular matrix and angiogenesis. FKN signaling occurs via CX3CR1, which is the only known member of the CX3C chemokine receptor subfamily. Signaling within the FKN-CX3CR1 axis plays an important role in many processes related to inflammation and the immune response, which often occur simultaneously and overlap. FKN is strongly upregulated by hypoxia and/or inflammation-induced inflammatory cytokine release, and it may act locally as a key angiogenic factor in the highly hypoxic tumor microenvironment. The importance of the FKN/CX3CR1 signaling pathway in tumorigenesis and cancer metastasis results from its influence on cell adhesion, apoptosis, and cell migration. This review presents the role of the FKN signaling pathway in the context of angiogenesis in inflammation and cancer. The mechanisms determining the pro- or anti-tumor effects are presented, which are the cause of the seemingly contradictory results that create confusion regarding the therapeutic goals.

Metastatic patterns and treatment options for head and neck cutaneous squamous cell carcinoma (Review).

According to current predictions, one-fifth of all Americans will develop skin cancer during their lifetime. Cutaneous squamous cell carcinoma (cSCC) most commonly occurs in the head and neck region, which is the area of the body with the highest level of sun exposure. High-risk head and neck cSCC (HNcSCC) is a broad category with numerous high-risk factors that are associated with unfavorable results. In cSCC staging systems, clinical and tumor traits that are likely to result in poor outcomes are identified. Metastasis occurs in ~2.5% of patients with cSCC, most often in the local lymph nodes, and there is some indication that lymph node metastasis has a distinct pattern based on the tumor site. Current findings on tumor molecular targets have suggested the use of systemic treatments, particularly immunotherapy (such as cemiplimab, pembrolizumab and nivolumab), over radiotherapy or chemotherapy for this type of metastasis. However, when used simultaneously with immunotherapy, radiotherapy may be beneficial in the treatment of metastatic HNcSCC by improving the efficacy of immunotherapy. The present review aims to assess the existing literature on metastatic HNcSCC pathways and treatment options, in order to define current and future directions. Notably, there is an urgent need to identify patients who may benefit from local or systemic cancer treatments. The treatment of lymph node metastasis presents a therapeutic challenge and requires comprehensive management.

Angiogenesis-Enabled Human Ovarian Tumor Microenvironment-Chip Evaluates Pathophysiology of Platelets in Microcirculation.

The tumor microenvironment (TME) promotes angiogenesis for its growth through the recruitment of multiple cells and signaling mechanisms. For example, TME actively recruits and activates platelets from the microcirculation to facilitate metastasis, but platelets may simultaneously also support tumor angiogenesis. Here, to model this complex pathophysiology within the TME that involves a signaling triad of cancer cells, sprouting endothelial cells, and platelets, an angiogenesis-enabled tumor microenvironment chip (aTME-Chip) is presented. This platform recapitulates the convergence of physiology of angiogenesis and platelet function within the ovarian TME and describes the contribution of platelets in promoting angiogenesis within an ovarian TME. By including three distinct human ovarian cancer cell-types, the aTME-Chip quantitatively reveals the following outcomes-first, introduction of platelets significantly increases angiogenesis; second, the temporal dynamics of angiogenic signaling is dependent on cancer cell type; and finally, tumor-educated platelets either activated exogenously by cancer cells or derived clinically from a cancer patient accelerate tumor angiogenesis. Further, analysis of effluents available from aTME-Chip validate functional outcomes by revealing changes in cytokine expression and several angiogenic and metastatic signaling pathways due to platelets. Collectively, this tumor microphysiological system may be deployed to derive antiangiogenic targets combined with antiplatelet treatments to arrest cancer metastasis.

TFE3/PI3K/Akt/mTOR Axis in Renal Cell Carcinoma Affects Tumor Microenvironment

The role of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway in renal cell carcinoma (RCC) progression, metastasis, and resistance to therapies has not been investigated thoroughly. Transcription factor E3 (TFE3) expression is related to a poorer prognosis and tumor microenvironment in patients with RCC. This study aimed to determine the relationship between TFE3 and the PI3K/Akt pathway. TFE3 down-regulation was achieved by transient transfection of siRNA and shRNA in UOK146 cells. TFE3 overexpression was induced by transient transfection with pcDNA3.1 encoding the constitutively active form of TFE3. The cells were treated with mammalian target of rapamycin (mTOR) and PI3K inhibitors. Western blot was performed to detect TFE3, programmed death-ligand 1, phospho-Akt, and Akt. Phospho-Akt expression increased significantly upon TFE3 down-regulation, and decreased significantly upon up-regulation. When RCC cells were treated with a PI3K inhibitor (LY294002), TFE3 expression increased and phospho-Akt expression decreased. Data from this study indicate that TFE3 plays a role in the PI3K/Akt pathway in RCC. The results of this study suggest that PI3K/Akt inhibitors may aid in the treatment of patients with RCC by affecting the tumor microenvironment.

The Molecular Landscape of Lung Metastasis in Primary Head and Neck Squamous Cell Carcinomas.

Background Lung metastasis in head and neck cancer (HNC) patients is a critical concern, often indicating an advanced disease stage and a poor prognosis. This study explores the molecular complexities of such metastases, identifying specific genes and pathways that may serve as valuable targets for diagnosis and treatment. The findings underscore the potential for significantly improved patient outcomes through targeted therapeutic strategies. Methodology In this research, we systematically collected raw gene expression data from head and neck squamous cell carcinoma (HNSCC) and lung squamous cell carcinoma (LSCC). By comparing tumorous and normal gene expression profiles from paired patient samples, we identified differentially expressed genes (DEGs). Network analysis helped visualize protein interactions and pinpoint crucial hub genes. Through validation and comparison across several datasets, we identified common DEGs. Additionally, we employed Kaplan-Meier analysis and log-rank testing to examine the relationship between gene expression patterns and patient survival. Result The study identified 145 overlapping DEGs in both HNSCC and LSCC, which are crucial for cancer progression and linked to lung metastasis, offering vital targets for personalized therapy by identifying key genes affecting disease development and patient survival. Pathway analyses linked these to lung metastasis, while protein-protein interaction network construction and hub gene identification highlighted genes crucial for development and patient survival, offering targets for personalized therapy. Conclusion Identifying key genes and pathways in lung metastasis from HNC, this study highlights potential targets for enhanced diagnosis and therapy. It underscores the crucial role of molecular insights in driving forward personalized treatment approaches and improving patient outcomes.

Elucidating the role of MICAL1 in pan-cancer using integrated bioinformatics and experimental approaches

ABSTRACT Molecule interacting with CasL 1 (MICAL1) is a crucial protein involved in cell motility, axon guidance, cytoskeletal dynamics, and gene transcription. This pan-cancer study analyzed MICAL1 across 33 cancer types using bioinformatics and experiments. Dysregulated expression, diagnostic potential, and prognostic value were assessed. Associations with tumor characteristics, immune factors, and drug sensitivity were explored. Enrichment analysis revealed MICAL1’s involvement in metastasis, angiogenesis, metabolism, and immune pathways. Functional experiments demonstrated its impact on renal carcinoma cells. These findings position MICAL1 as a potential biomarker and therapeutic target in specific cancers, warranting further investigation into its role in cancer pathogenesis.

Exploring the role of interleukin 11 in cancer progression, patient survival, and therapeutic insights.

The Interleukin (IL)-11 gene, which is one of the members of the cytokine family, has an oncogenic role in some cancers. The main goal of this study is to analyze IL-11 expression level in 14 prevalent cancers and highlights its role in patients' survival, drug resistance, and sensitivities. Also, an association of this gene with metastasis and inflammation pathways has been investigated. Using the cancer genome atlas (TCGA) data, the level of IL-11 expression and its role in prognosis and survival rate were evaluated in 13 common cancers. Then, confirming the obtained in-silico outcomes, the relative expression level of this gene in colorectal cancer (CRC) samples and their adjusted tissues were assayed by the RT-qPCR method. Furthermore, to examine the association between IL-11 expression and drug resistance and sensitivity, PharmacoGX data was applied. The co-expression network was used to recognize the pathways in which IL-11 was involved. The results from the TCGA dataset indicated that the expression level of IL-11 increased significantly in 13 prevalence cancers compared to the control groups. Interestingly, this enhanced expression level is associated with a high rate of mortality in patients with bladder, stomach, colorectal, and endometrial cancers. Also, the co-expression network analysis showed a strong correlation between IL-11 and the genes of metastasis pathway and the genes related to the inflammation process. Finally, regarding drug sensitivity, IL-11 expression level can be introduced as a remarkable biomarker for cancer detection due to area under curve (AUC). Altered expression of the IL-11 gene is observed in 13 common cancers and is associated with prognosis and mortality rate in patients. Moreover, this gene can be considered a prognostic biomarker in different types of cancer, such as CRC.

Estudio longitudinal de los diferentes patrones de progresión en el carcinoma cutáneo de células escamosas de alto riesgo

Background and objectiveCutaneous squamous cell carcinoma (cSCC) is the second leading cause of skin cancer mortality in Europe. Few studies have analyzed the different pathways of this tumor progression in its natural history. The main objective of this study was to analyze the different metastatic and progression pathways and their temporal occurrence in the evolution of cSCC. Material and methodWe conducted a multicenter, retrospective, and observational study of consecutive high-risk sSCCs included in the SQUAMATA project. ResultsA total of 222 out of the 1346 patients included relapsed. The most frequent route of progression was the lymphatic one (62.6%). A total of 20.2% of the cases with lymphatic progression developed distant metastases. Only 1 case (3.1%) of distant metastasis followed local recurrence without previous lymphatic metastasis. The median time to disease-related mortality was longer in patients who developed systemic metastases than in those who died of locoregional progression. ConclusionsThe mortality of patients with cSCC is mostly due to the regional progression of their lymphatic metastases. The appearance of distant metastases is practically always (96.9%) associated with previous lymphatic metastatic progression. Therefore, in the future, new studies will be needed to assess the regional management of cSCC in both surgical and adjuvant therapies.

Insights Into Colorectal Carcinoma: A Comprehensive Review of MicroRNA Expression Patterns.

Colorectal carcinoma (CRC) remains a significant contributor to cancer-related morbidity and mortality worldwide. MicroRNAs (miRNAs) have emerged as crucial regulators of gene expression and play critical roles in various biological processes, including carcinogenesis. This comprehensive review aims to elucidate the role of miRNAs in CRC by analyzing their expression patterns and functional implications. An extensive literature review identified dysregulated miRNAs associated with differentstages of CRC progression, from initiation to metastasis. These miRNAs modulate key signaling pathways in tumor growth, invasion, and metastasis. Furthermore, we discuss the potential of miRNAs as diagnostic biomarkers and therapeutic targets in CRC management. Future research directions include elucidating the functional significance of dysregulated miRNAs using advanced experimental models and computational approaches and exploring the therapeutic potential of miRNA-based interventions in personalized treatment strategies for CRC patients. Collaboration among researchers, clinicians, and industry partners will be essential to translate these findings into clinically impactful interventions that improve patient outcomes in CRC.

Abstract 674: Novel anti-cancer small molecule targeting the protein regulator of cytokinesis pathway

Abstract Aberrant migration and proliferation of cancer cells are the hallmark of metastatic tumors. Protein Regulator of Cytokinesis 1 (PRC1) is a scaffold protein that regulates metabolic pathways of cancer cell proliferation, survival, and metastasis. BKT300 is a small molecule (MW 399.33, C22H25NO6), that selectively inhibits the migration and survival of hematological and solid cancer cells. BKT300 inhibits, at high affinity, both actin and microtubule formation and organization leading to G2/M cell cycle arrest, mitotic catastrophe, and apoptosis through the caspase 3 pathway. These effects are selective to cancer cells and were not observed on normal cells. BKT300 was found to arrest PRC1 in a phosphorylated form at position T481, down regulates the expression of CDC25C and up regulate p21. Suppression of PRC1 using SiRNA ablation inhibited BKT300 induced apoptosis on several tumor cell lines and inhibited the effect of BKT300 on G2M cell cycle arrest. Furthermore, using Biacore SPR, microscale thermophoresis (MST), and ELISA assays we were able to demonstrate the direct binding of BKT300 to PRC1. Beyond its single agent activity, BKT300 was shown to synergize with standard of care (SoC) treatments, including anti PD-1 immunotherapy, BCL-2 inhibitor (Ventoclax), topoisomerase I inhibitor (Irinotecan) topoisomerase II inhibitor (Doxorubicin), anti-metabolite Fluorouracil (5-FU) and alkylating agent (Temozolomide). BKT300 has a remarkable safety profile when administered at high doses to mice rats, minipigs and non-human primates. Treatment with BKT300 showed almost no effects on hematopoiesis and biochemistry in doses far higher than the affective ones. The in-vivo activity of BKT300 was demonstrated in multiple Xenograft, Syngeneic and PDX models in mice. In-vivo treatment with BKT300 resulted in preventive (small tumors) as well as therapeutic (large tumors) activity in triple negative breast cancer (TNBC) xenograft, MDA-MB-453 and MDA-MB-468 models leading to over then 70%-90% reduction in tumor size following one to two treatments cycles. Similar results were obtained using PDX models derived from patients with Colon, Ovarian and Pancreatic cancer. BKT300 is a novel, first in class, targeted anti-cancer treatment that inhibits the activity of the scaffold protein PRC1. BKT300 is expected to provide a new treatment option to, the high unmet medical need of, advanced difficult to treat cancers, specifically ones that overexpress PRC1 and CDC25C. Citation Format: Amnon Peled, Michal Abraham, Hanna Wald, Rakefet Rosenfeld, Orly Eizenberg, Arnon Aharon. Novel anti-cancer small molecule targeting the protein regulator of cytokinesis pathway [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 674.

Abstract 7429: A web-based application to co-register elemental imaging with histopathology to enhance the study of metal bioaccumulation within tumors

Abstract Essential trace elements are crucial in various biological processes, including tumor growth, migration, and metastasis. For instance, copper ions are involved in mitochondrial metabolism, cell proliferation, tumor migration, metastasis and proangiongenic pathways. Localizing where metal ion homeostasis is disrupted is vital for understanding these processes beyond what bulk analysis can reveal. Elemental imaging offers high-resolution, quantifiable multi-elemental distribution maps, providing a more detailed analysis compared to traditional bulk measurements. However, aligning findings with tissue structures identified in histopathological data can be challenging. To address this, we developed a web-based application for the co-registration and analysis of Whole Slide Images (WSI) and elemental imaging data at the cellular level. The application integrates WSI with elemental maps generated by technologies such as ultrafast laser ablation and inductively coupled plasma time-of-flight mass spectrometry (LA-ICP-TOF-MS), as well as a range of other elemental imaging techniques. Hosted by the Biomedical National Elemental Imaging Resource, the current application allows for a streamlined data integration, co-registration, and analysis, including: 1) uploading WSI, elemental maps, and pathologist annotations, 2) automated preprocessing to identify tissue regions, 3) co-registration to merge WSI and elemental maps, 4) custom import, real-time overlay/editing of pathologist annotations transferred from WSI, 5) plotting, statistical tools and 6) data export. This application enables users to compare elemental abundance within/between tissue structures annotated on H&E or immunohistochemical slides. Derived data can pinpoint elements and mixtures whose abundance differs significantly across various pathological conditions. As an initial test, we applied the software on breast HER2/ER/PR tumors, varying in molecular subtype/histology. Preliminary results, using Bayesian lognormal regression models, adjusting for tumor subtypes, indicated a subtype-dependent variation in copper abundance. The relative difference in copper levels between tumor and stromal regions was notably less in HER2 tumors (p=0.026) compared to HR tumors (p<0.0001). Conversely, iron showed a consistently higher abundance in stromal areas across both subtypes (p<0.0001). Future enhancements include improved co-registration capabilities, hotspot analysis, machine learning, mixture effects, and cloud-based architecture. This method uniquely enables integration of elemental data with spatial transcriptomics, mapping gene transcripts on tissue slides. With current ST assays aligned with H&E slides, this tool facilitates co-registration, offering insights into biological pathways affected by metal homeostasis disruptions. Citation Format: Yunrui Lu, Ramsey Steiner, Serin Han, Matthew Chan, Tracy Punshon, Brian Jackson, Linda Vahdat, Jonathan Marotti, Joshua Levy. A web-based application to co-register elemental imaging with histopathology to enhance the study of metal bioaccumulation within tumors [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 7429.

Abstract 7569: Characterizing pathways for metastasis of retinoblastoma in forebrain organoids

Abstract Retinoblastoma (Rb) is a childhood cancer of the eye, which usually presents in children under the age of five. When detected early, Rb has favorable prognosis in developed countries, but metastasis into the central nervous system (CNS) through the optic nerve correlate with a high mortality rate. Even if detected early, the available harsh and unspecific treatments can cause serious side effects, and enucleations are still performed frequently. There is still uncertainty regarding the mechanism for metastasis into the central nervous system. We performed bioinformatic analysis of single cell sequencing data for retinoblastoma, retinal tissues, optic nerve, and brain, to identify potential pathways and cell-cell communications fostering Rb invasion into the optic nerve and brain. Our analysis revealed several potential metastatic drivers, including immune modulation involving MIF, secreted by the Rb cells, as well as cell-adhesion pathways promoting a change in cellular phenotype. Given that animal models do not faithfully recapitulate human Rb initiation and progression, we developed a novel human iPSC-derived forebrain organoid model to investigate mechanisms of Rb CNS metastasis. Microinjections of GFP-labelled Rb cells allow to track growth and spread. In this fully human model including microglial cells, we can, for the first time, study the Rb forebrain interactions and pathways involved. RNA-seq analysis of Rb cells growing in forebrain organoids shows increased activity in neuron projection development and neuron migration, overlapping with the observed phenotype switch of invading Rb. Together, we present a novel iPSC-derived human forebrain model to monitor growth and spread of metastatic Rb, and describe new pathways involved in invading and metastasizing Rb. Citation Format: Yulia Shtanko, Sara Rodriguez, Benjamin Schachner, Emily Adis, Samantha McLaughlin, Sebastian Borges, James Dollar, Ashley Zuniga, Gabriela Quintana, Sofia Lopez, Dawn Owens, Chloe Moulin, Steffanie Braun, Gulum Yenisehirli, Daniel Pelaez, J. William Harbour, Stefan Kurtenbach. Characterizing pathways for metastasis of retinoblastoma in forebrain organoids [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 7569.

Abstract 1560: In vivo CRISPR/Cas9 transcriptional activation screen leads to discovery of INAFM2, a novel driver of metastasis

Abstract Metastasis is the main cause of mortality in cancer patients and accounts for about 90% of cancer deaths, which has not changed in the past 50 years. This indicates a significant need for further study of metastasis pathways to find novel druggable targets. Ewing sarcoma (ES) is an aggressive childhood cancer that occurs in the bones or soft tissue. The five-year survival rate for Ewing sarcoma patients diagnosed with metastatic disease is still only 25%, making ES a good model for studying metastasis. We performed a genome-wide CRISPR/Cas9 transcriptional activation screen in a zebrafish xenograft model to discover genes whose activation leads to increased metastasis. The screen identified a number of genes that may accelerate ES metastasis; we chose INAFM2 for further study because multiple unique INAFM2-targeting gRNAs were enriched in the screen and we discovered a potential link to metastasis and patient survival in publicly available clinical data. Overexpression of INAFM2 in six different cancer cell lines (four ES, one hepatocellular carcinoma, and one stomach adenocarcinoma) caused increased cell migration and chemotaxis in vitro, and increased intravasation in zebrafish xenografts. INAFM2 knockdown in four cancer cell lines (two ES, one clear cell renal cell carcinoma, and one stomach adenocarcinoma) decreased chemotaxis and rescue of INAFM2 expression restored the cells’ ability to migrate. Analysis of patient tumor RNA sequencing data from many different types of cancers revealed correlations between INAFM2 expression and both an increased metastatic phenotype and poor patient survival. Further in vivo studies by tail vein injection of ES cells in immunodeficient mice demonstrated significantly increased gross metastases with INAFM2 overexpression and decreased gross metastases with INAFM2 knockdown compared to the respective control groups. This provides strong evidence for a substantial role of INAFM2 in cancer metastasis. Human INAFM2 is an unstudied gene and our preliminary work has revealed that the INAFM2 protein localizes to the cell membrane, is glycosylated, and may be involved in Wnt and/or MAPK pathway signaling. Inafm2 silencing by morpholino in zebrafish embryos lead to significant mortality, necrosis, and developmental delays/defects including severe cardiac edema. Current studies are focused on further discerning the molecular pathways that mediate the role of INAFM2 in promoting metastasis using in vitro and in vivo models as well as characterizing the functional significance of the direct protein binding partners and post-translational modifications for INAFM2. Citation Format: Anna Molotkova, Emre Deniz, Matthew Swift, Eric Glasgow, Jeffrey Toretsky, Aykut Üren. In vivo CRISPR/Cas9 transcriptional activation screen leads to discovery of INAFM2, a novel driver 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 1560.