Pro-tumor Role Research Articles

LINC00470 promotes malignant progression of testicular germ cell tumors.

Testicular germ cell tumor (TGCT) is a common malignant tumor in adolescents. Now, many long non-coding RNAs (LncRNAs) have been found to have an important function in TGCT. LINC00470 is specifically and highly expressed in TGCT, however, there is still no definite information concerning its role and underlying mechanism in TGCT. The purpose of this research was to look into the involvement of LINC00470 in TGCT and its intrinsic mechanism. UCSC and GEPIA2 databases were used to analyze the expression of LINC00470, and the BEST website was used to perform GSEA enrichment analysis, immune infiltration analysis, and drug susceptibility analysis. SiRNA transfection was used to silence LINC00470 in TCAM-2 and NCCIT cells. Clone formation and Transwell assays were performed in TGCT cells to confirm the effects of LINC00470 on clone formation, migration, and invasion. Western Blot was performed to determine the expression of proteins related to the EMT and AKT signaling pathways. LINC00470 was specifically highly expressed in TGCT, and played a role in promoting tumor cell clone formation and cell metastasis by affecting the TGF-β and PI3K-AKT-mTOR signaling pathways to regulate the epithelial-mesenchymal transition (EMT) process; LINC00470 may also play a pro-tumor role by negatively regulating immune infiltration; in addition, the expression of LINC00470 was negatively correlated with the chemosensitivity of cisplatin in TGCT patients. LINC00470 may play a significant role in the etiology and metastasis of TGCT through EMT and AKT-mediated signaling pathways.

Identification of HSPG2 as a bladder pro-tumor protein through NID1/AKT signaling

PurposeHeparan sulfate proteoglycans (HSPGs) are complex molecules found on the cell membrane and within the extracellular matrix, increasingly recognized for their role in tumor progression. This study aimed to investigate the involvement of Heparan sulfate proteoglycan 2 (HSPG2) in the progression of bladder cancer.MethodsWe identified HSPG2 as a promoter of bladder tumor progression using single-cell RNA sequencing and transcriptome analysis of sequencing data from seven patient samples obtained from the Gene Expression Omnibus (GEO) database (GSE135337). Transcript profiles of 28 normal tissues and 407 bladder tumor tissues were analyzed for HSPG2 expression and survival outcomes using the Sanger tools and cBioPortal databases. HSPG2-overexpressing T24 and Biu-87 cell lines were generated, and cell proliferation and migration were assessed using CCK-8 and Transwell assays. Western blotting and immunostaining were performed to evaluate the activation of Nidogen-1 (NID1)/protein kinase B (AKT) signaling. Mouse models with patient-derived tumor organoids (HSPG2high and HSPG2low) were established to assess anticancer effects.ResultsOur results demonstrated a marked upregulation of HSPG2 in malignant bladder tumors, which correlated significantly with poor patient prognosis. HSPG2 overexpression consistently enhanced bladder tumor cell proliferation and conferred chemotherapy resistance, as shown in both in vitro and in vivo experiments. Mechanistically, HSPG2 upregulated NID1 expression, leading to the activation of the AKT pro-survival signaling pathway and promoting sustained tumor growth in bladder cancer.ConclusionThis study highlights the critical pro-tumor role of HSPG2/NID1/AKT signaling in bladder cancer and suggests its potential as a therapeutic target in clinical treatment.

Spatial analyses revealed S100P + TFF1 + tumor cells in spread through air spaces samples correlated with undesirable therapy response in non-small cell lung cancer

Spread through air spaces (STAS) is a recognized aggressive pattern in lung cancer, serving as a crucial risk factor for postoperative recurrence. However, its phenotype and related spatial structure have remained elusive. To address these limitations, we conducted a comprehensive study based on spatial data, analyzing over 30,000 spots from 14 non-STAS samples and one STAS sample. We observed increased proliferation activities and angiogenesis in STAS, identifying S100P as a potential biomarker for STAS. Furthermore, our investigation into the heterogeneity of STAS tumor cells revealed a subset identified as S100P + TFF1 +, exhibiting a negative impact on patients' survival in public datasets. This subtype exhibited the highest activities in the TGFb and hypoxia, suggesting its potential pro-tumor role within the tumor microenvironment. To assess the role of S100P + TFF1 + tumor cells in therapy response, we included data from two clinical trial cohorts (BPI-7711 for EGFR-TKI therapy and ORIENT-3 for immunotherapy). The presence of S100P + TFF1 + tumor cells correlated with worse responses to both EGFR-TKI therapy and immunotherapy. Notably, TFF1 emerged as a serum marker for predicting EGFR-TKI response. Cell–cell communication analysis revealed that the TGFb signaling pathway was the most activated in S100P + TFF1 + tumor cells, with TGFB2-TGFBR2 identified as the main ligand-receptor pair. This was further validated by multiplex immunofluorescence performed on twenty NSCLC samples. In summary, our study identified S100P as the biomarker for STAS and highlighted the adverse role of S100P + TFF1 + tumor cells in survival outcomes.

High expression of COL8A1 predicts poor prognosis and promotes EMT in papillary thyroid cancer.

Collagen type VIII α 1 chain (COL8A1), a collagen type VIII protein, has been suggested to exert various functions in progression of multiple cancers. However, the effect of COL8A1 in papillary thyroid cancer (PTC) has not been elucidated. The Cancer Genome Atlas (TCGA) databases were applied to investigate the COL8A1 expression and its clinical significance in PTC. The COL8A1 expression level was further validated using Gene Expression Omnibus (GEO) data and clinical paired PTC tissues. Additionally, Kaplan-Meier curve was used to analyze the prognosis. The cell migrative and invasive abilities were evaluated by wound healing assay and Transwell assay. CCK8 assays were used to evaluate proliferation of PTC cells. Western blotting was conducted to explore the potential mechanisms involved in the pro-tumor role of COL8A1. The correlation between immune cell infiltration and COL8A1 was analyzed using Tumor Immune Estimation Resource (TIMER) database and the single-sample GSEA (ssGSEA) method. We found that COL8A1 was upregulated in PTC (P<0.05). High COL8A1 expression level was significantly associated with advanced T stage (P<0.01), N stage (P<0.001) and poor prognosis (P=0.0142) in PTC. Furthermore, cell migration and invasion were significantly reduced following COL8A1 knockdown (P<0.001). Mechanistic studies demonstrated that the epithelial-to-mesenchymal transition (EMT) related proteins (FN1, MMP9, MMP7, ZEB2 and Twist1) and phosphorylation of AKT and ERK were obviously down-regulated after COL8A1 knockdown (P<0.01). Moreover, COL8A1 expression was correlated with immune cell infiltration. Our study demonstrates that COL8A1 may function as an oncogene and a potential prognostic biomarker for PTC patients.

SKAP1 Expression in Cancer Cells Enhances Colon Tumor Growth and Impairs Cytotoxic Immunity by Promoting Neutrophil Extracellular Trap Formation via the NFATc1/CXCL8 Axis.

The mechanisms underlying the development and progression of colon cancer are not fully understood. Herein, Src kinase associated phosphoprotein 1 (SKAP1), an immune cell adaptor, is identified as a novel colon cancer-related gene. SKAP1 expression is significantly increased in colon cancer cells. High SKAP1 levels are independently predictive of poor survival in patients with colon cancer. Notably, SKAP1 expression in colon cancer cells exerted a significant tumor-promoting effect in vivo rather than in vitro. Screening of tumor-infiltrating immune cells revealed the involvement of neutrophils in SKAP1-induced colon tumor promotion. Enhanced formation of neutrophil extracellular traps (NETs) is found to be a key downstream event that contributed to the pro-tumor role of SKAP1. In colon cancer cells, SKAP1 increased the expression of C-X-C motif chemokine ligand 8 (CXCL8) via nuclear factor of activated T cells c1 (NFATc1). The blockade of CXCL8 or NFATc1 largely attenuated neutrophil infiltration, NET formation, and tumor promotion induced by SKAP1. Furthermore, inhibiting SKAP1-induced NET significantly enhanced the antitumor efficiency of adoptive natural killer cell therapy in colon tumor models. In conclusion, SKAP1 significantly promotes colon cancer growth via the cancer cell/neutrophil NFATc1/CXCL8/NET axis, suggesting that SKAP1 is a potential target for colon cancer therapy.

Single-cell transcriptional atlas of tumor-associated macrophages in breast cancer

BackgroundThe internal heterogeneity of breast cancer, notably the tumor microenvironment (TME) consisting of malignant and non-malignant cells, has been extensively explored in recent years. The cells in this complex cellular ecosystem activate or suppress tumor immunity through phenotypic changes, secretion of metabolites and cell-cell communication networks. Macrophages, as the most abundant immune cells within the TME, are recruited by malignant cells and undergo phenotypic remodeling. Tumor-associated macrophages (TAMs) exhibit a variety of subtypes and functions, playing significant roles in impacting tumor immunity. However, their precise subtype delineation and specific function remain inadequately defined.MethodsThe publicly available single-cell transcriptomes of 49,141 cells from eight breast cancer patients with different molecular subtypes and stages were incorporated into our study. Unsupervised clustering and manual cell annotation were employed to accurately classify TAM subtypes. We then conducted functional analysis and constructed a developmental trajectory for TAM subtypes. Subsequently, the roles of TAM subtypes in cell-cell communication networks within the TME were explored using endothelial cells (ECs) and T cells as key nodes. Finally, analyses were repeated in another independent publish scRNA datasets to validate our findings for TAM characterization.ResultsTAMs are accurately classified into 7 subtypes, displaying anti-tumor or pro-tumor roles. For the first time, we identified a new TAM subtype capable of proliferation and expansion in breast cancer-TUBA1B+ TAMs playing a crucial role in TAMs diversity and tumor progression. The developmental trajectory illustrates how TAMs are remodeled within the TME and undergo phenotypic and functional changes, with TUBA1B+ TAMs at the initial point. Notably, the predominant TAM subtypes varied across different molecular subtypes and stages of breast cancer. Additionally, our research on cell-cell communication networks shows that TAMs exert effects by directly modulating intrinsic immunity, indirectly regulating adaptive immunity through T cells, as well as influencing tumor angiogenesis and lymphangiogenesis through ECs.ConclusionsOur study establishes a precise single-cell atlas of breast cancer TAMs, shedding light on their multifaceted roles in tumor biology and providing resources for targeting TAMs in breast cancer immunotherapy.

The pro-tumoral and anti-tumoral roles of EphA4 on T regulatory cells and tumor associated macrophages during HNSCC tumor progression.

EphA4 in regulatory T cells has a pro-tumoral effect while EphA4 in macrophages plays an anti-tumoral role underscoring the necessity of developing biologically rational therapeutics.

Context-dependent roles for autophagy in myeloid cells in tumor progression.

Autophagy is known to suppress tumor initiation by removing genotoxic stresses in normal cells. Conversely, autophagy is also known to support tumor progression by alleviating metabolic stresses in neoplastic cells. Centered on this pro-tumor role of autophagy, there have been many clinical trials to treat cancers through systemic blocking of autophagy. Such systemic inhibition affects both tumor cells and non-tumor cells, and the consequence of blocked autophagy in non-tumor cells in the context of tumor microenvironment is relatively understudied. Here, we examined the effect of autophagy-deficient myeloid cells on the progression of autophagy-competent tumors. We found that blocking autophagy only in myeloid cells modulated tumor progression markedly but such effects were context dependent. In a tumor implantation model, the growth of implanted tumor cells was substantially reduced in mice with autophagy-deficient myeloid cells; T cells infiltrated deeper into the tumors and were responsible for the reduced growth of the implanted tumor cells. In an oncogene-driven tumor induction model, however, tumors grew faster and metastasized more in mice with autophagy-deficient myeloid cells. These data demonstrate that the autophagy status of myeloid cells plays a critical role in tumor progression, promoting or suppressing tumor growth depending on the context of tumor-myeloid cell interactions. This study indicates that systemic use of autophagy inhibitors in cancer therapy may have differential effects on rates of tumor progression in patients due to effects on myeloid cells and that this warrants more targeted use of selective autophagy inhibitors in a cancer therapy in a clinical setting.

NLRC5 promotes endometrial carcinoma progression by regulating NF-κB pathway-mediated mismatch repair gene deficiency

The innate immune molecule NLR family CARD domain-containing 5 (NLRC5) plays a significant role in endometrial carcinoma (EC) immunosurveillance. However, NLRC5 also plays a protumor role in EC cells. Mismatch repair gene deficiency (dMMR) can enable tumors to grow faster and also can exhibit high sensitivity to immune checkpoint inhibitors. In this study, we attempted to determine whether NLRC5-mediated protumor role in EC is via the regulation of dMMR. Our findings revealed that NLRC5 promoted the proliferation, migration, and invasion abilities of EC cells and induced the dMMR status of EC in vivo and in vitro. Furthermore, the mechanism underlying NLRC5 regulated dMMR was also verified. We first found NLRC5 could suppress nuclear factor-kappaB (NF-κB) pathway in EC cells. Then we validated that the positive effect of NLRC5 in dMMR was restricted when NF-κB was activated by lipopolysaccharides in NLRC5-overexpression EC cell lines. In conclusion, our present study confirmed the novel NLRC5/NF-κB/MMR regulatory mechanism of the protumor effect of NLRC5 on EC cells, thereby suggesting that the NLRC5-mediated protumor in EC was depend on the function of MMR.

Single cell RNA sequencing analysis of tumor infiltrating γδ T cells in murine and human glioblastoma.

Abstract Glioblastoma multiforme (GBM) is the most commonly occurring malignant brain tumor. The anatomical location and the immunosuppressive TME have brought disappointing results for multiple therapeutics approaches, including immune checkpoint inhibitors (ICIs). γδ T cells are an attractive target for adoptive cell therapies (ACTs) due to their MHCI-independent tumor killing mechanism and GVHD-Free nature. Thus, investigating GBM infiltrating γδ T cells can provide insight into the pro-tumoral and anti-tumoral roles of γδ T cells and provide potential targets for gene engineering prior to ACT. Our Single-cell RNA sequencing (scRNA-seq) and flow cytometric data demonstrate that GBM harbors subtypes of γδ T cells of different origins and functions. γδ T cells from the meninges produce IL-17A and are enriched for genes related to Rorγt programme and immune checkpoint molecules. On the contrary, blood-borne γδ T cells produce IFN-γ and express genes related to cytotoxicity, co-stimulation and activating NK receptors. Functional dichotomy of γδ T cell is also observed in human GBM, with Areg producing tumor-promoting subset and Gzmb producing cytotoxic subset. Interaction analysis reveals that these subtypes of γδ T cell strongly interact with myeloid cells through unique receptor-ligand interaction. These finding reveal the comprehensive role of γδ T cells during cancer progression and provide insights into potential gene-editing targets for effective ACTs.

The role of tumor-associated macrophages in hepatocellular carcinoma-from bench to bedside: A review.

Hepatocellular carcinoma is one of the most common cancers worldwide. Despite progress in treatment, recurrence after radical treatment is common, and the prognosis remains poor for patients with advanced disease. Therefore, there is a need to identify prognostic and predictive factors for the response to therapy or more intensive surveillance or treatment. Because the tumor microenvironment plays a crucial role in the development of cancer and metastasis, it is a crucial need to understand processes that are involved in carcinogenesis. Within the microenvironment, several immune cells with different roles are present. One of the most important of these is tumor-associated macrophages. These cells may exert either antitumor or protumor roles. Several studies have suggested that tumor-associated macrophages can be used as prognostic markers. Furthermore, they may be involved in resistance to immunotherapy or systemic treatment. As they play an important role in cancer development, tumor-associated macrophages are also a good target for therapy. In this review, we briefly summarize recent progress on knowledge regarding the basic molecular characteristics, impact on prognosis and potential clinical implications of tumor-associated macrophages in hepatocellular carcinoma.

Short-chain fatty acids induced lung tumor cell death and increased peripheral blood CD4+ T cells in NSCLC and control patients ex vivo.

Despite therapy advances, one of the leading causes of cancer deaths still remains lung cancer. To improve current treatments or prevent non-small cell lung cancer (NSCLC), the role of the nutrition in cancer onset and progression needs to be understood in more detail. While in colorectal cancer, the influence of local microbiota derived SCFAs have been well investigated, the influence of SCFA on lung cancer cells via peripheral blood immune system should be investigated more deeply. In this respect, nutrients absorbed via the gut might affect the tumor microenvironment (TME) and thus play an important role in tumor cell growth. This study focuses on the impact of the short-chain fatty acid (SCFA) Sodium Butyrate (SB), on lung cancer cell survival. We previously described a pro-tumoral role of glucose on A549 lung adenocarcinoma cell line. In this study, we wanted to know if SB would counteract the effect of glucose and thus cultured A549 and H520 in vitro with and without SB in the presence or absence of glucose and investigated how the treatment with SB affects the survival of lung cancer cells and its influence on immune cells fighting against lung cancer. In this study, we performed cell culture experiments with A549, H520 and NSCLC-patient-derived epithelial cells under different SB levels. To investigate the influence on the immune system, we performed in vitro culture of peripheral mononuclear blood cells (PBMC) from control, smoker and lung cancer patients with increasing SB concentrations. To investigate the effect of SB on lung tumor cells, we first analyzed the effect of 6 different concentrations of SB on A549 cells at 48 and 72 hours cell culture. Here we found that, SB treatment reduced lung cancer cell survival in a concentration dependent manner. We next focused our deeper analysis on the two concentrations, which caused the maximal reduction in cell survival. Here, we observed that SB led to cell cycle arrest and induced early apoptosis in A549 lung cancer cells. The expression of cell cycle regulatory proteins and A549 lung cancer stem cell markers (CD90) was induced. Additionally, this study explored the role of interferon-gamma (IFN-γ) and its receptor (IFN-γ-R1) in combination with SB treatment, revealing that, although IFN-γ-R1 expression was increased, IFN-γ did not affect the efficacy of SB in reducing tumor cell viability. Furthermore, we examined the effects of SB on immune cells, specifically CD8+ T cells and natural killer (NK) cells from healthy individuals, smokers, and NSCLC patients. SB treatment resulted in a decreased production of IFN-γ and granzyme B in CD8+ T cells and NK cells. Moreover, SB induced IFN-γ-R1 in NK cells and CD4+ T cells in the absence of glucose both in PBMCs from controls and NSCLC subjects. Overall, this study highlights the potential of SB in inhibiting lung cancer cell growth, triggering apoptosis, inducing cell cycle arrest, and modulating immune responses by activating peripheral blood CD4+ T cells while selectively inducing IFN-γ-R1 in NK cells in peripheral blood and inhibiting peripheral blood CD8+ T cells and NK cells. Thus, understanding the mechanisms of action of SB in the TME and its influence on the immune system provide valuable insights of potentially considering SB as a candidate for adjunctive therapies in NSCLC.

NCAPH serves as a prognostic factor and promotes the tumor progression in glioma through PI3K/AKT signaling pathway.

Non-SMC (Structural Maintenance of Chromosomes) condensin I complex subunit H (NCAPH) has been shown to facilitate progression and predict adverse prognostic outcome in many cancer types. However, the function of NCAPH in gliomas is still unclear. Series of experiments were taken to uncover the function of NCAPH in glioma. The expression of NCAPH and potential mechanism regulating progression of glioma was verified by bioinformatics analysis. Lentiviral transfection was used for establishment of loss-of-function and gain-of-function cell lines. CCK-8 assay and Colony-formation assay were used to evaluate proliferation. Transwell assay and Cell wound healing assay were used to assess migration and invasion. Cell cycle and apoptosis were measured by flow cytometry. Protein and RNA were quantified by WB and RT-PCR, respectively. The nude mice model of glioma was used to evaluate the effect of NCAPH in vivo. The expression of NCAPH increased significantly in glioma tissues and correlated with WHO grade, IDH wild-type and non-1p/19q codeletion. Glioma patients with high expression of NCAPH had an undesirable prognosis. Functionally, upregulated NCAPH promotes the malignant hallmarks of glioma cells in vivo and in vitro. NCAPH correlated with DNA damage repair ability of glioma cells and facilitated the proliferation, invasion, and migration of glioma cells by promoting the PI3K/AKT signaling pathway. This study identifies the important pro-tumor role of NCAPH in glioma and suggests that NCAPH is a potential therapeutic target.

Glioblastoma-instructed microglia transition to heterogeneous phenotypic states with phagocytic and dendritic cell-like features in patient tumors and patient-derived orthotopic xenografts

BackgroundA major contributing factor to glioblastoma (GBM) development and progression is its ability to evade the immune system by creating an immune-suppressive environment, where GBM-associated myeloid cells, including resident microglia and peripheral monocyte-derived macrophages, play critical pro-tumoral roles. However, it is unclear whether recruited myeloid cells are phenotypically and functionally identical in GBM patients and whether this heterogeneity is recapitulated in patient-derived orthotopic xenografts (PDOXs). A thorough understanding of the GBM ecosystem and its recapitulation in preclinical models is currently missing, leading to inaccurate results and failures of clinical trials.MethodsHere, we report systematic characterization of the tumor microenvironment (TME) in GBM PDOXs and patient tumors at the single-cell and spatial levels. We applied single-cell RNA sequencing, spatial transcriptomics, multicolor flow cytometry, immunohistochemistry, and functional studies to examine the heterogeneous TME instructed by GBM cells. GBM PDOXs representing different tumor phenotypes were compared to glioma mouse GL261 syngeneic model and patient tumors.ResultsWe show that GBM tumor cells reciprocally interact with host cells to create a GBM patient-specific TME in PDOXs. We detected the most prominent transcriptomic adaptations in myeloid cells, with brain-resident microglia representing the main population in the cellular tumor, while peripheral-derived myeloid cells infiltrated the brain at sites of blood–brain barrier disruption. More specifically, we show that GBM-educated microglia undergo transition to diverse phenotypic states across distinct GBM landscapes and tumor niches. GBM-educated microglia subsets display phagocytic and dendritic cell-like gene expression programs. Additionally, we found novel microglial states expressing cell cycle programs, astrocytic or endothelial markers. Lastly, we show that temozolomide treatment leads to transcriptomic plasticity and altered crosstalk between GBM tumor cells and adjacent TME components.ConclusionsOur data provide novel insights into the phenotypic adaptation of the heterogeneous TME instructed by GBM tumors. We show the key role of microglial phenotypic states in supporting GBM tumor growth and response to treatment. Our data place PDOXs as relevant models to assess the functionality of the TME and changes in the GBM ecosystem upon treatment.Graphical

Abstract 7041: Tumor-immune interactions and cisplatin resistance in localized muscle-invasive bladder cancer

Abstract Background: Cisplatin-based chemotherapy followed by radical cystectomy remains the standard-of-care treatment for patient with localized muscle-invasive bladder cancer (MIBC). Neoadjuvant clinical trials studying the combination of cisplatin-based chemotherapy and immune checkpoint blockade (ICB) in MIBC reported encouraging pathologic complete response rates. However, the molecular mechanisms and cellular programs involved in response and resistance to cisplatin and ICB in MIBC remain incompletely characterized. Our goal is to define cellular transcriptional programs and chromatin accessible regions in cancer cells and infiltrating immune cells associated with response and resistance to neoadjuvant systemic therapy. Methods: We performed single-cell Multiome ATAC and Gene expression sequencing (simultaneous identification of gene expression and open chromatin regions within the same cell) of tumors that had a complete response – “responders” – and tumors that had no response or progressed – “non-responders” – through neoadjuvant cisplatin-based chemotherapy (n=15). Single cell suspensions were prepared following the 10x Genomics protocol. Cell Ranger ARC was used for sequence alignment, peak calling, and generation of count matrices. Downstream single-cell gene expression analysis was performed with Scanpy and ATAC-seq with Seurat. Results: We identified two distinct urothelial cell populations in "responders" vs "non-responders" based on single cell gene expression and genome wide chromatin accessibility regions. Cancer cells in “non-responders” preferentially express basal cell gene expression signatures. “Non-responders” were significantly enriched in suppressive tumor-associated macrophages (TAMs). Interestingly, SPP1+ TAMs known to have a pro-tumoral role and promote resistance to ICB were exclusively identified in “non-responders”. Conclusion: Single cell and genome wide chromatin accessibility regions identify heterogenous urothelial cell populations expressing basal cell markers in tumors resistant to neoadjuvant cisplatin-based chemotherapy. “Non-responders” are significantly enriched with TAMs implicated in resistance to ICB. Future studies will better define the implications of these findings in cancer therapeutics for patients with MIBC. Citation Format: Filipe La Fuente De Carvalho, Jihyun Lee, Kevin Bi, Breanna Titchen, Konrad Stawiski, Jihye Park, Amanda Garza, Daniel Michaud, Jennifer Guerriero, Elizabeth Mittendorf, Kent Mouw, Eliezer Van Allen. Tumor-immune interactions and cisplatin resistance in localized muscle-invasive bladder 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 7041.

Abstract 5408: SERPINB3 impact on cervical cancer cell mobility and migration

Abstract Cervical cancer is the fourth most common cancer in women, with 311,000 deaths worldwide in 2018. About two thirds of patients with cervical cancer exhibit elevated serum levels of squamous cell carcinoma antigen (SCCA), also known as SERPINB3. SERPINB3 is associated with advanced stage and lymph node metastasis and we have previously shown it has a protective role against radiation treatment and chemotherapy induced cell death. We have also shown that SERPINB3 promotes tumor growth in both immunocompromised and immune competent murine models, yet the molecular mechanism is unknown. We have recently shown that SERPINB3 expression results in activation of signal transducer of transcription 3 (STAT3) and extracellular signal regulated kinase (ERK) in cervical cancer cells. These signaling cascades promote cell proliferation, survival, invasion, and metastasis. Proximity labeling using biotin-ligase fused to SERPINB3 revealed STAT3 and ERK as potential interacting partners. We aim to identify the molecular mechanism by which SERPINB3 promotes tumor progression, and determine if SERPINB3 drives invasion and metastasis in-vivo. We hypothesized that SERPINB3 directly interacts with and activates STAT3 and ERK leading to downstream signaling promoting invasion and metastasis. To test this hypothesis, we used cervical tumor cell lines engineered with SERPINB3 or empty vector control and determined migration by wound healing assay and downstream activation of STAT3 and ERK. Co-immunoprecipitation (co-IP) with western blot was performed to determine an interaction between SERPINB3 and STAT3 and ERK. E6/E7-expressing TC-1, and Lewis Lung Carcinoma cells, LL2, to develop a method for injecting the cervix of immunocompetent c57BL/6 albino mice (no available murine cervical carcinoma line). Wound healing assays revealed that knocking out SERPINB3 shows a decrease in wound closure in both SW756 and HT3 cervical cancer cells compared to control cells, respectively. Co-IP of STAT3 in HT3 cells revealed the presence of SERPINB3, providing further evidence for an interaction between STAT3 and SERPINB3. We were able to grow tumors in the cervix of the C57/BL6 mice and identified potential metastatic deposits in the lung. Moving forward, we plan to identify the minimal functional domain of the interaction between SERPINB3 and STAT3/ERK using a step-wise genetic approach for protein domain deletion. To determine the role of SERPINB3 on metastasis in vivo, we will use, LL2 and TC-1 engineered with the mouse orthologue mSERPINB3a to determine effect of SERPINB3 on invasion and metastasis. Conducting this study will provide insights as to how SERPINB3 may function in a pro-tumor role in cervical cancer and allow the development of SERPINB3-targeted drugs. Citation Format: Marlene L. Campos Guerrero, Li-Yun Chen, Stephanie Markovina. SERPINB3 impact on cervical cancer cell mobility and migration [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 5408.

Abstract 1597: CD144+cancer-associated fibroblasts drive the malignancy of pancreatic cancer via stimulating inflammatory paracrine

Abstract Purpose: Cancer-associated fibroblasts (CAFs) are highly heterogeneous in pancreatic ductal adenocarcinoma (PDAC). We previously reported that CAFs can acquire an endothelial phenotype under stress. Thus, we hypothesized that there are CAFs highly expressing endothelial markers and substantiated a novel subtype of CAFs promoting tumor progression. Experimental design: We performed single-cell RNA sequencing to identify the existence of CD144+CAFs. The prospective and retrospective analysis were combined to figure out the correlation between CD144+CAFs proportion and clinical outcome. The proliferation and invasion assay of cell lines, organoids and pancreatic cancer xenograft model were used to detect the effect of CD144+CAFs on tumor progression. Cytokine array assay, RNA-sequencing, IP-mass spectrum, Ch-IP and luciferase analysis were conducted to elucidate the underlying mechanism. siRNA delivery nanosystem was designed and administrated to precisely target CD144+CAFs in vivo. Results: CD144+CAFs were present in tumor microenvironment of PDAC and breast cancer and patients with a higher CD144+CAFs proportion have worse prognosis in PDAC. CD144+CAFs can promote the metastasis of PDAC through enhancing epithelial-mesenchymal transition. CD144-β-catenin-STAT3 signaling axis was activated and downstream inflammatory secreted cytokines were transcriptionally upregulated to maintain the functions and phenotype of CD144+CAFs. A novel CAF-targeting siRNA delivery nanosystem, loading FAPα and siCDH5, was designed and administrated to precisely target CD144+CAFs, which substantially inhibited the protumoral roles in vivo. Conclusion: CD144+CAFs can promote the growth and metastasis of PDAC by activating CD144-β-catenin-STAT3 signaling axis and stimulating inflammatory secretion. CAF-targeting siRNA delivery nanosystem can inhibit tumor progression by precisely targeting CD144+CAFs. Citation Format: Xugang Sun, Jun Yu, Tiansuo Zhao, Jihui Hao. CD144+cancer-associated fibroblasts drive the malignancy of pancreatic cancer via stimulating inflammatory paracrine [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 1597.

Abstract 5932: Immunomodulatory and anticancer effects of cyclical gemcitabine/nab-paclitaxel with continuous dosing of tinodasertib (AUM001) in preclinical KPC model

Abstract Background: PDAC has a poor 5-year survival outcome, and its aggressiveness limits the treatment efficacy. This is often driven by dysregulated mRNA translation of pro-tumorigenic genes that contribute to the proliferative and metastatic nature of PDAC. Synergizing chemotherapy with inhibition of oncogenic mRNA translation via MNK-coupled phosphorylation of eIF4E could potentially be viable. To support this hypothesis, we combined AUM001, a MNK inhibitor, with gemcitabine (GEM)-nab-paclitaxel (nPTX) with the aim of modulating tumor growth kinetics and maintaining it in a stable state. Methods: PDAC KPC cells were subcutaneously inoculated into BL6 mice and randomized to five treatment groups. Chemotherapy involved administering GEM-nPTX intraperitoneally on days 1, 4, and 8. AUM001 was given orally daily for 8 days for both mono- and combination therapies and was further administered for an additional 3 weeks in continuous dosing group. IHC staining for Ki67, MVD, and stromal markers was conducted on FFPE sections as well as Western blot analysis of tumor tissues. Apoptosis was assessed using Annexin V. Immune profiling were evaluated by flow cytometric assay and GeoMx spatial transcriptomics analysis. Results: The administration of GEM-nPTX and AUM001 (GPA) or GEM-nPTX alone (GP) led to an average 38% reduction in net tumor growth with no observed toxicity. Notably, continuous dosing of AUM001 (GPAc) following the cessation of chemotherapy exhibited a 45% decrease in net tumor growth (P=0.056) when compared to GPA. EIF4E phosphorylation and Myc expression were markedly inhibited in GPAc with lower recovery rate for MVD as compared to the GPA group at EOO (P &amp;lt; 0.05). A higher fraction of apoptotic cells was detected in the GPAc group at EOO than GPA group in reference to GP group (P=NS). GPA also resulted in a lower frequency of NK cells which was partially lifted in GPAc during observation phase. Macrophages which are known to have a pro-tumor role initially increased in frequency upon addition of AUM during chemotherapy, but subsequently decreased during observation. These modulations in infiltrating immune cells correlated with the differential expression of several immune related genes in the tumor associated CD45+ immune cells, revealed by the GeoMx analysis. Conclusions: This study showed that sustained administration of AUM001 alongside cyclical GEM-nPTX is well-tolerated with good anti-tumor outcomes. Additionally, the maintenance of AUM001 may enhance the decrease in pro-oncogenic protein expression and remodel the tumor immune microenvironment. In conclusion, continuous dosing of AUM001 following the cessation of chemotherapy could stabilize the tumor state, potentiating its application in clinical settings as maintenance therapy for PDAC. Citation Format: Wai Fook Leong, Sze Sing Lee, Jyue Yuan Lim, Siwen Sylvialin Chen, Fara Faye Desacola Regis, Romana Vidergar, Subhra K. Biswas, Balram Chowbay. Immunomodulatory and anticancer effects of cyclical gemcitabine/nab-paclitaxel with continuous dosing of tinodasertib (AUM001) in preclinical KPC model [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 5932.

Abstract 3335: A novel and potent IRE1α RNase inhibitor, HM100168 as a promising therapeutic strategy in solid cancers

Abstract Protein folding homeostasis in the endoplasmic reticulum (ER) is regulated by a signaling network called the unfolded protein response (UPR). Inositol-requiring enzyme 1α (IRE1α) is an ER membrane-resident kinase/RNase that mediates signal transmission in the most evolutionarily conserved way of the UPR. The IRE1α-XBP1 pathway, one of the most essential branches of the UPR, is activated in various types of cancer. When IRE1α is activated, the spliced form of XBP1 (XBP1s) is generated to activate the transcription of many pro-survival genes to prevent cellular death and relieve cellular stress. IRE1α plays an instrumental pro-tumoral role in various cancers, such as glioblastoma, myeloma, lung adenocarcinoma, and breast cancers, as well as high IRE1α activity is associated with poor prognoses. Moreover, IRE1-XBP1 plays an essential role in the UPR and ER stress responses, which have been implicated in the development of drug resistance. Thereby, blockade of the IRE1α-XBP1 pathway is crucial as it is considered as a potential anti-cancer strategy. Here, we developed a potent and selective IRE1α RNase inhibitor, which have a hydroxy-aryl-aldehydes (HAA) moiety that binds to IRE1α within the RNase catalytic site. Our lead compound, HM100168, potently inhibited the RNase activity of human IRE1α at nanomolar concentrations without affecting its kinase activity. Experiments were performed on MCF7 breast cancer cells to confirm the inhibitory effect in the mRNA and protein levels of XBP1s, which was increased by tunicamycin (TM), a ER stress inducer. HM100168 strongly inhibited ​​XBP1s levels in a dose-dependent manner, displaying the potent IC50 values. We tested cell growth inhibition activity in 3D as well as 2D cell culture condition of various cancer cell lines. Interestingly, HM100168 effectively inhibited cell proliferation of diverse cancer cell lines in both 2D and 3D culture condition. In addition, we explored the promising anti-cancer effects of HM100168 and its synergistic potential when combined with anti-tumorigenic agents. As a result, HM100168 in combination with anti-tumorigenic agents significantly enhanced suppression of tumor growth in human cancer cell transplanted mice. In conclusion, we have identified a novel compound with unique properties that specifically blocked the endonuclease activity of IRE1α. HM100168 inhibited IRE1α endonuclease activity, without affecting its kinase activity after endoplasmic reticulum stress in vitro. The identification of this novel IRE1α inhibitor supports the hypothesis that the IRE1α-XBP1 axis is a promising target for anticancer therapy. Also, co-targeting of IRE1α-XBP1 in combination with anti-tumorigenic agents can be considered a novel approach to overcome cancer resistance. Further preclinical studies will be performed and reported soon after the establishment of a preclinical candidate. Citation Format: Jisook Kim, Seung Hyun Jung, Minjeong Kim, Wongi Park, Jooyun Byun, Soonki Park, Miyoung Lee, Yu-Yon Kim, Junghwa Park, Seokhyun Hong, Minhwa Kim, Young Gil Ahn. A novel and potent IRE1α RNase inhibitor, HM100168 as a promising therapeutic strategy in solid cancers [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 3335.

Pumilio RNA binding family member 1 deficiency activates anti-tumor immunity in hepatocellular carcinoma via restraining M2 macrophage polarization

ABSTRACT Pumilio RNA-binding family member 1 (PUM1) has been implicated in both the progression of colorectal cancer and the regulation of inflammation. The role of PUM1 in the polarization of tumor-associated macrophages (TAMs) into the M2 phenotype has not yet been reported in hepatocellular carcinoma. Using the PUM1-knockout mice model, flow cytometry, and IHC, we validated the role of PUM1 in hepatocellular carcinoma (HCC) TAMs. One-way analysis of variance (ANOVA) or student’s t-tests was used to compare the experimental groups. We found that PUM1 inhibited anti-tumor immunity in HCC through TAM-mediated inhibition of CD8+ T cells. We also showed that PUM1 promotes the transformation of TAMs into pro-tumorigenic M2-like phenotypes by activating cAMP signaling pathway. This study emphasized the potential of PUM1 as a target for immunotherapy in HCC through TAMs. The present study revealed the molecular mechanism underlying the pro-tumor role of PUM1 in HCC.