Aggressive Tumor Phenotype Research Articles

Effects of senescence on the tumour microenvironment and response to therapy.

Cellular senescence is a state of durable cell arrest that has been identified both in vitro and in vivo. It is associated with profound changes in gene expression and a specific secretory profile that includes pro-inflammatory cytokines, growth factors and matrix-remodelling enzymes, referred to as the senescence-associated secretory phenotype (SASP). In cancer, senescence can have anti- or pro-tumour effects. On one hand, it can inhibit tumour progression in a cell autonomous manner. On the other hand, senescence can also promote tumour initiation, progression, metastatic dissemination and resistance to therapy in a paracrine manner. Therefore, despite efforts to target senescence as a potential strategy to inhibit tumour growth, senescent cancer and microenvironmental cells can eventually lead to uncontrolled proliferation and aggressive tumour phenotypes. This can happen either through overcoming senescence growth arrest or through SASP-mediated effects in adjacent tumour cells. This review will discuss how senescence affects the tumour microenvironment, including extracellular matrix remodelling, the immune system and the vascular compartment, to promote tumourigenesis, metastasis and resistance to DNA-damaging therapies. It will also discuss current approaches used in the field to target senescence: senolytics, improving the immune clearance of senescent cells and targeting the SASP.

THU479 FOXA1 Loss Remodels The Tumor Immune Microenvironment In Late-stage Prostate Cancer

Abstract Disclosure: L.T. Brea: None. H. Shi: None. V. Keo: None. W. Xie: None. X. Lu: None. G. Gritsina: None. X. Wang: None. S.H. Park: None. J. Zhao: None. J. Yu: None. Background: Castration resistant prostate cancer (CRPC) has shown a poor response to immune checkpoint inhibitors due to its immunosuppressive nature. CRPC is infiltrated by tumor-associated macrophages (TAMs) and T regulatory cells (Tregs), which induce an immunosuppressive tumor immune microenvironment (TIME) and promote tumor progression. A deeper understanding of tumor intrinsic mechanisms shaping the TIME in prostate cancer (PCa) is needed to harness immunotherapies for PCa patients. Previous studies have found that epithelial transcription factor FOXA1 is downregulated in CRPC. In accordance, we have reported that FOXA1 loss induces tumor cell invasion and macrophage recruitment in vitro through HIF1A-CCL2 signaling. However, the extent to which FOXA1 loss regulates the TIME in PCa remains unclear, largely due to the lack of immune-proficient mouse models for the study of FOXA1 function in the context of PCa. Methods: To examine how FOXA1 loss regulates the PCa TIME, we generated a novel genetically engineered mouse model (GEMM) with prostate-specific deletion of Pten and Foxa1 (PbCre4:Ptenf/fFoxa1f/f) to compare with the established Pten-null PCa mouse model (PbCre4:Ptenf/f). We performed immunohistochemistry analyses on the GEMM prostate tumor tissues to characterize histopathology changes and assess tumor immune infiltration. We also performed spatial transcriptomics and scRNA-seq analyses of the GEMMs to comprehensively evaluate the effect of Foxa1 loss on downstream pathways and the surrounding TIME. Finally, we performed bioinformatics analysis of PCa patient datasets to validate the clinical relevance. Results: We found PbCre4:Ptenf/fFoxa1f/f mouse prostate tumors exhibited a more aggressive histopathologic tumor phenotype compared to age-matched PbCre4:Ptenf/f. Notably, immunohistochemistry staining and spatial transcriptomic analysis of the GEMMs revealed striking remodeling of the TIME upon Foxa1 loss, with marked increases in tumor infiltration by Tregs and TAMs. Moreover, CIBERSORTx analysis of patient datasets revealed FOXA1 expression negatively correlates with Treg and M2-macrophage infiltration. We also found pro-tumorigenic inflammatory cytokine signaling genes, linked to the recruitment of immunosuppressive cells, were upregulated in PbCre4:Ptenf/fFoxa1f/f prostate tumors. Furthermore, we confirmed inflammatory pathways were upregulated among FOXA1-low patient samples. Additional studies are ongoing to further elucidate the mechanisms by which FOXA1 regulates inflammatory cytokine signaling and the TIME in PCa.Conclusion: This study presents a novel GEMM for the study of FOXA1 function in an immunocompetent setting. Our data supports FOXA1 as a critical tumor intrinsic regulator of the TIME. This will have important implications on the design of immunotherapeutic approaches for late-stage PCa often with FOXA1 loss. Presentation: Thursday, June 15, 2023

2403P CDKN2A deletions are a mechanism for p16 inactivation in urothelial bladder carcinomas and are associated with an aggressive tumor phenotype

2403P CDKN2A deletions are a mechanism for p16 inactivation in urothelial bladder carcinomas and are associated with an aggressive tumor phenotype

Replication stress and defective checkpoints make fallopian tube epithelial cells putative drivers of high-grade serous ovarian cancer

Clinical and molecular evidence indicates that high-grade serous ovarian cancer (HGSOC) primarily originates from the fallopian tube, not the ovarian surface. However, the reasons for this preference remain unclear. Our study highlights significant differences between fallopian tube epithelial (FTE) and ovarian surface epithelial (OSE) cells, providing the molecular basis for FTEs as site of origin of HGSOC. FTEs, unlike OSEs, exhibit heightened replication stress (RS), impaired repair of stalled forks, ineffective G2/M checkpoint, and increased tumorigenicity. BRCA1 heterozygosity exacerbates these defects, resulting in RS suppression haploinsufficiency and an aggressive tumor phenotype. Examination of human and mouse sections reveals buildup of the RS marker 53BP1 primarily in the fallopian tubes, particularly at the fimbrial ends. Furthermore, menopausal status influences RS levels. Our study provides a mechanistic rationale for FTE as the site of origin for HGSOC, investigates the impact of BRCA1 heterozygosity, and lays the groundwork for targeting early HGSOC drivers.

Anatomic Variants in the Anterior Inferior Cerebellar Artery Encountered During Resection of Vestibular Schwannomas.

Vestibular schwannomas (VS) are often phenotypically benign lesions that may be technically challenging to resect because of involvement of neurovascular structures. The anterior inferior cerebellar artery (AICA) is commonly identified near VS, with variable position in relation to the tumor; however, little published literature describes anatomic and pathologic variants of AICA observed during VS resection. A prospectively maintained cohort of surgically managed VS with available operative reports and clinical/radiographic follow-up was queried and reviewed for noted aberrations. We identified 66 cases with noted AICA abnormalities among 880 reviewer cases, including 20 loops extending into the internal auditory canal (2.3%), 18 arteries embedded in dura (2.0%), 15 AICA branches directly within VS (1.7%), 8 main trunk arteries coursing between cranial nerves 7 and 8 (0.9%), 3 arteries embedded in temporal bone (0.2%), 1 aneurysm (0.1%), and 1 artery bifurcating cranial nerve 6 (0.1%). The median age of AICA-variant patients was 55 years (range 19-74), and 29 were female (45%). Compared with the other AICA variants, tumors embedded with AICA tended to be larger lesions on maximal axial diameter (2.9 vs 1.6 cm; P = .006), they more commonly underwent less than total resection (73% vs 28%; P = .0001), and they had higher rates postoperative House-Brackmann scores >2 (47% vs 20%; P = .005). Two patients had radiographic and symptomatic postoperative cerebral ischemia or hemorrhage-1 from a bone-encased AICA and 1 from a dural embedded variant. Anatomic variants of AICA occur in approximately 7% of VS operations. Most aberrations do not affect surgical or clinical outcomes, and the rate of major vascular injury was low. However, certain types variably add operative time and in the case of AICA encasement in the tumor, likely indicate a more aggressive tumor phenotype with lower rates of gross total resection and high incidences of facial nerve weakness.

247. TARGETING HAUS7/GAMMA-TUBULIN INTERACTION WITH HTIP1 SUPPRESSES TUMORIGENESIS IN ESOPHAGEAL SQUAMOUS CELL CARCINOMA

Abstract Background Uncontrolled cell division is a hallmark of cancer, making targeting deregulated cell cycle molecules with prognostic significance a promising approach to combat tumorigenesis. Our research focuses on HAUS7, a molecule highly relevant to esophageal squamous cell carcinoma (ESCC), and its interaction with gamma-tubulin. We designed a novel peptide, HTIP1, to target this interaction and suppress tumorigenesis. Methods We investigated the clinical relevance of HAUS7 upregulation in ESCC cohorts, studied its physiological microtubule-regulating and tumorigenic functions in vitro and/or in vivo, and employed a multidisciplinary approach to decode the mechanism underlying HAUS7 suppression. HTIP1 was designed by structure modelling and docking simulation. Its properties, cancer selectivity, anti-tumor efficacy, off-target effect and mechanisms of action were comprehensively evaluated. Patient-derived tumor xenograft models were used to provide additional preclinical evidence. Results Our results show that HAUS7 is the most essential HAUS member for regulating microtubule dynamics in ESCC, and its overexpression is correlated with aggressive tumor phenotypes and poor prognosis. Mechanistically, HAUS7 suppression releases gamma-tubulin, which upon phosphorylation, seizes LMAN1 and together they re-localize to the nucleus. The removal of LMAN1 from its innate location triggers endoplasmic reticulum stress and apoptosis. We designed HTIP1 to perturb HAUS7/gamma-tubulin interaction, which demonstrated anti-tumor effects similar to HAUS7 suppression, with high tumor specificity, negligible off-target effects, and more potency than relevant compounds. Conclusions Our findings provide a new strategy to counter tumorigenesis and pave the way for the development of a new drug targeting the HAUS7/gamma-tubulin interaction.

NOX1 and PRDX6 synergistically support migration and invasiveness of hepatocellular carcinoma cells through enhanced NADPH oxidase activity

The NADPH oxidase 1 (NOX1) complex formed by proteins NOX1, p22phox, NOXO1, NOXA1, and RAC1 plays an important role in the generation of superoxide and other reactive oxygen species (ROS) which are involved in normal and pathological cell functions due to their effects on diverse cell signaling pathways. Cell migration and invasiveness are at the origin of tumor metastasis during cancer progression which involves a process of cellular de-differentiation known as the epithelial-mesenchymal transition (EMT). During EMT cells lose their polarized epithelial phenotype and express mesenchymal marker proteins that enable cytoskeletal rearrangements promoting cell migration, expression and activation of matrix metalloproteinases (MMPs), tissue remodeling, and cell invasion during metastasis. In this work, we explored the importance of the peroxiredoxin 6 (PRDX6)-NOX1 enzyme interaction leading to NOXA1 protein stabilization and increased levels of superoxide produced by NOX in hepatocarcinoma cells. This increase was accompanied by higher levels of N-cadherin and MMP2, correlating with a greater capacity for cell migration and invasiveness of SNU475 hepatocarcinoma cells. The increase in superoxide and the associated downstream effects on cancer progression were suppressed when phospholipase A2 or peroxidase activities of PRDX6 were abolished by site-directed mutagenesis, reinforcing the importance of these catalytic activities in supporting NOX1-based superoxide generation. Overall, these results demonstrate a clear functional cooperation between NOX1 and PRDX6 catalytic activities which generate higher levels of ROS production, resulting in a more aggressive tumor phenotype.

Whole transcriptome analysis of canine pheochromocytoma and paraganglioma.

Pheochromocytomas and paragangliomas (PPGLs) are neuroendocrine tumors arising from the chromaffin cells in the adrenal medulla and extra-adrenal paraganglia, respectively. Local invasion, concurrent disorders, and metastases prevent surgical removal, which is the most effective treatment to date. Given the current lack of effective medical treatment, there is a need for novel therapeutic strategies. To identify druggable pathways driving PPGL development, we performed RNA sequencing on PPGLs (n = 19) and normal adrenal medullas (NAMs; n = 10) of dogs. Principal component analysis (PCA) revealed that PPGLs clearly clustered apart from NAMs. In total, 4,218 genes were differentially expressed between PPGLs and NAMs. Of these, 232 had a log2 fold change of >3 or < -3, of which 149 were upregulated in PPGLs, and 83 were downregulated. Compared with NAMs, PPGLs had increased expression of genes related to the cell cycle, tumor development, progression and metastasis, hypoxia and angiogenesis, and the Wnt signaling pathway, and decreased expression of genes related to adrenal steroidogenesis. Our data revealed several overexpressed genes that could provide targets for novel therapeutics, such as Ret Proto-Oncogene (RET), Dopamine Receptor D2 (DRD2), and Secreted Frizzled Related Protein 2 (SFRP2). Based on the PCA, PPGLs were classified into 2 groups, of which group 1 had significantly higher Ki67 scores (p = 0.035) and shorter survival times (p = 0.04) than group 2. Increased expression of 1 of the differentially expressed genes between group 1 and 2, pleiotrophin (PTN), appeared to correlate with a more aggressive tumor phenotype. This study has shed light on the transcriptomic profile of canine PPGL, yielding new insights into the pathogenesis of these tumors in dogs, and revealed potential novel targets for therapy. In addition, we identified 2 transcriptionally distinct groups of PPGLs that had significantly different survival times.

A novel classifier combining G protein-coupled receptors and the tumor microenvironment is associated with survival status in glioblastoma.

Background: Numerous studies have highlighted the crucial role of G protein-coupled receptors (GPCRs) in tumor microenvironment (TME) remodeling and their correlation with tumor progression. However, the association between GPCRs and the TME in glioblastoma (GBM) remains largely unexplored. Methods: In this study, we investigated the expression profile of GPCRs in GBM using integrated data from single-cell RNA sequencing and bulk sequencing. Surgical samples obtained from meningioma and GBM patients underwent single-cell RNA sequencing to examine GPCR levels and cell-cell interactions. Tumor microenvironment (TME) score is calculated by the infiltrated immune cells with CIBERSORT. Results: Our findings revealed a predominantly increased expression of GPCRs in GBM, and demonstrated that the classification of GPCRs and TME is an independent risk factor in GBM. Patients with high GPCR expression in the tumor tissue and low TME score exhibited the worst outcomes, suggesting a potentially aggressive tumor phenotype. On the other hand, patients with low GPCR expression in the tumor tissue and high TME score showed significantly better outcomes, indicating a potentially more favorable tumor microenvironment. Furthermore, the study found that T cells with high GPCR levels displayed extensive cell-cell connections with other tumor and immune cells in the single cell RNA analysis, indicating their potential involvement in immune escape. Conclusion: In conclusion, GPCRs in combination with TME classification can serve as prognostic markers for GBM. GPCRs play an essential role in tumor progression and the TME in GBM.

Cytoplasmic HIF-2α as tissue biomarker to identify metastatic sympathetic paraganglioma

Pheochromocytomas (PCCs) and paragangliomas (PGLs) are rare neuroendocrine tumors. PGLs can further be divided into sympathetic (sPGLs) and head-and-neck (HN-PGLs). There are virtually no treatment options, and no cure, for metastatic PCCs and PGLs (PPGLs). Here, we composed a tissue microarray (TMA) consisting of 149 PPGLs, reflecting clinical features, presenting as a useful resource. Mutations in the pseudohypoxic marker HIF-2α correlate to an aggressive tumor phenotype. We show that HIF-2α localized to the cytoplasm in PPGLs. This subcompartmentalized protein expression differed between tumor subtypes, and strongly correlated to proliferation. Half of all sPGLs were metastatic at time of diagnosis. Cytoplasmic HIF-2α was strongly expressed in metastatic sPGLs and predicted poor outcome in this subgroup. We propose that higher cytoplasmic HIF-2α expression could serve as a useful clinical marker to differentiate paragangliomas from pheochromocytomas, and may help predict outcome in sPGL patients.

Stromal Tumor-Infiltrating Lymphocytes Associated with Immunohistopathology and Molecular Subtypes of Breast Cancer in Vietnam.

Breast cancer is a heterogeneous disease with varied symptoms and pathogenesis, as well as variable prognosis and therapeutic outcomes. Stromal tumor-infiltrating lymphocytes, one of the tumor microenvironment factors, has been recognized as an important immunological biomarker that reflected the antitumor immune response in breast cancer. We analyzed 207 invasive breast cancer patients who had lumpectomy or mastectomy and have not received any pre-operative treatment. Clinicopathological characteristics, immunohistochemistry characteristics, molecular subtypes classification and stromal TILs evaluation were investigated. Stromal TILs correlated with well-established prognostic markers. Tumor grade showed significantly higher sTILs percentages in high-grade tumors than in low-grade tumors (p<0.001). There was a statistically significant association between intermediate and high levels of sTILs and a high Ki-67 index (p< 0.001). ER/PR negative was significantly related to high sTILs. Mean sTILs score was significantly higher in TNBC (40.1±31.6%) compared to others, statistically significant (p<0.001). In HER2-negative breast cancer, sTILs were significantly associated with histologic grade, ER status, PR status, and Ki67 index. sTILs played an important role, associated with unfavorable factors in breast cancer. Our findings support the use of stromal sTILs to identify a more aggressive phenotype of tumors.

Differential Gene Expression Signatures and Cellular Signaling Pathways induced by Lamin A/C Transcript Variants in MCF7 Cell Line.

Lamins are the major component of nuclear lamina. Alternative splicing of the 12 exons comprising lamin A/C gene creates five known transcript variants, lamin A, lamin C, lamin AΔ10, lamin AΔ50, and lamin C2. The main objective for this study was to examine the association of critical pathways, networks, molecular and cellular functions regulated by each Lamin A/C transcript variants. Ion AmpliSeq Transcriptome Human Gene Expression analysis was performed on MCF7 cells stably transfected with lamin A/C transcript variants. Lamin A or lamin AΔ50 upregulation was associated with activation of cell death and inactivation of carcinogenesis while both lamin C or lamin AΔ10 upregulation activated carcinogenesis and cell death. Data suggest anti-apoptotic and anti-senescence effects of lamin C and lamin AΔ10 as several functions, including apoptosis and necrosis functions are inactivated following lamin C or lamin AΔ10 upregulation. However, lamin AΔ10 upregulation is associated with a more carcinogenic and aggressive tumor phenotype. Lamin A or lamin AΔ50 upregulation is associated with a predicted activation of increased cell death and inactivation of carcinogenesis. Thus, different signaling pathways, networks, molecular and cellular functions are activated/inactivated by lamin A/C transcript variants resulting in a large number of laminopathies.

Venetoclax is a potent hepsin inhibitor that reduces the metastatic and prothrombotic phenotypes of hepsin-expressing colorectal cancer cells.

Introduction: Hepsin is a type II transmembrane serine protease and its expression has been linked to greater tumorigenicity and worse prognosis in different tumors. Recently, our group demonstrated that high hepsin levels from primary tumor were associated with a higher risk of metastasis and thrombosis in localized colorectal cancer patients. This study aims to explore the molecular role of hepsin in colorectal cancer. Methods: Hepsin levels in plasma from resected and metastatic colorectal cancer patients were analyzed by ELISA. The effect of hepsin levels on cell migration, invasion, and proliferation, as well as on the activation of crucial cancer signaling pathways, was performed in vitro using colorectal cancer cells. A thrombin generation assay determined the procoagulant function of hepsin from these cells. A virtual screening of a database containing more than 2000 FDA-approved compounds was performed to screen hepsin inhibitors, and selected compounds were tested in vitro for their ability to suppress hepsin effects in colorectal cancer cells. Xenotransplantation assays were done in zebrafish larvae to study the impact of venetoclax on invasion promoted by hepsin. Results: Our results showed higher plasma hepsin levels in metastatic patients, among which, hepsin was higher in those suffering thrombosis. Hepsin overexpression increased colorectal cancer cell invasion, Erk1/2 and STAT3 phosphorylation, and thrombin generation in plasma. In addition, we identified venetoclax as a potent hepsin inhibitor that reduced the metastatic and prothrombotic phenotypes of hepsin-expressing colorectal cancer cells. Interestingly, pretreatment with Venetoclax of cells overexpressing hepsin reduced their invasiveness in vivo. Discussion: Our results demonstrate that hepsin overexpression correlates with a more aggressive and prothrombotic tumor phenotype. Likewise, they demonstrate the antitumor role of venetoclax as a hepsin inhibitor, laying the groundwork for molecular-targeted therapy for colorectal cancer.

Circular RNA circTNIK promotes tumor progression and metastasis in gastric cancer by regulating ZEB2.

Tumor progression and distant metastasis are the main causes of deaths in gastric cancer. Growing evidence revealed that circular RNAs (circRNAs) play critical role in the pathology of malignant disease, the role of circRNAs in gastric cancer progression and metastasis is still unknown. Differentially expressed circRNAs was identified by circRNA microarray and validated by quantitative reverse transcription polymerase reaction. The biological function of circTNIK was evaluated by in vitro and in vivo experiments after ectopic expression or siRNA mediated knockdown of circTNIK. The interaction between circTNIK and miR-138-5p was determined by luciferase activity assay, RNA immunoprecipitation, and fluorescence in situ hybridization assays. circTNIK rather than linear TINK mRNA was significantly upregulated in gastric cancer tissues, cell lines compared with normal controls. Higher circTNIK expression was correlated with aggressive tumor phenotypes and poor overall survival in gastric cancer patients. Ectopic circTNIK expression promoted cell proliferation, invasion, tumorigenesis, and metastasis in gastric cancer cells whereas knockdown of circTNIK inhibited cell proliferation, invasion, tumorigenesis, and metastasis in gastric cancer cells. Importantly, circTNIK functions as a molecular sponge for miR-138-5p to regulate the expression of ZEB2. Overall, our study demonstrates how circTNIK regulates gastric cancer progression and metastasis by sponging miR-138-5p to modulate the expression of ZEB2. CircTNIK might be used as a prognostic biomarker in gastric cancer patients.

NAD+ Metabolism Generates a Metabolic Vulnerability in Endocrine-Resistant Metastatic Breast Tumors in Females.

Approximately 70% of human breast cancers express estrogen receptor-α (ERα), providing a potential target for endocrine therapy. However, 30% to 40% of patients with ER+ breast cancer still experience recurrence and metastasis, with a 5-year relative overall survival rate of 24%. In this study, we identified nicotinamide phosphoribosyltransferase (NAMPT), an important enzyme in nicotinamide adenine dinucleotide (NAD+) metabolism, to be increased in metastatic breast cancer (MBC) cells treated with fulvestrant (Fulv). We tested whether the blockade of NAD+ production via inhibition of NAMPT synergizes with standard-of-care therapies for ER+ MBC in vitro and in vivo. A synergistic effect was not observed when KPT-9274 was combined with palbociclib or tamoxifen or when Fulv was combined with other metabolic inhibitors. We show that NAMPT inhibitor KPT-9274 and Fulv works synergistically to reduce metastatic tumor burden. RNA-sequencing analysis showed that NAMPT inhibitor in combination with Fulv reversed the expression of gene sets associated with more aggressive tumor phenotype, and metabolomics analysis showed that NAMPT inhibition reduced the abundance of metabolites associated with several key tumor metabolic pathways. Targeting metabolic adaptations in endocrine-resistant MBC is a novel strategy, and alternative approaches aimed at improving the therapeutic response of metastatic ER+ tumors are needed. Our findings uncover the role of ERα-NAMPT crosstalk in MBC and the utility of NAMPT inhibition and antiestrogen combination therapy in reducing tumor burden and metastasis, potentially leading to new avenues of MBC treatment.

Identification and Clinical Significance of Pancreatic Cancer Stem Cells and Their Chemotherapeutic Drug Resistance.

Pancreatic cancer ranks in the 10th-11th position among cancers affecting men in Taiwan, besides being a rather difficult-to-treat disease. The overall 5-year survival rate of pancreatic cancer is only 5-10%, while that of resectable pancreatic cancer is still approximately 15-20%. Cancer stem cells possess intrinsic detoxifying mechanisms that allow them to survive against conventional therapy by developing multidrug resistance. This study was conducted to investigate how to overcome chemoresistance and its mechanisms in pancreatic cancer stem cells (CSCs) using gemcitabine-resistant pancreatic cancer cell lines. Pancreatic CSCs were identified from human pancreatic cancer lines. To determine whether CSCs possess a chemoresistant phenotype, the sensitivity of unselected tumor cells, sorted CSCs, and tumor spheroid cells to fluorouracil (5-FU), gemcitabine (GEM), and cisplatin was analyzed under stem cell conditions or differentiating conditions. Although the mechanisms underlying multidrug resistance in CSCs are poorly understood, ABC transporters such as ABCG2, ABCB1, and ABCC1 are believed to be responsible. Therefore, we measured the mRNA expression levels of ABCG2, ABCB1, and ABCC1 by real-time RT-PCR. Our results showed that no significant differences were found in the effects of different concentrations of gemcitabine on CSCs CD44+/EpCAM+ of various PDAC cell line cultures (BxPC-3, Capan-1, and PANC-1). There was also no difference between CSCs and non-CSCs. Gemcitabine-resistant cells exhibited distinct morphological changes, including a spindle-shaped morphology, the appearance of pseudopodia, and reduced adhesion characteristics of transformed fibroblasts. These cells were found to be more invasive and migratory, and showed increased vimentin expression and decreased E-cadherin expression. Immunofluorescence and immunoblotting experiments demonstrated increased nuclear localization of total β-catenin. These alterations are hallmarks of epithelial-to-mesenchymal transition (EMT). Resistant cells showed activation of the receptor protein tyrosine kinase c-Met and increased expression of the stem cell marker cluster of differentiation (CD) 24, CD44, and epithelial specific antigen (ESA). We concluded that the expression of the ABCG2 transporter protein was significantly higher in CD44+ and EpCAM+ CSCs of PDAC cell lines. Cancer stem-like cells exhibited chemoresistance. Gemcitabine-resistant pancreatic tumor cells were associated with EMT, a more aggressive and invasive phenotype of numerous solid tumors. Increased phosphorylation of c-Met may also be related to chemoresistance, and EMT and could be used as an attractive adjunctive chemotherapeutic target in pancreatic cancer.

Abstract 5852: Vimentin content associated with shear wave speed in hepatocellular carcinoma: Non-invasive identification of more aggressive tumor phenotypes

Abstract Tumor stromal heterogeneity can negatively impact immune surveillance and treatment outcomes. Current cross-sectional imaging tools rely solely on tumor size and perfusion to monitor therapy and fail to characterize tumor biomechanics. Shear-wave elastography has emerged as a non-invasive technique that can quantify stromal elasticity. The goal of the present study is to characterize the molecular underpinnings associated with shear wave elastography measurements in liver cancer. To this end, rat hepatoma cell line McA-RH7777 stably expressing green fluorescent protein genes (fluc/GFP) were implanted in the left liver lobe of Buffalo rats. Three weeks after tumor implantation, adequate tumor size was verified by B-mode ultrasound. A 7-color panel was developed to assess the percentage of vimentin, α-smooth muscle actin, IBA-1, S100A4, TGF-β, and glial fibrillary acidic protein (GFAP) in tumor sections. Shear wave elastography data was used to stratify tumor nodules as stiff or soft according to the mean value of the shear wave (3.05 versus 2,26 m/s). Our results showed two distinct tumor phenotypes. Stiffer tumors, as determined by higher shear wave speed measurements on ultrasound, were characterized by higher percentages of vimentin-positive cells, low S100A4 and IBA-1 macrophages expressing TGF-b, and intermediate expression of IBA-1 macrophages and GFAP cells. Slow shear wave speed, characteristic of softer tumors, displayed the opposite phenotype (two-factor ANOVA; P≤0.001). The most remarkable difference between stiffness phenotypes was the percentage of cells expressing vimentin, with a mean percentage in stiffer tumors of 26.54 compared with 13.68% in softer tumors (P&amp;lt;0.003). Our results indicate that high expression of vimentin is highly correlated with increased shear wave speeds. Given the association between vimentin expression and epithelial-mesenchymal transition a marker of aggressive characteristics of tumor cells (increased migration and invasion), our findings suggest that longitudinal quantification of shear wave speed can be useful to assess functional changes in the stromal component and identify more aggressive tumor phenotypes with a higher likelihood of metastatic potential. Citation Format: Andrea C. Cortes, Kiyoyuki Minamiguchi, Patricia L. Da Costa Lopez, Simone Anfossi, Williams Malea L, Maria S. Stenkamp, Rony Avritscher, Natalie W. Fowlkes. Vimentin content associated with shear wave speed in hepatocellular carcinoma: Non-invasive identification of more aggressive tumor phenotypes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5852.

Abstract 3710: Serum tryptophan metabolites mediate constitutive AHR activity in head and neck squamous cell carcinoma cells

Abstract The aryl hydrocarbon receptor (AHR) is transcription factor, that is activated upon binding to various exogenous and endogenous ligands. AHR activation leads to induction of target gene expression, including the members of cytochrome P450 enzyme family that metabolize many xenobiotics. Numerous studies have shown that the AHR is involved in multiple regulatory pathways important during carcinogenesis. High level of AHR protein and activation was observed in head and neck squamous cell carcinoma (HNSCC) and has been correlated with aggressive tumor phenotypes and migratory potential. Endogenous compounds derived from host tryptophan metabolism such as kynurenine, as well as the derivatives of indole derived from bacterial tryptophan metabolism such as indole-3-propionic acid are known to activate AHR. A fundamental question not previously addressed is whether these circulating tryptophan (Tryp) metabolites can mediate persistent AHR activation within tumor tissues and its effect on tumorigenesis. The focus of our research is to determine whether the continuous exposure of Tryp metabolites at relevant concentrations leads to sustained AHR activation in HNSCC cells and its subsequent effect on tumor cell phenotypes. Here, we present data demonstrating sustained AHR activation by Tryp metabolites in two HNSCC cell lines. A total of 6 tryptophan (Tryp) metabolites present at significant levels in serum with known AHR agonist activity were identified and quantified from 40 healthy individuals on a controlled diet by LC/MS/MS. HN30 and OSC19 cells were treated with a representative pool of 6 Tryp metabolites in a cell culture system to achieve continuous exposure to the Tryp metabolites, thus mimicking blood circulation in vivo. Treated cells were collected at six different time points and AHR activation within cells was determined by measuring mRNA and protein expression of AHR target genes. The effect of Tryp pool on HN30 and OSC19 cell viability was determined. The presence of Tryp metabolites within the cells and media was quantitated by LC/MS/MS at six time points over the course of 24 h. Continuous exposure of Tryp metabolites resulted in sustained AHR activation in HN30 and OSC19 cells, confirmed by induced mRNA expression of target genes and subsequent protein levels. The LC/MS data revealed the presence of all six Tryp metabolites in media collected at all time points, suggesting no spontaneous degradation of metabolites. Tryp metabolites were detected inside the cells starting from 2 h post-treatment to the last time point 24 h. Viability assays indicated no cytotoxic effects of Tryp metabolites on cells. Overall, our work has demonstrated a pattern of sustained AHR activation in HNSCC cells resulting from continuous exposure of Tryp metabolites, supporting the concept that endogenous Tryp metabolites in HNSCC patients would impact tumor progression, metastasis, and treatment outcomes. Citation Format: Dhwani Patel, Ethan W. Morgan, Fangcong Dong, Iain A. Murray, Gary H. Perdew. Serum tryptophan metabolites mediate constitutive AHR activity in head and neck squamous cell carcinoma cells. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3710.

Abstract 749: The impact of multimorbidity on the relationship between breast cancer tumor characteristics and survivorship care experiences among older women: A SEER-CAHPS analysis

Abstract Our study examined the associations between breast cancer tumor characteristics and self-reported survivorship care experiences stratified by multimorbidity among older female breast cancer survivors in the United States. Utilizing the National Cancer Institute’s Surveillance, Epidemiology, and End Results (SEER) program and the Centers for Medicare and Medicaid Services’ Consumer Assessment of Healthcare Providers and Systems (CAHPS) survey data linkage from 2000-2019. Breast cancer (BC) survivors (N=19,017) were aged ≥65 years at CAHPS survey who completed a survey after diagnosis of primary site invasive BC. Adjusted multivariable linear regression models were used to estimate beta (β) and standard error (SE) coefficients of the relationships between tumor characteristics (extent of disease: localized, regional, distant; estrogen receptor [ER] status: ER+, ER-, missing/unknown) and several CAHPS composite and rating outcomes, overall and stratified by multimorbidity (BC only, 2-5 chronic conditions, ≥6 chronic conditions). Women were on average 76.3 years (SD=7.14) of age at survey and 4.65 years (SD=3.51) since BC diagnosis. Most survivors were non-Hispanic white (78.1%), had ≥6 chronic conditions (38.1%), were diagnosed with localized BC (80.5%) and/or ER+ tumors (56.9%). Survivors with regional BC at diagnosis reported significantly higher mean scores on Getting Needed Care (β=1.00, SE=0.46, p=0.03), and with distant BC reported significantly lower mean scores on Physician Communication (β=-1.92, SE=0.92, p=0.03) compared to those with localized BC. When stratified by multimorbidity, survivors with distant stage compared to localized, and with 2-5 chronic conditions reported significantly higher mean scores for Getting Needed Care (β=3.85, SE=1.96, p=0.04), while this association was inverse and not statistically significant (β=-2.72, SE=1.87, p=0.14) among survivors with BC only (p-interaction=0.01). While the overall associations between ER status and the CAHPS outcomes were not statistically significant, we observed significant statistical interactions by multimorbidity for the following association: among survivors with BC only, survivors with ER- tumors reported lower Health Plan Ratings (β=-1.89, SE=0.73, p=0.009) compared to survivors with ER+ tumors, while this association was positive however, marginally statistically significant (β=1.63, SE=0.86, p=0.05) among survivors with ≥6 conditions (p-interaction=0.002). BC survivors who are diagnosed with advanced stage and aggressive tumor phenotypes, such as ER- BC, experience different survivorship care experiences when they are also managing multiple chronic conditions. Remarkably, our results demonstrate that women with distant stage breast cancer and multimorbidity reported higher score on getting the care they need. Citation Format: Kate E. Dibble, Zhengyi Deng, Avonne E. Connor. The impact of multimorbidity on the relationship between breast cancer tumor characteristics and survivorship care experiences among older women: A SEER-CAHPS analysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 749.

Abstract 271: MYC mediates enhanced lactate reutilization and resistance to anti-angiogenesis therapy in preclinical models of LKB1-deficient NSCLC

Abstract Deletion or loss of function mutations of the STK11/LKB1 tumor suppressor are associated with primary resistance to immunotherapy in KRAS-mutant lung adenocarcinoma (LUAD) and drive metabolic reprogramming of tumor cells. We observed that LKB1-deficient tumors were resistant to anti-angiogenic therapy in the hypoxic and nutrient-depleted or acidic tumor microenvironment (TME). We determined that MYC which is elevated in LKB1-deficient cells, regulated the expression of the lactate transporter, MCT4. Moreover, knockdown of MYC decreases glycolysis and cell proliferation. Therefore, we hypothesize that metabolic changes in LKB1-deficient tumor cells is at least partially driven by MYC upregulation, and that depletion of MYC or targeting of key enzymes or transporters downstream of MYC such as MCT4 may abrogate lactate reutilization and sensitize LKB1-deficient tumors to anti-angiogenic therapy. To explore the impact of MYC on lactate metabolism in LKB1-deficient NSCLC cells, we performed isotope tracing in KRAS mutant (K) and KRAS mutant LKB1 knockout (KL) syngeneic murine lung cancer cells. 13C3-lactate tracing revealed that isotopologues were significantly enriched in TCA components such as pyruvate, citrate and a-ketoglutarate in KL cells. Meanwhile, lactate-treated KL cells shows less reactive oxygen species (ROS), suggesting that LKB1-deficient tumor cells reutilize lactate as an energy source more efficiently than LKB1-intact cells. MCT4 KO significantly abrogated lactate incorporation into TCA cycle. Similarly, MYC knockdown or MCT4 KO decreased lactate-induced oxygen consumption, but increased ROS levels. In contrast, exhausted T cells were inefficient at lactate reutilization, and high levels of lactate increased ROS in T cells. Hence, LKB1-deficient tumors have a survival advantage over T cells in the lactate-rich TME. We queried scRNAseq data from K, KL and KL MCT4KO syngeneic models and similarly observed that KL tumor cells exhibited elevated hypoxia and angiogenesis gene expression signatures, which was reversed by MCT4 KO. However, ROS detoxification was decreased in T cells from KL tumors but increased in MCT4 KO tumors. Finally, we injected KL murine tumor cells into immunocompetent mice, and randomly treated them with vehicle or the VEGF blocking antibody, DC101. MYC knockdown or MCT4 KO sensitized KL tumors to VEGF inhibition, and significantly increased overall survival. Collectively, our data indicates that in LKB1-deficient tumors, upregulation of MYC promotes tumor cell metabolic reprogramming and that targeting MYC or MCT4 can inhibit lactate reutilization and enhance the efficacy of anti-angiogenic agents. These findings provide insight into the mechanisms driving the aggressive phenotype of KRAS-mutant LKB1-deficient tumors and identify a novel therapeutic strategy for targeting this patient population. Citation Format: Yu Qian, David Molkentine, Chendong Yang, Ana Galan Cobo, Irene Guijarro, Minghao Dang, Alissa Poteete, Peixin Jiang, Ferdinandos Skoulidis, Linghua Wang, Alexandre Reuben, John D. Minna, Ralph J. DeBerardinis, John V. Heymach. MYC mediates enhanced lactate reutilization and resistance to anti-angiogenesis therapy in preclinical models of LKB1-deficient NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 271.