Adenocarcinoma Mouse Research Articles

Veillonella parvula promotes the proliferation of lung adenocarcinoma through the nucleotide oligomerization domain 2/cellular communication network factor 4/nuclear factor kappa B pathway

Enrichment of Veillonella parvula in the lung microbiota is strongly associated with non-small cell lung cancer (NSCLC) and induces the progression of lung adenocarcinoma in vivo, but its actual role and mechanism remain unexplored. This study analyzed the correlation between NSCLC and V. parvula abundance based on 16 s rRNA sequencing results. The effects of V. parvula on the progression of lung adenocarcinoma were observed in vivo and in vitro using a C57 bl/6j mouse tumor-bearing model, a bacterial cell co-culture model, combined with transcriptome sequencing, and a TCGA database to explore and validate the growth promotion of lung adenocarcinoma by V. parvula and its molecular mechanism. 16 s rRNA sequencing revealed that V. parvula was significantly enriched in lung adenocarcinoma. In vivo, V. parvula promoted the growth of lung adenocarcinoma in mice by suppressing the infiltration of tumor-associated T lymphocytes and peripheral T lymphocytes. It showed a higher affinity for lung adenocarcinoma in vitro and promoted lung adenocarcinoma cell proliferation through adhesion or intracellular invasion. Further analysis of differential gene expression and KEGG enrichment by transcriptome sequencing revealed that V. parvula induced CCN4 expression and activated NOD-like receptor and NF-κB signaling pathway in lung adenocarcinoma cells. Further analysis clarified that V. parvula promoted activation of the NF-κB pathway via Nod2/CCN4 signaling, which promoted lung adenocarcinoma cell proliferation. Thus, V. parvula mediates activation of the Nod2/CCN4/NF-κB signaling pathway to promote non-small cell lung adenocarcinoma progression, thereby providing a potential target for diagnosing and treating lung adenocarcinoma.Graphical

Intralesional Chemotherapy for Prostate Cancer: In vivo Proof of Principle

Introduction: Prostate cancer (PCA) is one of the most common cancers in the world, and current therapies are debilitating to patients. To develop a novel modality for the treatment of PCA, we evaluated the efficacy of intralesional administration of the Sirt3 activator Honokiol (HK) and the NADPH oxidase inhibitor Dibenzolium (DIB). Methods: We used a well-established transgenic adenocarcinoma mouse prostate (TRAMP-C2) model of hormone-independent PCA. MTS assay, apoptosis assay, wound healing assay, transwell invasion assay, RT-qPCR, and Western blotting were conducted in vitro, and HK and DIB were intratumorally administered to mice bearing TRAMP-C2 tumors. Tumor size and weight were observed over time. After removing tumors, H-E staining and immunohistochemistry (IHC) staining were conducted. Results: Treatment by HK or DIB showed an inhibitory effect on cell proliferation and migration in PCA cells. Poor ability to induce apoptosis in vitro, insufficient expression of caspase-3 on IHC staining, and increased necrotic areas on H-E staining indicated that necrosis plays an important role in cell death in treating groups by HK or DIB. RT-PCR, Western blotting, and IHC staining for epithelial mesenchymal transition (EMT) markers suggested that EMT was suppressed by HK and DIB individually. In addition, HK induced activation of CD3. Mouse experiments showed safe antitumor effects in vivo. Conclusions: HK and DIB suppressed PCA proliferation and migration. Further research will explore the effects of HK and DIB at the molecular level to reveal new mechanisms that can be exploited as therapeutic modalities.

NFIC regulates ribosomal biology and ER stress in pancreatic acinar cells and restrains PDAC initiation

Pancreatic acinar cells rely on PTF1 and other transcription factors to deploy their transcriptional program. We identify NFIC as a NR5A2 interactor and regulator of acinar differentiation. NFIC binding sites are enriched in NR5A2 ChIP-Sequencing peaks. Nfic knockout mice have a smaller, histologically normal, pancreas with reduced acinar gene expression. NFIC binds and regulates the promoters of acinar genes and those involved in RNA/protein metabolism, and Nfic knockout pancreata show defective ribosomal RNA maturation. NFIC dampens the endoplasmic reticulum stress program through binding to gene promoters and is required for resolution of Tunicamycin-mediated stress. NFIC is down-regulated during caerulein pancreatitis and is required for recovery after damage. Normal human pancreata with low levels of NFIC transcripts display reduced expression of genes down-regulated in Nfic knockout mice. NFIC expression is down-regulated in mouse and human pancreatic ductal adenocarcinoma. Consistently, Nfic knockout mice develop a higher number of mutant Kras-driven pre-neoplastic lesions.

Increased Production of Interleukin-10 and Decreased Production of Tumor Necrosis Factor-α by Lacticaseibacillus rhamnosus PL60

Lacticaseibacillus rhamnosus PL60, originally isolated from healthy baby feces, has beneficial activities including resistance to acid and bile acids, immune-enhancing activity, inhibition of various intestinal pathogens, anti-oxidation activity, adherence to the intestines, susceptibility to antimicrobials, an anti-obesity effect, and an anti-fatty liver effect. In the present study, the binding activity of L. rhamnosus PL60 to the HT-29 human colorectal adenocarcinoma cell line and mouse intestines was established. L. rhamnosus PL60 increased interleukin-10 production and decreased tumor necrosis factor-α production in a dose-dependent manner in HT-29 cells treated with lipopolysaccharide. Similar results were observed in an in vivo experiment with BALB/c mice fed with L. rhamnosus PL60. Taken together, results from previous studies and the present study establish that L. rhamnosus PL60 is a probiotic candidate for inflammation-related diseases.

A New Type of Endometrial Cancer Models in Mice Revealing the Functional Roles of Genetic Drivers and Exploring their Susceptibilities.

Endometrial cancer (EC) is the most common female reproductive tract cancer and its incidence has been continuously increasing in recent years. The underlying mechanisms of EC tumorigenesis remain unclear, and efficient target therapies are lacking, for both of which feasible endometrial cancer animal models are essential but currently limited. Here, an organoid and genome editing-based strategy to generate primary, orthotopic, and driver-defined ECs in mice is reported. These models faithfully recapitulate the molecular and pathohistological characteristics of human diseases. The authors names these models and similar models for other cancers as organoid-initiated precision cancer models (OPCMs). Importantly, this approach can conveniently introduce any driver mutation or a combination of driver mutations. Using these models,it is shown that the mutations in Pik3ca and Pik3r1 cooperate with Pten loss to promote endometrial adenocarcinoma in mice. In contrast, the Kras G12D mutati led to endometrial squamous cell carcinoma. Then, tumor organoids are derived from these mouse EC models and performed high-throughput drug screening and validation. The results reveal distinct vulnerabilities of ECs with different mutations. Taken together, this study develops a multiplexing approach to model EC in mice and demonstrates its value for understanding the pathology of and exploring the potential treatments for this malignancy.

Simultaneous activation of Kras-Akt and Notch pathways induces extrahepatic biliary cancer via the mTORC1 pathway.

Biliary tract cancer (BTC) has poor prognosis. The Notch receptor is aberrantly expressed in extrahepatic cholangiocarcinoma (eCCA). However, the role of Notch signaling in the initiation and progression of eCCA and gallbladder (GB) cancer remains unknown. Therefore, we investigated the functional role of Notch signaling during tumorigenesis of the extrahepatic bile duct (EHBD) and GB. Activation of Notch signaling and oncogenic Kras resulted in the development of biliary intraepithelial neoplasia (BilINs) in the EHBD and GB, which were premalignant lesions that progressed to adenocarcinoma in mice. The expression of genes involved in the mTORC1 pathway was increased in biliary spheroids from Hnf1b-CreERT2; KrasLSL-G12D ; Rosa26LSL-NotchIC mice and inhibition of the mTORC1 pathway suppressed spheroid growth. Additionally, simultaneous activation of the PI3K-AKT and Notch pathways in EHBD and GB induced biliary cancer development in mice. Consistent with this, we observed a significant correlation between activated NOTCH1 and phosphorylated Ribosomal Protein S6 (p-S6) expression in human eCCA. Furthermore, inhibition of the mTORC1 pathway suppressed the growth of Notch-activated human biliary cancer cells in vitro and in vivo. Mechanistically, the Kras/Notch-Myc axis activated mTORC1 through TSC2 phosphorylation in mutant biliary spheroids. These data indicate that inhibition of the mTORC1 pathway could be an effective treatment strategy for Notch-activated human eCCA. © 2023 The Pathological Society of Great Britain and Ireland.

Polymerase θ inhibition steps on the cGAS pedal.

Deficiencies in homologous recombination (HR) repair lead to an accumulation of DNA damage and can predispose individuals to cancer. Polymerase theta (Pol θ, encoded by POLQ) is overexpressed by HR-deficient cancers and promotes cancer cell survival by mediating error-prone double-stranded break (DSB) repair and facilitating resistance against poly-ADP ribose polymerase inhibitor treatment. In this issue of the JCI, Oh, Wang, et al. report on the impact of Pol θ inhibition on activation of antitumor immunity. The authors used pancreatic ductal adenocarcinoma (PDAC) cell and mouse models characterized by HR-associated gene alterations and POLQ overexpression. POLQ knockdown showed synthetic lethality in combination with gene mutations involving DNA repair, including BRCA1, BRCA2, and ATM. Notably, Pol θ deficiency or inhibition suppressed tumor growth, increased the accumulation of unrepaired DNA damage, and enhanced T cell infiltration via the cGAS/STING pathway. These findings suggest a broader scope for Pol θ inhibition in HR-deficient cancers.

Expression of ductal and acinar immunohistochemical markers in preneoplastic lesions of a pancreatic ductal adenocarcinoma mouse model

Expression of ductal and acinar immunohistochemical markers in preneoplastic lesions of a pancreatic ductal adenocarcinoma mouse model

If you give a mouse a mutation: comparing the therapeutic utility of renowned mouse models of human cancers

Cancers of the breast, prostate and intestinal tract account for most cancer-associated deaths in humans and represent several of the highest incidence human neoplasms. Therefore, understanding the underlying pathophysiology, including the formation and propagation of these cancers, is key to designing potential treatments. Over the last 50 years or more, genetically engineered mouse models (GEMMs) have been instrumental platforms to our discovery of neoplastic disease as many follow near-identical molecular and histological progression as human tumours. In this mini review, we summarize three key preclinical models and focus on some of the major findings in relation to clinical care. We discuss the MMTV-PyMT (polyomavirus middle T antigen) mouse, TRAMP (transgenic adenocarcinoma mouse prostate) mouse and APCMin (multiple intestinal neoplasm mutation of APC gene) mouse, which mimic breast, prostate and intestinal cancers, respectively. We aim to describe the significant contributions these GEMMs have made to our collective understanding of high-incidence cancers as well as briefly discuss the limitations of each model as a device for therapeutic discovery.

Specific Targeting and Labeling of Colonic Polyps in CPC-APC Mice with Mucin 5AC Fluorescent Antibodies: A Model for Detection of Early Colon Cancer.

Poor visualization of polyps can limit colorectal cancer screening. Fluorescent antibodies to mucin5AC (MUC5AC), a glycoprotein upregulated in adenomas and colorectal cancer, could improve screening colonoscopy polyp detection rate. Adenomatous polyposis coli flox mice with a Cdx2-Cre transgene (CPC-APC) develop colonic polyps that contain both dysplastic and malignant tissue. Mice received MUC5AC-IR800 or IRdye800 as a control IV and were sacrificed after 48 h for near-infrared imaging of their colons. A polyp-to-background ratio (PBR) was calculated for each polyp by dividing the mean fluorescence intensity of the polyp by the mean fluorescence intensity of the background tissue. The mean 25 μg PBR was 1.70 (±0.56); the mean 50 μg PBR was 2.64 (±0.97); the mean 100 μg PBR was 3.32 (±1.33); and the mean 150 μg PBR was 3.38 (±0.87). The mean PBR of the dye-only control was 2.22 (±1.02), significantly less than the 150 μg arm (p-value 0.008). The present study demonstrates the ability of fluorescent anti-MUC5AC antibodies to specifically target and label colonic polyps containing high-grade dysplasia and intramucosal adenocarcinoma in CPC-APC mice. This technology can potentially improve the detection rate and decrease the miss rate of advanced colonic neoplasia and early cancer at colonoscopy.

Abstract 5458: TONSL is an immortalizing oncogene of the chromosome 8q24.3 amplicon and new therapeutic target in breast cancer

Abstract Study of genomic aberrations leading to immortalization of epithelial cells has been technically challenging due to lack of an appropriate isogenic model system. To address this technical challenge, we utilized primary breast luminal epithelial cells propagated from healthy donors of different genetic ancestry and their hTERT-immortalized counterparts to identify functional gene expression changes associated with immortalization. We identified elevated expression of TONSL (Tonsoku Like, DNA Repair Protein) as one of the earliest events during immortalization. TONSL is located on chromosome 8q24.3 and amplified in ~20% of breast cancers with significantly higher amplification in metastatic tumors. TONSL forms a complex with FACT and MMS22L1 to modulate multiple cellular pathways including DNA replication, repair through homologous recombination (HR) and functions as a post replicative histone/chromatin reader. TONSL alone immortalized primary breast epithelial cells and increased telomerase activity. While TONSL overexpression alone was insufficient for neoplastic transformation, TONSL-immortalized primary cells modified to overexpress defined oncogenes generated estrogen receptor-positive adenocarcinomas in NSG mice. Analysis of breast tumor microarray with ~500 tumors revealed poor overall and progression free survival of patients with TONSL-overexpressing tumors. TONSL increased chromatin accessibility to pro-oncogenic transcription factors including NF-κB and limited access to the tumor suppressor p53. Most importantly, TONSL overexpression resulted in significant changes in the expression of genes associated with DNA repair hubs, including upregulation of several genes in HR and Fanconi Anemia pathways. Consistent with the effects of TONSL on HR-associated genes, TONSL overexpressing primary cells exhibited upregulated DNA repair via HR. Moreover, TONSL was an essential gene for growth of TONSL-amplified breast cancer cell lines in vivo. Breast cancer cell lines with TONSL/chr8q24.3 amplification were sensitive to TONSL-FACT complex inhibitor CBL0137, both in vitro and in vivo. To our knowledge, TONSL is the only gene other than telomerase with immortalizing function and represents a new therapeutic target for breast cancer with chr8q24.3 amplification. Citation Format: Aditi S. Khatpe, Rebecca Dirks, Poornima Bhat-Nakshatri, Henry Mang, Katie Batic, Sarah Swiezy, Jacob Olson, Xi Rao, Yue Wang, Hiromi Tanaka, Sheng Liu, Jun Wan, Duojiao Chen, Yunlong Liu, Fang Fang, Sandra Althouse, Emily Hilsey, Maggie M. Granatir, Rebekah Addison, Constance J. Temm, George Sandusky, Audrey Lee-Gosselin, Kenneth Nephew Nephew, Kathy D. Miller, Harikrishna Nakshatri. TONSL is an immortalizing oncogene of the chromosome 8q24.3 amplicon and new therapeutic target in breast cancer. [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 5458.

Abstract 16: The role of innate and adaptive immunity in the sex bias of LKB1-mutant lung adenocarcinoma

Abstract The tumor suppressor gene, LKB1/STK11, is one of the most frequently mutated genes in lung adenocarcinoma (LUAD). A multidimensional analysis of TCGA-LUAD patient data indicates that LKB1 mutation is one of the top sex-biased mutations, being more frequent in males than in females. A similar pattern was observed in other large LUAD cohorts. However, the underlying mechanism was unknown, partially due to a lack of an appropriate mouse model to represent this clinical phenomenon. We previously demonstrated a similar sex bias in a Lenti-Cre-induced KRAS/LKB1-mutant genetically engineered mouse model (GEMM) and a syngeneic tail-vein injection model. Here, we demonstrated that intravenous injection of the same cell line in male and female NOD scid gamma (NSG) mice does not exhibit a sex-bias pattern of lung metastasis formation (P=0.9746). However, sex bias was observed when we used CB17-SCID mice. This data suggest that innate immunity is partly responsible for the sex bias of LKB1-mutant lung cancer formation. We also characterized immune cells from the lung with LKB1-mutant tumors. Monocytes/macrophages decreased from 23% to 19%, and neutrophils increased from 15% to 19%. Lymphoid cells remained the same at 58%, and T-cells account for 34% of all CD45+ cells. Interestingly, T-cells doubled to account for 81% of all CD45+ cells from the lung in female mice resistant to the formation of lung metastasis. Our data indicated that both the innate and the adaptive immunity are responsible for eliminating LKB1-mutant lung adenocarcinoma in female mice. Citation Format: Yijian Fan, Lenore Monterroza, Rui Jin, Xiuju Liu, Periasamy Selvaraj, Rabindra Tirouvanziam, Wei Zhou. The role of innate and adaptive immunity in the sex bias of LKB1-mutant lung adenocarcinoma [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 16.

Abstract 1962: Oncologic anthropology-impact of Duffy adaptive trait on breast epithelial and tumor cell biology

Abstract Adaptation to diverse environments during human evolution including those conditions that favor certain infectious diseases to become endemic in a region has resulted in natural selection of genetic variants. These variants remain fixed in current day populations despite changes in the environment. At least 20 such variants, also called adaptive traits, have been described to date and Duffy phenotype is one among them. Individuals in sub-Saharan Africa carry an inactivating mutation in a gene called atypical cytokine receptor 1 (ACKR1 or DARC) and this phenotype is called Duffy-phenotype. ACKR1 is a decoy receptor for CCL2 and IL-8 (CXCL8) and limits the activity of these cytokines. The Duffy-null phenotype confers protection against malarial infection. However, recent studies suggest that breast cancers in Duffy-null women display a distinct immune profile and have worse outcomes, possibly due to altered signaling by cytokines/chemokines. Using our institutional resource of breast tissues from >5000 healthy women, we have initiated studies to examine the impact of adaptive traits on normal breast and breast cancer biology. Towards this goal, we have created breast epithelial cell lines from Duffy wild type and Duffy-null/heterozygous carriers, all of African ancestry. ACKR1 is expressed in breast epithelial cells of Duffy-wild type but not in Duffy-null carriers. Breast epithelial cell lines as well as their transformed variants from Duffy-null/heterozygous carriers contained elevated levels of phosphorylated cMET(Y1349) and pERK(T202/Y204) compared to cell lines from Duffy-wild type carriers. Transformed Duffy-wild type and Duffy-heterozygous cells generated adenocarcinomas in NSG mice, which indicates that our model system closely recapitulates breast cancers in human. Cell lines created from Duffy-heterozygous tumors compared to Duffy-wild tumors displayed elevated pERK levels. Duffy phenotype also affects parainflammatory signals from transformed cells as tumor-derived cells from Duffy-null/heterozygous carriers expressed higher levels of IGFBP2 compared to tumor-derived cells from non-carriers. Additional studies are underway to determine whether Duffy phenotype alters sensitivity to chemo and targeted therapies. Collectively, these results suggest that adaptive traits alter epithelial cell intrinsic signaling pathways, which may affect tumor growth patterns, tumor microenvironment, and potentially sensitivity to specific therapies. Citation Format: Poornima Bhat-Nakshatri, Brijesh Kumar, Madhura S. Shukla, Katie Batic, Henry Mang, Rana German, Anna Maria Storniolo, Harikrishna Nakshatri. Oncologic anthropology-impact of Duffy adaptive trait on breast epithelial and tumor cell biology [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 1962.

Abstract 5139: Chemerin suppresses prostate tumor growth by modulating the recruitment of NK and CD8+ T-cells to tumor microenvironment

Abstract Infiltration of immune cells into the tumor microenvironment (TME) can regulate growth and survival of malignant cells, thereby affecting tumorigenesis and tumor progression. While immune checkpoint inhibitors and agonism of costimulatory molecules can act to stimulate leukocytes, the lack of adequate recruitment of effector cells into TME often results in suboptimal immune responses. Chemerin (retinoic acid receptor responder 2, RARRES2) is an endogenous chemoattractant that is widely expressed by many tissues and recruits innate leukocytes through its receptor, CMKLR1 which is expressed on NK cells, some dendritic cells, and macrophages. Previous studies show that RARRES2 is downregulated across multiple tumor types including prostate cancer. Here, using a syngeneic transgenic adenocarcinoma mouse prostate (TRAMP-C1) model, we show that forced overexpression of RARRES2 by tumor cells results in significant tumor suppression and recruitment of NK and CD8+ T-cells within the TME. Male C57/BL/6 mice were inoculated with either RARRES2-overexpressing, vector control (VC) or mixed (50:50; VC: RARRES2) TRAMP-C1 cells. Chemerin overexpression in TRAMP-C1 cells did not alter their phenotypic behavior in vitro, but significantly suppressed tumor growth in vivo in mice. Furthermore, we observed a significantly higher influx of NK (CD3-CD19-NK1.1+) and CD8+ T-cells (CD3+CD8+) in tumor-infiltrating leukocytes (TILs) from mice with mixed tumors compared with VC tumors. Depletion of NK cells, CD4+, or CD8+ T cells showed that NK cells and CD8+ T-cells, but not CD4+ T-cells, were required for chemerin-dependent suppression of TRAMP-C1 tumor growth. We found significant increases in splenic NK and CD8 T cells, and a concomitant significant decrease in splenic G-MDSC in mice with chemerin-expressing tumors compared to controls, suggesting chemerin expression within the TME has an effect on systemic immune responses. Treatment of control tumors with anti-PD1 checkpoint inhibition did not result in significant tumor suppression, while anti-PD1 treatment of chemerin-expressing tumors significantly reduced tumor growth, doubling the complete response rate in these unresponsive tumors. Thus, for the first time we have demonstrated that increasing chemerin expression within the prostate TME can suppress growth by augmenting recruitment of NK and CD8+ T-cells and has the potential to be combined with checkpoint inhibitors in order to improve response rates in non-responsive tumors. Overall, these data suggest that a tumor-targeted chemerin therapeutic may have potential to treat human prostate cancers. Citation Format: Muhammad A. Saeed, Kevin Kim, Russell K. Pachynski. Chemerin suppresses prostate tumor growth by modulating the recruitment of NK and CD8+ T-cells to tumor microenvironment. [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 5139.

Abstract 3082: Altered levels of corticosteroid-binding globulin: SerpinA6 and progression of prostate cancer

Abstract SerpinA6 is a main transport protein for cortisol binding, which helps in cortisol tissue delivery particularly at inflammatory sites. Cortisol plays a vital role in adaptation to stress, modulating fuel metabolism, and inflammation. SerpinA6 releases cortisol specifically at inflammatory sites upon cleavage by human neutrophil elastase between residues 344 and 345. Castration induce inflammation in the prostate increases glucocorticoids (GCs) level and elevates pro-inflammatory cytokine IL-6 levels. European and Asian populations represented association between single nucleotide polymorphisms (SNPs) in CYP17A1 and SERPINA6/A1 and circulating glucocorticoid concentrations. We propose increasing IL-6 levels after castration alters SerpinA6 levels in prostate may results in prostate cancer disease progression. Deficient SerpinA6 may not be sufficient to keep steroids inactive, due to the increasing GCs. Our data suggests IL-6 treatment of androgen independent prostate cancer (PC-3) cells decreases SerpinA6 level and conversely increases glucocorticoid receptor (GR) expression in a dose (1-10ng/ml) and time (30-180 minutes) dependent fashion. Moreover, IL-6 dose dependent treatment in PC-3 cells decreases FOXO3a expression, a major apoptotic protein dysregulated in prostate cancer. Next, we determine the role of SerpinA6 (30µg/ml) and IL-6 (5ng/ml) independently and in combination to PC-3 cells. We observed IL-6 and SerpinA6 co-treatment to PC-3 cells (charcoal stripped FBS media) for 6 hours reduced Neutrophil elastase levels significantly. Silencing IL-6 in PC-3 cells increased SerpinA6 expression. Moreover, silencing SerpinA6 in metastatic variant of PC-3 cells increased phospho-Akt-(ser-473), glucocorticoid receptor and increased apoptotic protein FOXO3a expressions. Using dexamethasone (15µM), which is known to suppress PC-3 cell migration, we found increased expression of SerpinA6. Conversely, Mifepristone (15µM), which inhibits PC-3 cell growth, deceases SerpinA6 expression after 24 hours of treatment. In humans the normal range of circulatory SerpinA6 level are 19-45µg/ml. We treated normal prostate epithelial cells (RWPE1) and prostate cancer (PC-3) cells with human recombinant protein 18 and 36µg/ml of doses for 24 hours; RWPE1 cells represented a SerpinA6 dose dependent increase in cellular viability, whereas PC-3 cells demonstrated no change in the viability after a dose response treatment with SerpinA6 for 24 hours. In-vivo data represented altered SerpinA6 levels in castrated TRansgenic Adenocarcinoma Mouse Prostate model after dexamethasone (GR agonist) and Mifepristone (GR antagonist) treatment. We suggest decreasing levels of SerpinA6 in prostate cancer patients may have potential role in disease progression. We need to have an effective agent, to prevent maintain SerpinA6 level to inhibit the progression of prostate cancer. Citation Format: Mohammed Al-Toubat, Jatinder Kumar, Shiva Gautam, Kethandapatti Balaji, Sanjeev Shukla. Altered levels of corticosteroid-binding globulin: SerpinA6 and progression of prostate cancer [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 3082.

Abstract 4904: Glutamine antagonist DRP-104 enhances anti-tumor responses in KEAP1 mutant lung cancer

Abstract KEAP1 is mutated in approximately 20% of non-small cell lung cancer (NSCLC) and is associated with poor response rates to checkpoint blockade. Given the therapeutic challenge presented by KEAP1 mutant tumors and the great potential held by immunotherapeutic approaches, there is an urgent need to identify the mechanisms that mediate immune evasion in patients with KEAP1 mutant lung adenocarcinoma. We previously demonstrated that metabolic rewiring in KEAP1 mutant tumors leads to increased dependency on glutamine as a substrate for multiple anabolic pathways. DRP-104 (Sirpiglenastat) is a novel glutamine antagonist which inhibits all glutamine consuming reactions. Using pre-clinical mouse models and patient derived xenograft models we demonstrate that DRP-104 robustly impairs the growth of KEAP1 mutant tumors. Furthermore, using an immunocompetent orthotopic lung adenocarcinoma mouse model we find that treatment with DRP-104 increases survival of Keap1 mutant tumor bearing mice treated with anti-PD1 therapy. We have dissected the mechanism by which DRP-104 impacts KEAP1 mutant tumors and the surrounding immune microenvironment. Notably, DRP-104 impairs nucleotide synthesis in KEAP1 mutant tumors and thereby inhibits cancer cell growth. In addition, DRP-104 reduces exhausted T cell and T regulatory populations within the tumor microenvironment as well as increases effector T cell function. Overall, we find that DRP-104 impairs the growth of KEAP1 mutant tumors through both targeting of tumor intrinsic metabolic vulnerabilities and through tumor extrinsic immunostimulatory mechanisms such as promoting the expansion of functional anti-tumor T cell populations. This data provides additional rationale for the ongoing phase I/II clinical trial (NCT04471415) using DRP-104 in NSCLC with mutations in KEAP1 as well as for combining DRP-104 with checkpoint blockade. Citation Format: Ray Pillai, Sarah LeBoeuf, Ali Rashidfarrokhi, Shih Ming Huang, Triantafyllia Karakousi, Anastasia-Maria Zavitsanou, Warren Wu, Volkan Sayin, Robert Wild, Sergei Koralov, Thales Papagiannakopoulos. Glutamine antagonist DRP-104 enhances anti-tumor responses in KEAP1 mutant lung cancer. [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 4904.

Abstract 1456: An expression study of circadian clock-associated transcription factors and nuclear receptors in mouse prostate gland and tumor

Abstract The circadian clock lies in the suprachiasmatic nucleus of the hypothalamus and is regulated directly by the daily light/dark cycles, generating rhythmic expression of core circadian-controlled genes (CCCG) some belonging to the nuclear receptors (NR) superfamily. Recent advances show that the disruption of the circadian clock is identified as a risk factor for endocrine cancers, such as prostate cancer which is the most common hormone-related male cancer globally. Whether the prostate gland has its own circadian expression of CCCG and NR, and how the dysregulated circadian clock would influence the fate of the prostate tumors is not well studied. Thus, the present study aims to determine the circadian expression patterns of CCCG and NR in normal mouse prostate and further validate their disrupted circadian expression in a transgenic mouse adenocarcinoma prostate cancer model (TGMAP) with neuroendocrine phenotypes and suppressed androgen gene expression (AR), over the 12 hours light/dark cycle. We establish the rhythmic expression patterns of multiple CCCG and the full class of NR by quantitative polymerase-chain reaction analysis in normal mouse prostate (C57BL/6 strain) and advanced prostate tumors derived from TGMAP in a 12-hours light/dark cycle with 4-hours interval. Our results showed that 30 out of 49 NR and several CCCG follow rhythmic expression patterns, followed by 10 NR with unidentifiable expression patterns and 9 NR showing very low expression in the mouse prostate tissue. The circadian expression of these transcription factors including the AR gene, could be divided into 2 categories with single peak or double peaks at specific time points throughout the light/dark cycle, linking them to the regulating function of the circadian clock in the normal prostate gland. In the advanced prostate tumors derived from TGMAP model that exhibit androgen independence and neuroendocrine trans-differentiation phenotype, the rhythmic cycles of the NR and CCCG showed disruption, and several transcription factors were upregulated in overall circadian expression in comparison to the normal prostate tissue. In summary, this study establishes two categories of rhythmic expression patterns in normal mouse prostate tissue that link the circadian expression of these transcription factors in the peripheral clock to that of androgen signaling axis that is responsible for the maintenance of prostate gland while disruption of such circadian expression patterns was demonstrated in the TGMAP tumors of which the AR signaling was either lost or disrupted. The upregulated circadian expression patterns as revealed in TGMAP model also provide insight into the significance of the dysregulation of circadian rhythm as regulated by CCCG and NR in the advanced prostate cancer. (Study supported by a Direct Grant for Research 2021-2022 Project No. 2021.062, CUHK) Citation Format: Ria Chopra, Franky Leung Chan. An expression study of circadian clock-associated transcription factors and nuclear receptors in mouse prostate gland and tumor [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 1456.

Design and preclinical evaluation of a novel apelin-based PET radiotracer targeting APJ receptor for molecular imaging of angiogenesis

APJ has been extensively described in the pathophysiology of angiogenesis and cell proliferation. The prognostic value of APJ overexpression in many diseases is now established. This study aimed to design a PET radiotracer that specifically binds to APJ. Apelin-F13A-NODAGA (AP747) was synthesized and radiolabeled with gallium-68 ([68Ga]Ga-AP747). Radiolabeling purity was excellent (> 95%) and stable up to 2 h. Affinity constant of [67Ga]Ga-AP747 was measured on APJ-overexpressing colon adenocarcinoma cells and was in nanomolar range. Specificity of [68Ga]Ga-AP747 for APJ was evaluated in vitro by autoradiography and in vivo by small animal PET/CT in both colon adenocarcinoma mouse model and Matrigel plug mouse model. Dynamic of [68Ga]Ga-AP747 PET/CT biodistributions was realized on healthy mice and pigs for two hours, and quantification of signal in organs showed a suitable pharmacokinetic profile for PET imaging, largely excreted by urinary route. Matrigel mice and hindlimb ischemic mice were submitted to a 21-day longitudinal follow-up with [68Ga]Ga-AP747 and [68Ga]Ga-RGD2 small animal PET/CT. [68Ga]Ga-AP747 PET signal in Matrigel was significantly more intense than that of [68Ga]Ga-RGD2. Revascularization of the ischemic hind limb was followed by LASER Doppler. In the hindlimb, [68Ga]Ga-AP747 PET signal was more than twice higher than that of [68Ga]Ga-RGD2 on day 7, and significantly superior over the 21-day follow-up. A significant, positive correlation was found between the [68Ga]Ga-AP747 PET signal on day 7 and late hindlimb perfusion on day 21. We developed a new PET radiotracer that specifically binds to APJ, [68Ga]Ga-AP747 that showed more efficient imaging properties than the most clinically advanced tracer of angiogenesis, [68Ga]Ga-RGD2.

Antioxidant and Protective Effects of the Polyphenolic Glycoconjugate from Agrimonia eupatoria L. Herb in the Prevention of Inflammation in Human Cells

Herein, structural and biological studies of a complex biopolymer (polyphenolic glycoconjugate) isolated from the flowering parts of Agrimonia eupatoria L. (AE) are presented. Spectroscopic analyses (UV-Vis and 1H NMR) of the aglycone component of AE confirmed that it consists mainly of aromatic and aliphatic structures characteristic of polyphenols. AE showed significant free radical elimination activity, i.e., ABTS+ and DPPH·, and was an effective copper reducing agent in the CUPRAC test, eventually proving that AE is a powerful antioxidant. AE was nontoxic to human lung adenocarcinoma cells (A549) and mouse fibroblasts (L929) and was nongenotoxic to S. typhimurium bacterial strains TA98 and TA100. Moreover, AE did not induce the release of proinflammatory cytokines such as interleukin 6 (IL-6) and tumor necrosis factor (TNF-α) by human pulmonary vein (HPVE-26) endothelial cells or human peripheral blood mononuclear cells (PBMCs). These findings correlated with the low activation of the transcription factor NF-κB in these cells, which plays an important role in the regulation of the expression of genes responsible for inflammatory mediator synthesis. The AE properties described here suggest that it may be useful for protecting cells from the adverse consequences of oxidative stress and could be valuable as a biomaterial for surface functionalization.

Effects of Monoamino-Oxidase-A (MAO-A) Inhibition on Skeletal Muscle Inflammation and Wasting through Pancreatic Ductal Adenocarcinoma in Triple Transgenic Mice.

Cancer cachexia describes a syndrome of muscle wasting and lipolysis that is still largely untreatable and negatively impacts prognosis, mobility, and healthcare costs. Since upregulation of skeletal muscle monoamine-oxidase-A (MAO-A), a source of reactive oxygen species, may contribute to cachexia, we investigated the effects of the MAO-inhibitor harmine-hydrochloride (HH, intraperitoneal, 8 weeks) on muscle wasting in a triple-transgenic mouse model of pancreatic ductal adenocarcinoma (PDAC) and wild type (WT) mice. Gastrocnemius and soleus muscle cryo-cross-sections were analyzed for fiber type-specific cross-sectional area (CSA), fraction and capillarization using ATPase- and lectin-stainings. Transcripts of pro-apoptotic, -atrophic, and -inflammatory signals were determined by RT-qPCR. Furthermore, we evaluated the integrity of neuromuscular junction (NMJ, pre-/post-synaptic co-staining) and mitochondrial ultrastructure (transmission electron microscopy). MAO-A expression in gastrocnemius muscle was increased with PDAC vs. WT (immunohistochemistry: p < 0.05; Western blot: by trend). PDAC expectedly reduced fiber CSA and upregulated IL-1β in both calf muscles, while MuRF1 expression increased in soleus muscle only. Although IL-1β decreased, HH caused an additional 38.65% (p < 0.001) decrease in gastrocnemius muscle (IIBX) fiber CSA. Moreover, soleus muscle CSA remained unchanged despite the downregulation of E3-ligases FBXO32 (p < 0.05) and MuRF1 (p < 0.01) through HH. Notably, HH significantly decreased the post-synaptic NMJ area (quadriceps muscle) and glutathione levels (gastrocnemius muscle), thereby increasing mitochondrial damage and centronucleation in soleus and gastrocnemius type IIBX fibers. Moreover, although pro-atrophic/-inflammatory signals are reversed, HH unfortunately fails to stop and rather promotes PDAC-related muscle wasting, possibly via denervation or mitochondrial damage. These differential adverse vs. therapeutic effects warrant studies regarding dose-dependent benefits and risks with consideration of other targets of HH, such as the dual-specificity tyrosine phosphorylation regulated kinases 1A and B (DYRK1A/B).