Adenocarcinoma Mouse Research Articles

Abstract A006: The NALCN channel regulates metastasis and non-malignant cell dissemination

Abstract We identify the Sodium Leak Channel Non-Selective Protein (NALCN) as a key regulator of cancer metastasis and non-malignant cell dissemination. Among 10,022 human cancers, NALCN loss-of-function mutations were enriched in gastric and colorectal cancers. Deletion of Nalcn from gastric (Prom1CreERT2/LacZ;KrasG12D;Trp53Flx/Flx; n=269), intestinal (Villin1-CreERT2;KrasG12D;Trp53Flx/Flx; n=141) or pancreatic adenocarcinomas (Pdx1-Cre;KrasG12D;Trp53Flx/+; n=55) in mice did not alter tumor incidence, but markedly increased the number of circulating tumor cells (CTCs) and metastases. Treatment of these mice (Villin1-CreERT2;KrasG12D;Trp53Flx/Flx; n=28) with gadolinium–an imaging contrast agent and NALCN channel blocker–similarly increased CTCs and metastasis. Deletion of Nalcn from mice that lacked oncogenic mutations and never developed cancer(Prom1CreERT2/LacZ; n=174), caused shedding of epithelial cells into the blood at levels equivalent to those seen in tumor-bearing animals. These cells trafficked to distant organs to form normal structures including lung epithelium and kidney glomeruli and tubules. Thus, NALCN regulates cell shedding from solid tissues independent of cancer, divorcing this process from tumorigenesis and unmasking a potential new target for anti-metastatic therapies. Citation Format: Eric Rahrmann, David Shorthouse, Amir Jassim, Linda Hu, Mariaestela Ortiz, Betania Mahler-Araujo, Peter Vogel, Marta Paez-Ribes, Atefeh Fatemi, Gregory Hannon, Radhika Iyer, Jay Blundon, Filipe Lourenço, Jonathan Kay, Rosaylnn Nazarian, Benjamin Hall, Stanislav Zakharenko, Douglas Winton, Liqin Zhu, Richard Gilbertson. The NALCN channel regulates metastasis and non-malignant cell dissemination [abstract]. In: Proceedings of the AACR Special Conference: Cancer Metastasis; 2022 Nov 14-17; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_2):Abstract nr A006.

Retracted: In Vivo Growth Inhibition of Human Caucasian Prostate Adenocarcinoma in Nude Mice Induced by Amygdalin with Metabolic Enzyme Combinations.

[This retracts the article DOI: 10.1155/2022/4767621.].

RNF43 induces the turnover of protease-activated receptor 2 in colon cancer.

Post-translational modification of G-protein coupled receptors (GPCRs) plays a central role in tissue hemostasis and cancer. The molecular mechanism of post-translational regulation of protease-activated receptors (PARs), a subgroup of GPCRs is yet understudied. Here we show that the cell-surface transmembrane E3 ubiquitin ligase ring finger 43 (RNF43) is a negative feedback regulator of PAR2 , impacting PAR2 -induced signaling and colon cancer growth. RNF43 co-associates with PAR2 , promoting its membrane elimination and degradation as shown by reduced cell surface biotinylated PAR2 levels and polyubiquitination. PAR2 degradation is rescued by R-spondin2 in the presence of leucine-rich repeat-containing G-protein-coupled receptor5 (LGR5). In fact, PAR2 acts jointly with LGR5, as recapitulated by increased β-catenin levels, transcriptional activity, phospho-LRP6, and anchorage-independent colony growth in agar. Animal models of the chemically induced AOM/DSS colon cancer of wt versus Par2/f2rl1 KO mice as also the 'spleen-liver' colon cancer metastasis, allocated a central role for PAR2 in colon cancer growth and development. RNF43 is abundantly expressed in the Par2/f2rl1 KO-treated AOM/DSS colon tissues while its level is very low to nearly null in colon cancer adenocarcinomas of the wt mice. The same result is obtained in the 'spleen-liver' model of spleen-inoculated cells, metastasized to the liver. High RNF43 expression is observed in the liver upon shRNA -Par2 silencing. "Limited-dilution-assay" performed in mice in-vivo, assigned PAR2 as a member of the cancer stem cell niche compartment. Collectively, we elucidate an original regulation of PAR2 oncogene, a member of cancer stem cells, by RNF43 ubiquitin ligase. It impacts β-catenin signaling and colon cancer growth.

A newseries of thiazole-hydrazone hybrids for Akt-targeted therapy of non-small cell lung cancer.

In an attempt to identify potent antitumor agents for the fight against non-small cell lung cancer, new thiazolyl hydrazones (2a-n) were synthesized and examined for their in vitro cytotoxic effects on A549 human lung adenocarcinoma and L929 mouse embryonic fibroblast cells by means of theMTT assay. Furthermore, the effects of the most potent anticancer agents on apoptosis and Akt inhibition were investigated. 2-[2-((Isoquinolin-5-yl)methylene)hydrazinyl]-4-(4-methylsulfonylphenyl)thiazole (2k) (IC50 = 1.43 ± 0.12 µM) and 2-[2-((isoquinolin-5-yl)methylene)hydrazinyl]-4-(1,3-benzodioxol-5-yl)thiazole (2l) (IC50 = 1.75 ± 0.07 µM) displayed more pronounced anticancer activity than cisplatin (IC50 = 3.90 ± 0.10 µM) on A549 cell lines; 2-[2-((isoquinolin-5-yl)methylene)hydrazinyl]-4-(4-methoxyphenyl)thiazole (2j) (IC50 = 3.93 ± 0.06 µM) showed anticancer activity close to cisplatin. These compounds were found to induce apoptosis in A549 cells. Compound 2j (IC50 = 3.55 ± 0.64 µM) showed stronger Akt inhibitory activity than GSK690693 (IC50 = 4.93 ± 0.06 µM), while compounds 2k and 2l did not cause Akt inhibition at IC50 concentrations (1.43 and 1.75 µM, respectively). To comprehensively elucidate the binding pose of compound 2j and to provide a detailed understanding on the ligand' binding mechanism, induced-fit docking calculations were also conducted. Both in vitro and in silico studies suggest that compound 2j shows its cytotoxic and apoptotic effects on A549 cell linesvia Akt inhibition. However, it is understood that compounds 2k and 2l exert their strong anticancer effects on A549 cells through different pathways.

Overexpression of Estrogen Receptor α in Mammary Glands of Aging Mice Is Associated with a Proliferative Risk Signature and Generation of Estrogen Receptor α–Positive Mammary Adenocarcinomas

Age is a risk factor for human estrogen receptor-positive breast cancer, with highest prevalence following menopause. While transcriptome risk profiling is available for human breast cancers, it is not yet developed for prognostication for primary or secondary breast cancer development utilizing at-risk breast tissue. Both estrogen receptor α (ER) and aromatase overexpression have been linked to human breast cancer. Herein, conditional genetically engineered mouse models of estrogen receptor 1 (Esr1) and cytochrome P450 family 19 subfamily A member 1 (CYP19A1) were used to show that induction of Esr1 overexpression just before or with reproductive senescence and maintained through age 30 months resulted in significantly higher prevalence of estrogen receptor-positive adenocarcinomas than CYP19A1 overexpression. All adenocarcinomas tested showed high percentages of ER+cells. Mammary cancer development was preceded by a persistent proliferative transcriptome risk signature initiated within 1 week of transgene induction that showed parallels to the Prosigna/Prediction Analysis of Microarray 50 human prognostic signature for early-stage human ER+breast cancer. CYP19A1 mice also developed ER+mammary cancers, but histology was more divided between adenocarcinoma and adenosquamous, with one ER- adenocarcinoma. Results demonstrate that, like humans, generation of ER+adenocarcinoma in mice was facilitated by aging mice past the age of reproductive senescence. Esr1 overexpression was associated with a proliferative estrogen pathway-linked signature that preceded appearance of ER+mammary adenocarcinomas.

Extensive preclinical validation of combined RMC-4550 and LY3214996 supports clinical investigation for KRAS mutant pancreatic cancer

Over 90% of pancreatic cancers present mutations in KRAS, one of the most common oncogenic drivers overall. Currently, most KRAS mutant isoforms cannot be targeted directly. Moreover, targeting single RAS downstream effectors induces adaptive resistance mechanisms. We report here on the combined inhibition of SHP2, upstream of KRAS, using the allosteric inhibitor RMC-4550 and of ERK, downstream of KRAS, using LY3214996. This combination shows synergistic anti-cancer activity invitro, superior disruption of the MAPK pathway, and increased apoptosis induction compared with single-agent treatments. Invivo, we demonstrate good tolerability and efficacy of the combination, with significant tumor regression in multiple pancreatic ductal adenocarcinoma (PDAC) mouse models. Finally, we show evidence that 18F-fluorodeoxyglucose (FDG) positron emission tomography (PET) can be used to assess early drug responses in animal models. Based on these results, we will investigate this drug combination in the SHP2 and ERK inhibition in pancreatic cancer (SHERPA; ClinicalTrials.gov: NCT04916236) clinical trial, enrolling patients with KRAS-mutant PDAC.

Influences of calorie restriction and lipopolysaccharide therapy on inflammation, cytokine response, and cell proliferation in pancreatic adenocarcinoma mouse model.

The study aimed to investigate the effects of lipopolysaccharide (LPS) alone and in combination with calorie restriction (CR) on the pancreatic tissues in C57BL/6 mice modeled with pancreatic ductal adenocarcinoma (PDAC). Forty male C57BL/6 mice (10-13 weeks old) were divided into five groups; LPS,LPS + CR, PDAC, PDAC + LPS, and PDAC + LPS + CR. Nuclear factor kappa B (NF-κβ), interleukin-6 (IL-6), and c-Jun N-terminal kinases (JNK) mRNA expression levels were measured in pancreatic tissues. NF-κβ, IL-6, JNK, and proliferating cell nuclear antigen(PCNA) peptide levels were determined by immunohistochemistry. Oxidative stress markers and antioxidant enzyme activities were determined spectrophotometrically. TH1/TH2 cytokine measurements were determined by a flow cytometer. It was detected that the number of PCNA immune + cells in the PDAC + LPS + CR group was significantly lower than in the PDAC and PDAC + LPS groups (p < 0.01, p < 0.05 respectively). PDAC + LPS + CR group's plasma interferon-gamma(IFN-γ), IL-6, IL-2, tumor necrosis factor-alpha, IL-3, and IL-4 levels were found to be significantly lower than the PDAC group (p < 0.01, p < 0.001, p < 0.01, p < 0.05, p < 0.01, and p < 0.05 respectively). According to our findings, the combination of low-dose LPS and 40% CR was found to be more effective in PDAC model mice.

TNF-α-dependent lung inflammation upregulates PD-L1 in monocyte-derived macrophages to contribute to lung tumorigenesis.

Chronic inflammation, which is dominated by macrophage-involved inflammatory responses, is an instigator of cancer initiation. Macrophages are the most abundant immune cells in healthy lungs, and associated with lung tumor development and promotion. PD-L1 is a negative molecule in macrophages and correlated with an immunosuppressive function in tumor environment. Macrophages expressing PD-L1, rather than tumor cells, exhibits a critical role in tumor growth and progression. However, whether and how PD-L1 in macrophages contributes to inflammation-induced lung tumorigenesis requires further elucidation. Here, we found that higher expression of PD-L1 in CD11b+ CD206+ macrophages was positively correlated with tumor progression and PD-1+ CD8+ T cells population in human adenocarcinoma patients. In the urethane-induced inflammation-driven lung adenocarcinoma (IDLA) mouse model, the infiltration of circulating CD11bhigh F4/80+ monocyte-derived macrophages (MoMs) was increased in pro-tumor inflamed lung tissues and lung adenocarcinoma. PD-L1 was mainly upregulated in MoMs associated with enhanced T cells exhaustion in lung tissues. Anti-PD-L1 treatment can reduce T cells exhaustion at pro-tumor inflammatory stage, and then inhibit tumorigenesis in IDLA. The pro-tumor lung inflammation depended on TNF-α to upregulate PD-L1 and CSN6 expression in MoMs, and induced cytokines production by alveolar type-II cells (AT-II). Furthermore, inflammatory AT-II cells could secret TNF-α to upregulate PD-L1 expression in bone-marrow driven macrophages (BM-M0). Inhibition of CSN6 decreased PD-L1 expression in TNF-α-activated macrophage in vitro, suggesting a critical role of CSN6 in PD-L1 upregulation. Thus, pro-tumor inflammation can depend on TNF-α to upregulate PD-L1 in recruited MoMs, which may be essential for lung tumorigenesis.

Epithelial cell size dysregulation in human lung adenocarcinoma.

Human cells tightly control their dimensions, but in some cancers, normal cell size control is lost. In this study we measure cell volumes of epithelial cells from human lung adenocarcinoma progression in situ. By leveraging artificial intelligence (AI), we reconstruct tumor cell shapes in three dimensions (3D) and find airway type 2 cells display up to 10-fold increases in volume. Surprisingly, cell size increase is not caused by altered ploidy, and up to 80% of near-euploid tumor cells show abnormal sizes. Size dysregulation is not explained by cell swelling or senescence because cells maintain cytoplasmic density and proper organelle size scaling, but is correlated with changes in tissue organization and loss of a novel network of processes that appear to connect alveolar type 2 cells. To validate size dysregulation in near-euploid cells, we sorted cells from tumor single-cell suspensions on the basis of size. Our study provides data of unprecedented detail for cell volume dysregulation in a human cancer. Broadly, loss of size control may be a common feature of lung adenocarcinomas in humans and mice that is relevant to disease and identification of these cells provides a useful model for investigating cell size control and consequences of cell size dysregulation.

Midostaurin Modulates Tumor Microenvironment and Enhances Efficacy of Anti-PD-1 against Colon Cancer.

Simple SummaryColon cancer is one of the most common types of cancer worldwide. Immune checkpoint inhibitors have promising effects on various types of cancers with limited efficacy in colon cancer. Midostaurin (PKC412) is currently used for the treatment of patients with acute myeloid leukemia harboring FLT3-mutation. The aim of this study was to assess the potential effect of midostaurin on the modulation of TME and the efficacy of anti-PD-1 against colon cancer. We showed midostaurin inhibited colorectal adenocarcinoma cell growth and induced multinucleation and micronuclei formation. Midostaurin inhibited colorectal adenocarcinoma cell growth associated with the formation of dsDNA and ssDNA; the up-regulation of mRNA expression of cGAS, STING, IRF3, and IFNAR1; the down-regulation of Trex-1, c-Kit, and Flt3 protein expression. The tumor-implanted model displayed a combination of midostaurin-enhanced efficacy of anti-PD-1 to suppress tumor growth. In TME, midostaurin diminished Treg cells and increased M1 macrophage. The expressions of STING and INFβ proteins were elevated in the tumor specimens. Our results suggest that midostaurin may have the potential to enhance immunotherapy in clinical practice.Immunotherapy modulating the tumor microenvironment (TME) immune function has a promising effect on various types of cancers, but it remains as a limited efficacy in colon cancer. Midostaurin (PKC412) has been used in the clinical treatment of fms-like tyrosine kinase 3 (FLT3)-mutant acute myeloid leukemia and has demonstrated immunomodulatory activity. We aimed to evaluate the effect of midostaurin on the modulation of TME and the efficacy of anti-programmed cell death protein 1 (PD-1) against colon cancer. Midostaurin inhibited the growth of murine CT26 and human HCT116 and SW480 cells with multinucleation and micronuclei formation in morphology examination. The cell cycle arrested in the G2/M phase and the formation of the polyploid phase was noted. The formation of cytosolic DNA, including double-strand and single-strand DNA, was increased. Midostaurin increased mRNA expressions of cGAS, IRF3, and IFNAR1 in colorectal adenocarcinoma cells and mouse spleen macrophages. The protein expressions of Trex-1, c-KIT, and Flt3, but not PKCα/β/γ and VEGFR1, were down-regulated in midostaurin-treated colorectal adenocarcinoma cells and macrophages. Trex-1 protein expression was abrogated after FLT3L activation. In vivo, the combination of midostaurin and anti-PD-1 exhibited the greatest growth inhibition on a CT26-implanted tumor without major toxicity. TME analysis demonstrated that midostaurin alone decreased Treg cells and increased neutrophils and inflammatory monocytes. NKG2D+ and PD-1 were suppressed and M1 macrophage was increased after combination therapy. When combined with anti-PD-1, STING and INFβ protein expression was elevated in the tumor. The oral administration of midostaurin may have the potential to enhance anti-PD-1 efficacy, accompanied by the modulation of cytosolic DNA-sensing signaling and tumor microenvironment.

The NALCN channel regulates metastasis and nonmalignant cell dissemination

We identify the sodium leak channel non-selective protein (NALCN) as a key regulator of cancer metastasis and nonmalignant cell dissemination. Among 10,022 human cancers, NALCN loss-of-function mutations were enriched in gastric and colorectal cancers. Deletion of Nalcn from gastric, intestinal or pancreatic adenocarcinomas in mice did not alter tumor incidence, but markedly increased the number of circulating tumor cells (CTCs) and metastases. Treatment of these mice with gadolinium—a NALCN channel blocker—similarly increased CTCs and metastases. Deletion of Nalcn from mice that lacked oncogenic mutations and never developed cancer caused shedding of epithelial cells into the blood at levels equivalent to those seen in tumor-bearing animals. These cells trafficked to distant organs to form normal structures including lung epithelium, and kidney glomeruli and tubules. Thus, NALCN regulates cell shedding from solid tissues independent of cancer, divorcing this process from tumorigenesis and unmasking a potential new target for antimetastatic therapies.

Reelin Protects against Colon Pathology via p53 and May Be a Biomarker for Colon Cancer Progression.

Simple SummaryColon cancer is a multifactorial disease involving genetic, environmental and lifestyle risk factors. Despite being one of the most common malignancies and a leading cause of cancer-related death worldwide, its underlying molecular mechanisms are scarcely known, highlighting the need to identify novel biomarkers for the clinical detection of its initiation and progression. The aim of the current study was to elucidate the role of the protein reelin in colon cancer initiation and progression using mouse models and human samples that extend from colitis or precancerous lesions to colon cancer. The results might contribute to understanding the mechanisms involved in colon cancer, and suggest that reelin may be a biomarker of colon pathology progression.Previous observations made in human and mouse colons suggest that reelin protects the colon from pathology. In this study, we evaluated reelin expression during the transition from either colitis or precancerous lesions to colon cancer and tried to elucidate reelin regulation under these transition processes. Samples of healthy and pathological colons from humans and mice treated with either azoxymethane/dextran sulfate sodium (DSS) or azoxymethane alone were used. The relative abundances of reelin, DNMT-1 and ApoER2 mRNAs were determined by PCR in the colon samples cited above and in the tissue adjacent to mouse colon polyps and adenocarcinomas. In both, humans and mice, reelin mRNA abundance increased significantly in ulcerative colitis and slightly in polyps and decreased in adenomas and adenocarcinomas. Reelin expression was higher in the tissue adjacent to the colon adenocarcinoma and lower in the lesion itself. The reelin expression changes may result, at least in part, from those in DNMT-1 and appear to be independent of ApoER2. Lack of reelin downregulated p-Akt and p53 in healthy colon and prevented their increases in the inflamed colon, whereas it increased GSK-3β in DSS-untreated mice. In conclusion, reelin mRNA abundance depends on the severity of the colon pathology, and its upregulation in response to initial injuries might prevent the beginning of colon cancer, whereas reelin repression favors it. Increased p53 expression and activation may be involved in this protection. We also propose that changes in colon reelin abundance could be used to predict colon pathology progression.

Self-healing pectin/cellulose hydrogel loaded with limonin as TMEM16A inhibitor for lung adenocarcinoma treatment

Lung cancer as one of the highest incident malignant tumors did not receive satisfactory chemotherapy due to lack of specific drug targets and targeted drugs. This study screened a new effective lung tumor inhibitor limonin from herbal medicine, which inhibited proliferation and promoted apoptosis of lung adenocarcinoma cells by targeting specific high expressed TMEM16A ion channel. Moreover, a novel biodegradable self-healing hydrogel was prepared from acylhydrazide functionalized carboxymethyl cellulose (CMC-AH) and oxidized pectin (pec-CHO) to reduce the side effects of the limonin to the body. The hydrogels showed fast gelation, good biocompatibility and sustained limonin release property. The limonin-loaded hydrogel significantly inhibited the growth of lung adenocarcinoma in xenografts mice because the limonin inhibited the proliferation, migration and promoted apoptosis of LA795 cells, and eliminated the acute toxicity through sustained release from the hydrogel. Combined the antitumor performance of the limonin and sustained release of pec-CHO/CMC-AH hydrogel, this limonin/hydrogel system achieved satisfactory antitumor effect and eliminated side effects in vivo. Therefore, this system has great potential application for enhanced lung adenocarcinoma therapy.

ID1 marks the tumorigenesis of pancreatic ductal adenocarcinoma in mouse and human

Pancreatic Ductal Adenocarcinoma (PDAC) is a deadly disease that has an increasing death rate but no effective treatment to now. Although biological and immunological hallmarks of PDAC have been frequently reported recently, early detection and the particularly aggressive biological features are the major challenges remaining unclear. In the current study, we retrieved multiple scRNA-seq datasets and illustrated the genetic programs of PDAC development in genetically modified mouse models. Notably, the transcription levels of Id1 were elevated specifically along with the PDAC development. Pseudotime trajectory analysis revealed that Id1 was closely correlated with the malignancy of PDAC. The gene expression patterns of human PDAC cells were determined by the comparative analysis of the scRNA-seq data on human PDAC and normal pancreas tissues. ID1 levels in human PDAC cancer cells were dramatically increased compared to normal epithelial cells. ID1 deficiency in vitro significantly blunt the invasive tumor-formation related phenotypes. IPA analysis on the differentially expressed genes suggested that EIF2 signaling was the core pathway regulating the development of PDAC. Blocking EFI2 signaling remarkably decreased the expression of ID1 and attenuated the tumor-formation related phenotypes. These observations confirmed that ID1 was regulated by EIF2 signaling and was the critical determinator of PDAC development and progression. This study suggests that ID1 is a potential malignant biomarker of PDAC in both mouse models and human and detecting and targeting ID1 may be a promising strategy to treat or even rescue PDAC.

Therapeutic potential of human serum albumin nanoparticles encapsulated actinonin in murine model of lung adenocarcinoma

Non-small cell lung cancer comprises 85% of the global lung cancer cases. Conventional chemotherapeutics possess certain limitations like systemic toxicity and drug resistance that requires the development of new therapeutic agents for successful treatment of lung cancer. Actinonin, a human peptide deformylase inhibitor, has demonstrated anti-cancerous properties in various leukemias and solid cancer types. However, it has limited therapeutic application because of its low bioavailability and systemic toxicity if administered in free form. This limitation can be overcome by using nano-delivery systems that will increase the therapeutic efficacy of actinonin. In the present study, human serum albumin actinonin nanoparticles were prepared using a desolvation technique and folic acid was conjugated to lysine residues of albumin for effective delivery to the lung. The lung adenocarcinoma model was established 24 weeks after intraperitoneal administration of urethane and chemotherapeutic efficacy of free as well as nanoencapsulated actinonin was evaluated. This study demonstrated anti-proliferative potential of folic acid conjugated human serum albumin nanoparticles encapsulating actinonin. The intraperitoneally administered nanoformulation exhibited sustain release profile of actinonin with longer half-life and mean retention time. The reduced dose frequency resulted in therapeutic efficacy comparable to free drug in vivo in terms of 100% survival and reduced tumor burden along with downregulation of epidermal growth factor receptor, folate receptor α and peptide deformylase expression in lung adenocarcinoma mice model. Therefore, actinonin encapsulated albumin nanoparticles-based therapy holds great potential as an alternative strategy to improve its anti-cancerous activity against lung adenocarcinoma.

Sitagliptin Potentiates the Anti-Neoplastic Activity of Doxorubicin in Experimentally-Induced Mammary Adenocarcinoma in Mice: Implication of Oxidative Stress, Inflammation, Angiogenesis, and Apoptosis

Sitagliptin (STG) is a highly selective dipeptidyl peptidase-4 inhibitor recently used in the treatment of type 2 diabetes. The current study aimed to investigate the anti-neoplastic effect of STG alone and in combination with Doxorubicin (Dox), a known chemotherapeutic agent but with ominous side effects. After intramuscular inoculation of 2 × 106 Ehrlich tumor cells, Female Swiss mice were divided into tumor-bearing control, STG-treated, Dox-treated, and a combination of STG and Dox-treated groups. The results showed a significant reduction in the tumor growth of the treated animals in comparison with those of the positive control group with a more prominent effect in the co-treated group. Where, the anti-proliferative and apoptotic effect of STG, and its chemo-sensitizing ability, when used in combination with Dox, was mediated by modulation of oxidative stress (MDA and GSH), attenuation of tumor inflammation (IL-6 and IL-1β), and angiogenesis (VEGF), suppressing proliferation (β-catenin and cyclin-D1) and enhancement of apoptosis (survivin, p53, caspase 3). Thus, in conclusion, STG as adjunctive therapy for Dox could be a strategy for the treatment of breast cancer patients, by their ability in hindering cell proliferation and minimizing the associated oxidative and inflammatory adverse reactions.

Development of a traditional Chinese medicine-based agent for the treatment of cancer cachexia.

BackgroundDespite recent advances in understanding the pathophysiology of cancer cachexia, prevention/treatment of this debilitating disease remains an unmet medical need.MethodsWe developed an integrated, multi‐tiered strategy involving both in vitro and in vivo muscle atrophy platforms to identify traditional Chinese medicine (TCM)‐based anti‐cachectic agents. In the initial screening, we used inflammatory cytokine‐induced atrophy of C2C12 myotubes as a phenotypic screening platform to assess the protective effects of TCMs. The selected TCMs were then evaluated for their abilities to protect Caenorhabditis elegans from age‐related reduction of mobility and contractility, followed by the C‐26 colon adenocarcinoma mouse model of cachexia to confirm the anti‐muscle atrophy effects (body/skeletal muscle weights, fibre size distribution, grip strengths, and serum IL‐6). Transcriptome analysis, quantitative real‐time polymerase chain reaction, and immunoblotting were performed to gain understanding of the potential mechanism(s) by which effective TCM protected against C26 tumour‐induced muscle atrophy.ResultsOf 29 widely used TCMs, Dioscorea radix (DR) and Mu Dan Pi (MDP) showed a complete protection (all P values, 0.0002) vis‐à‐vis C26 conditioned medium control in the myotube atrophy platform. MDP exhibited a unique ability to ameliorate age‐associated decreases in worm mobility, accompanied by improved total body contractions, relative to control (P < 0.0001 and <0.01, respectively), which, however, was not noted with DR. This differential in vivo protective effect between MDP and DR was also confirmed in the C‐26 mouse model. MDP at 1000 mg/kg (MDP‐H) was effective in protecting body weight loss (P < 0.05) in C‐26 tumour‐bearing mice without changing food or water intake, accompanied by the restoration of the fibre size distribution of hindleg skeletal muscles (P < 0.0001) and the forelimb grip strength (P < 0.05). MDP‐treated C‐26‐tumour‐bearing mice were alert, showed normal posture and better body conditions, and exhibited lower serum IL‐6 levels (P = 0.06) relative to vehicle control. This decreased serum IL‐6 was associated with the in vitro suppressive effect of MDP (25 and 50 μg/mL) on IL‐6 secretion into culture medium by C26 cells. RNA‐seq analysis, followed by quantitative real‐time polymerase chain reaction and/or immunoblotting, shows that MDP's anti‐cachectic effect was attributable to its ability to reverse the C‐26 tumour‐induced re‐programming of muscle homoeostasis‐associated gene expression, including that of two cachexia drivers (MuRF1 and Atrogin‐1), in skeletal muscles.ConclusionsAll these findings suggest the translational potential of MDP to foster new strategies for the prevention and/or treatment of cachexia. The protective effect of MDP on other types of muscle atrophy such as sarcopenia might warrant investigations.

Abstract 1128: MTORC1/2 and HDAC1/2 inhibition promote tumor response through inhibition of MYC

Abstract Background: The identification of treatment strategies targeting PIK3CA mutant colorectal cancer (CRC) are of great clinical interest. Previous work from our lab has identified MTORC1/2 and HDAC1/2 inhibition with copanlisib (cop; PI3K/MTOR) and romidepsin (romi; HDAC1/2) as a potential therapeutic strategy. We hypothesized that changes in c-MYC protein levels and c-MYC target gene (CTG) alterations might be a potential mechanism of action for this combination. Methods: Known CTGs were identified and their expression levels were examined in PIK3CA mutant vs WT CRCs using the cBioPortal Colorectal Adenocarcinoma (TCGA, PanCancer Atlas) dataset. Murine derived cancer organoids (MDCO) were generated from adenocarcinomas of Apc and Pik3ca mutant transgenic mice (F1 (FVB x B6) Apcfl/+ Pik3caH1047R/+). MDCO results were corroborated using the human 2D isogenic cell lines SW48 and SW48PIK3CA-H1047R (SW48PK) and RAS/RAF WT patient derived cancer organoids (PDCO) generated from CRC patient samples under approved IRB protocols. Immunoblots (IB) were used to assess c-MYC levels after treatment with cop, romi, and the combination across all models. RNA sequencing was conducted on PDCOs and SW48PK cells and changes in 16 CTGs were examined. An aggregate score was created for each treatment group where a statistically significantly altered CTG with log fold change ≥1.5 added one point and ≤-1.5 subtracted one point. All others were scored 0. Results: c-MYC target genes were assessed for differential expression in PIK3CA mutant CRC vs PIK3CA WT CRC with only 1/16 genes decreased in PIK3CA mutant CRC (GADD45A: log ratio -0.21, q=0.01). Next, MDCOs treated with the combination showed a decrease in total c-MYC levels in the combination therapy. These results were corroborated in two human 2D CRC cell lines, SW48 and SW48PK and a panel of PDCOs. Interestingly, c-MYC levels decreased in both romi alone and the combination in both the SW48 and SW48PK cell lines after 24 hours of treatment. However, the extent to which c-MYC levels were decreased was not as substantial in the panel of PDCOs. Across all in vitro models a decrease in PI3K signaling, as illustrated by decreased pRPS6 and p4EBP1 in response to cop treatment and an increase in H3K27 acetylation in response to romi, was observed in the single agent and combination therapies. RNA sequencing demonstrated that cop, romi, and combo had a CTG score of 0, -4, and -7 respectively in the PIK3CA mutant PDCO. Similar results were seen in an additional RAS/RAF WT PDCO (0, 1, and -4, respectively) and SW48PK cells (0, -3, and -7, respectively). Conclusion: A potential mechanism by which cop and romi treatment promote tumor response is through a decrease in c-MYC protein levels and expression of downstream c-MYC target genes. This regimen deserves further mechanistic investigations in vivo. Citation Format: Rebecca A. DeStefanis, Autumn M. Olson, Alyssa K. DeZeeuw, Susan N. Payne, Cheri A. Pasch, Linda Clipson, Dustin A. Deming. MTORC1/2 and HDAC1/2 inhibition promote tumor response through inhibition of MYC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1128.

Abstract 3711: Modulation and inhibition of prostate cancer cells by microbial metabolites and resveratrol

Abstract The chemotherapeutic compounds resveratrol (RES) and short-chain fatty acids (SCFAs) are potent inhibitors of cancer cell proliferation during in vitro testing. These compounds are naturally occurring and present relatively low toxicity to normal cells and tissues, making them an attractive cancer treatment alternative. SCFAs are the chemical by-products of microbial activity known as fermentation. RES is a polyphenolic compound found in select fruits and vegetables and has been known for its versatility in preventing and reversing many diseases, including several cancers. Butyric and propionic acid are considered the most potent of the SCFAs found within the intestinal lumen. The prime goal of this research is to understand if the RES and SCFA compounds use a shared biological pathway(s) to fight and inhibit cancer. We used TRAMP C1 and C3 prostate cancer cells (transgenic adenocarcinoma mouse prostate) as an experimental model. These cells were treated with the RES and SCFAs both singularly and synergistically for 24hrs. The cells were analyzed to understand the potential cell toxicity and proliferation, the modulation of apoptotic and proapoptotic proteins/genes, and any change in mitochondrial membrane potential pathways. The result demonstrated that the expression of apoptogenic proteins induces programmed cell death in a mitochondria-independent manner. Therefore, the result suggests that RES and SCFAs can facilitate many critical biological functions within the host's body, including cellular respiration, nutrient assimilation, and various immunological responses to inhibit cancer cell proliferation. Citation Format: James Stokes, Rajesh Singh, Manoj K. Mishra. Modulation and inhibition of prostate cancer cells by microbial metabolites and resveratrol [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3711.

Abstract 210: Development and characterization of a novel immunocompetent mouse model of radioresistant lung cancer

Abstract Standard treatment for inoperable or unresectable stage I-III NSCLC includes stereotactic ablative radiotherapy (SABR) and concurrent chemo and radiotherapy, but locoregional recurrence occurs in 10%-30% of the patients. In the absence of distant metastasis, re-irradiation is the main curative-intent treatment option available for these patients, but the risk of severe adverse effects and tumor resistance of heavily irradiated patients are challenges to this therapy. On the other hand, the combination between re-irradiation with chemotherapy or immunotherapy is a scenario not extensively studied in the literature. Current existing models have been developed in immunodeficient mice, where the effect of immunotherapy cannot be assessed. To address this issue, we have developed and characterized a lung cancer model of radiation resistance, which can be used in syngeneic mice with fully functional immune system. To this aim, we have generated a mouse adenocarcinoma radioresistant cell line (Lacun3 RR) from previously characterized Lacun3 adenocarcinoma cells. This radioresistant cell line was generated after several rounds of radiation (8 Gy) administered to the parental Lacun3 cells in vitro. Clonogenic and apoptotic assays confirmed that Lacun3 RR were more resistant to radiotherapy than the parental cell line, within the 2-4 Gy range. In addition, in vivo assays performed by subcutaneous injection of these cells showed that Lacun3 RR were completely refractory to 10 Gy irradiation, whereas a significant reduction in tumor volume (~60%) was found for parental Lacun3-derived tumors. Phenotypic characterization of these cells showed that Lacun3 RR cells had increased properties of cancer stem cells (CSC), with significantly higher ability to form tumorspheres. Lacun3 RR cells also showed increased CSC markers (ALDH1, NOTCH1) and slightly lower proliferation rates in vitro than Lacun3 cells. Cell signaling analysis revealed an increase in phosphorylated (p)p38 MAPK and pAKT basal and post-irradiation levels, and lower pH2AX (suggesting less DNA damage) in Lacun3 RR cells. RNAseq and DNAseq (Whole Exome Sequencing) were performed in both cell lines. As expected, we found that a substantial number of genes differentially expressed were significantly related to DNA repair, cell cycle regulation and spindle formation. Mutational burden was estimated from the DNAseq, revealing a similar global tumor mutational burden for both cell lines. However, some mutations were exclusively found in Lacun3 RR cells. The tumor microenvironment and tumor immune populations are currently being investigated in this model. In conclusion, we have developed a mouse model to study radiation resistance in a context of complete immunity, which could be highly valuable to study possible synergistic effects of radiotherapy and immunotherapy of re-irradiated NSCLC patients. Citation Format: Sergio León, Diego Serrano, Esther Redín, Jennifer Barranco, Maeva Houry, Francisco Expósito, Ibón Tamayo, Luis Montuenga, Alfonso Calvo, Jose Javier Aristu. Development and characterization of a novel immunocompetent mouse model of radioresistant lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 210.