Reduced Lung Metastasis Research Articles

PDLIM2 Suppression Inhibit Proliferation and Metastasis in Kidney Cancer.

Simple SummaryKidney cancer is a common malignant tumor in both men and women and accounts for approximately 5% of all cancer incidences. Advances in imaging technology and the increasing use of health care facilities have led to the early detection of kidney cancer cases. However, many individuals are still diagnosed with metastatic kidney cancer. If the cancer is accompanied by metastases at the time of diagnosis, the 5-year survival rate is 12%. Despite the beneficial effects of anticancer drug treatment on the survival of patients with metastatic kidney cancer, survival may be less than a year. PDLIM2 plays an essential role in cancer formation and inhibition. To verify oncogenic function of the PDLIM2, we conducted several experiments and animal experiments. Our findings indicating that PDLIM2 may be a new therapeutic target for metastatic kidney cancer.We evaluated the expression of PDLIM2 in human kidney cancer cell lines from primary or metastatic origins and found that PDLIM2 expression was highly elevated in metastatic kidney cancers. We evaluated the effect of PDLIM2 inhibition by RNA interference method. PDLIM2 knockdown showed the decreased proliferation and metastatic character in human metastatic kidney cancer cells. By repeated round of orthotopic injection of RenCa mouse kidney cancer cell line, we obtained metastatic prone mouse kidney cancer cell lines. PDLIM2 expression was highly expressed in these metastatic prone cells comparing parental cells. In addition, we evaluated the in vivo efficacy of PDLIM2 knockout on the tumor formation and metastasis of kidney cancer cells using a PDLIM2 knockout mice. The experimental metastasis model with tail vein injection and orthotopic metastasis model injected into kidney all showed reduced lung metastasis cancer formation in PDLIM2 knockout mice comparing control Balb/c mice. Overall, our findings indicate that PDLIM2 is required for cancer formation and metastasis in metastatic kidney cancer, indicating that PDLIM2 may be a new therapeutic target for metastatic kidney cancer.

Long non-coding RNA LINC01559 serves as a competing endogenous RNA accelerating triple-negative breast cancer progression

BackgroundLong non-coding RNA (lncRNA) is an endogenous RNA over 200 nt in length involved in gene regulation. LINC01559 is a novel lncRNA that has been identified as a fundamental player in human cancer. However, its role in triple-negative breast cancer (TNBC) remains unknown. Here, we explored the expression, function and clinical implication of LINC01559 in TNBC. MethodsRNA expression was detected by qRT-PCR analysis. Cell Counting Kit-8 (CCK-8), 5-Ethynyl-2′-deoxyuridine (EdU), wound healing and Transwell assays were used to test cell viability, DNA synthesis rate, migration and invasion, respectively. The competing endogenous RNA (ceRNA) axis involved in LINC01559 was determined by RNA pull-down and luciferase reporter assays. The xenograft model was used to verify the function of LINC01559 in vivo. ResultsLINC01559 was significantly increased in TNBC tissues as compared to matched normal tissues, which was due to high levels of H3K4Me3 and H3K27Ac in the promoter region. Knockdown of LINC01559 inhibited TNBC cell proliferation, migration and invasion in vitro, and also retarded tumor growth and reduced lung metastasis in vivo. Mechanistically, LINC01559 served as a ceRNA that sponged miR-370-3p, miR-485-5p and miR-940, resulting in increasing the expression of a cohort of oncogenes, thus accelerating TNBC progression. ConclusionsOur data provide a comprehensive analysis of LINC01559 in TNBC, we found that LINC01559 functioned as a carcinogenic ceRNA via sponging miRNAs. Targeting of LINC01559 may be a potential treatment for TNBC patients.

The receptor for advanced glycation endproducts (RAGE) decreases survival of tumor-bearing mice by enhancing the generation of lung metastasis-associated myeloid-derived suppressor cells

Metastatic cancer has a poor prognosis. Novel pharmacologic targets need to be identified. The receptor for advanced glycation endproducts (RAGE) is a pattern recognition receptor constitutively expressed in the lungs. Absence of overt disease in RAGE null mice suggests that RAGE is unnecessary or redundant in health. We report that RAGE null tumor-bearing mice have reduced lung metastasis and improved survival. Bone marrow chimera studies suggest that hematopoietic cell RAGE is an important contributor to these effects. Deletion of RAGE reduces both the quantity and suppressive activity of tumor-induced MDSC. Protein and mRNA studies suggest that RAGE contributes to the generation and function of MDSC including expression of the alarmins S100A8/A9 and activity of inducible nitric oxide synthase, arginase-1, and NF-κB. These findings demonstrate the important role of RAGE in determining the quantity and function of tumor-associated MDSC and suggest RAGE as a pharmacologic target for patients with metastatic disease.

Abstract LT021: Targeting AXL favors an anti-tumorigenic tumor microenvironment that enhances immunotherapy responses by decreasing HIF-1a levels in cancer cells

Abstract Breast cancer-related deaths are predominantly associated to complications arising from metastases. Hypoxia is an important biological process that fuels metastasis and contributes to deregulate the tumor microenvironment (TME) of solid tumors. Hypoxia also promotes resistance to therapies by a plethora of mechanisms including enhancing tumoral angiogenesis, metabolic reprogramming, epithelial-to-mesenchymal transition (EMT) and immune evasion. The expression of the receptor tyrosine kinase AXL, a hypoxia-induced gene, is associated with poor clinical prognosis and metastasis in various cancers, including breast cancer. Here, we hypothesize that genetic or pharmacological interference of AXL may alter hypoxic responses and associated modulation of the TME offering an ideal setting for immunotherapy. In a HER2 mouse model of breast cancer (MMTV-NIC), we found that the mammary gland epithelial cells specific deletion of AXL led to a reduction of lung metastases and improved the TME by reducing the hypoxic response in tumor cells. When breast cancer cells were subjected to hypoxic conditions, we found that AXL inhibition reduced the levels of the hypoxia transcription factor HIF-1a which resulted in an altered hypoxic response. Specifically, AXL deletion led to a reduction of hypoxia-induced EMT and invasion, and a reduction of the secretion of key cytokines for macrophages recruitment and polarization. We demonstrate in vivo that AXL pharmacological inhibition in the MMTV-NIC breast cancer model generated an anti-inflammatory TME that enhanced an anti-PD-1 immune checkpoint blockade response and decreased the metastatic burden. Collectively, these results suggest that targeting AXL could be a powerful approach to improve immunotherapy response by generating an anti-tumoral microenvironment therefore limiting the metastatic burden of breast cancer. Citation Format: Marie-Anne Goyette, Jean-Philippe Gratton, Jean-François Côté. Targeting AXL favors an anti-tumorigenic tumor microenvironment that enhances immunotherapy responses by decreasing HIF-1a levels in cancer cells [abstract]. In: Proceedings of the AACR Virtual Special Conference on the Evolving Tumor Microenvironment in Cancer Progression: Mechanisms and Emerging Therapeutic Opportunities; in association with the Tumor Microenvironment (TME) Working Group; 2021 Jan 11-12. Philadelphia (PA): AACR; Cancer Res 2021;81(5 Suppl):Abstract nr LT021.

Abstract LT010: Pleiotrophin drives pro-metastatic immune niche within breast tumor microenvironment

Abstract Background: Pleiotrophin (PTN), a heparin binding cytokine important during embryonic development, is expressed at elevated levels in several cancers, including breast cancer. High PTN expression in serum and tumor tissues of breast cancer patients correlates with worse outcome. Previous studies indicate PTN contributes to angiogenesis, hematopoiesis, and inflammation; however, the function of PTN in breast cancer progression and metastasis has not been elucidated. Methods: The effect of anti-PTN therapy (mAb; 3B10) on breast tumor progression and pulmonary metastasis was evaluated in four preclinical models of breast cancer: 4T1, E0771, Met-1, MMTV-PyMT. In addition, the effect of global loss of PTN using Ptn-deficient mice was evaluated in two murine models of breast cancer. Tissues from these experiments were analyzed by RNAseq and histology for changes in angiogenesis, hematopoiesis, immune cell infiltration, and cytokine/chemokine expression. Findings: TCGA data shows high PTN expression correlates with poor survival of Stage IV breast cancer patients. Furthermore, our data indicate that: a) blocking PTN pharmacologically or genetically results in a significant reduction of lung metastasis in multiple preclinical mouse models of breast cancer, b) scRNAseq and dual FISH-IHC (fluorescence in-situ hybridization and immunohistochemistry) demonstrates that endothelial cells and select population of tumor cells express PTN in the tumor microenvironment (TME), c) inhibition of PTN results in a decrease in overall inflammation with a particular decrease in neutrophil populations at primary and secondary tumor sites, d) in a genetically engineered mouse model (GEMM) of breast cancer, MMTV-PyMT, PTN expression is enriched at the metastatic site. Conclusion: Our data suggests that PTN is important in driving a pro-metastatic immune niche within the TME that promotes tumor cell escape from the primary tumor and survival of metastatic cancer cells in secondary sites. Additionally, our studies highlight that inhibition of PTN has potential to reduce metastatic burden in breast cancer and suggest that future studies testing the combination of PTN inhibition with standard chemotherapy or immune therapy are warranted. Citation Format: Debolina Ganguly, Noah Sorrelle, Adrian Dominguez, Jason Toombs, Marcel Schmidt, Fa Valdes Mora, David Gallego Ortega, Anton Wellstein, Rolf A. Brekken. Pleiotrophin drives pro-metastatic immune niche within breast tumor microenvironment [abstract]. In: Proceedings of the AACR Virtual Special Conference on the Evolving Tumor Microenvironment in Cancer Progression: Mechanisms and Emerging Therapeutic Opportunities; in association with the Tumor Microenvironment (TME) Working Group; 2021 Jan 11-12. Philadelphia (PA): AACR; Cancer Res 2021;81(5 Suppl):Abstract nr LT010.

Identification of molecular anti-metastasis mechanisms of lycorine in colorectal cancer by RNA-seq analysis

BackgroundColorectal cancer (CRC) is one of the most common malignancies worldwide. Metastasis is the major cause of death in patients with CRC. Lycorine, the phenanthridine alkaloid most commonly found in spp of the Amaryllidaceae family, has shown promising anticancer activities with minor side effects. However, the effects and the detailed mechanism of lycorine against metastasis of CRC remains unclear. Study design/methodsThe purpose of this study was to investigate the effects of lycorine on CRC and characterize the molecular mechanisms observed in lycorine-treated CRC cells using RNA-sequencing. MTT assay, colony formation assay, acridine orange/ethidium bromide (AO/EB) staining and Annexin V-FITC/Propidium iodide (PI) staining were conducted to examine the effects of lycorine on cell proliferation and apoptosis in CRC cells. RNA sequencing, real-time PCR assays and western blot were performed. Migration and invasion abilities of lycorine-treated CRC cells were investigated by wound healing and transwell invasion assays. The mouse CRC lung metastasis model was established and was used to detect the effect of lycorine on CRC in vivo. ResultsOur results demonstrated that lycorine inhibited the proliferation and colony formation of CRC cells in a concentration-dependent manner. AO/EB staining and Annexin V-FITC/PI staining showed that lycorine induced apoptosis in a concentration-dependent manner. Lycorine also reduced lung metastasis of CRC in vivo. Moreover, transcriptomic analysis suggested that lycorine regulated the expression of 3556 genes. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway was implicated according to the differentially expressed genes (DEGs), and multiple pathways including those of mitogen-activated protein kinase (MAPK), relaxin, Ras, phosphatidylinositol 3‑kinase (PI3K)-protein kinase B (Akt) and Wnt/β-catenin were selected by functional enrichment analyses. Furthermore, based on transcriptomic analysis, we found that the tumor necrosis factor (TNF) pathway and endoplasmic reticulum stress were responsible for lycorine-induced apoptosis. ConclusionsThese results obtained in this study demonstrated that lycorine has the potential to suppress CRC in vitro and in vivo through the lycorine-regulated multiple signaling pathways.

Leronlimab, a humanized monoclonal antibody to CCR5, blocks breast cancer cellular metastasis and enhances cell death induced by DNA damaging chemotherapy

BackgroundTriple-negative breast cancer (BCa) (TNBC) is a deadly form of human BCa with limited treatment options and poor prognosis. In our prior analysis of over 2200 breast cancer samples, the G protein-coupled receptor CCR5 was expressed in > 95% of TNBC samples. A humanized monoclonal antibody to CCR5 (leronlimab), used in the treatment of HIV-infected patients, has shown minimal side effects in large patient populations.MethodsA humanized monoclonal antibody to CCR5, leronlimab, was used for the first time in tissue culture and in mice to determine binding characteristics to human breast cancer cells, intracellular signaling, and impact on (i) metastasis prevention and (ii) impact on established metastasis.ResultsHerein, leronlimab was shown to bind CCR5 in multiple breast cancer cell lines. Binding of leronlimab to CCR5 reduced ligand-induced Ca+ 2 signaling, invasion of TNBC into Matrigel, and transwell migration. Leronlimab enhanced the BCa cell killing of the BCa chemotherapy reagent, doxorubicin. In xenografts conducted with Nu/Nu mice, leronlimab reduced lung metastasis of the TNBC cell line, MB-MDA-231, by > 98% at 6 weeks. Treatment with leronlimab reduced the metastatic tumor burden of established TNBC lung metastasis.ConclusionsThe safety profile of leronlimab, together with strong preclinical evidence to both prevent and reduce established breast cancer metastasis herein, suggests studies of clinical efficacy may be warranted.

Nicotine promotes breast cancer metastasis by stimulating N2 neutrophils and generating pre-metastatic niche in lung

Smoking has a profound impact on tumor immunity, and nicotine, which is the major addictive component of smoke, is known to promote tumor progression despite being a non-carcinogen. In this study, we demonstrate that chronic exposure of nicotine plays a critical role in the formation of pre-metastatic niche within the lungs by recruiting pro-tumor N2-neutrophils. This pre-metastatic niche promotes the release of STAT3-activated lipocalin 2 (LCN2), a secretory glycoprotein from the N2-neutrophils, and induces mesenchymal-epithelial transition of tumor cells thereby facilitating colonization and metastatic outgrowth. Elevated levels of serum and urine LCN2 is elevated in early-stage breast cancer patients and cancer-free females with smoking history, suggesting that LCN2 serve as a promising prognostic biomarker for predicting increased risk of metastatic disease in female smoker(s). Moreover, natural compound, salidroside effectively abrogates nicotine-induced neutrophil polarization and consequently reduced lung metastasis of hormone receptor-negative breast cancer cells. Our findings suggest a pro-metastatic role of nicotine-induced N2-neutrophils for cancer cell colonization in the lungs and illuminate the therapeutic use of salidroside to enhance the anti-tumor activity of neutrophils in breast cancer patients.

EGFR inhibition blocks cancer stem cell clustering and lung metastasis of triple negative breast cancer.

Triple-negative breast cancer (TNBC) is one of the most aggressive and metastatic breast cancer subtypes lacking targeted therapy. Our recent work demonstrated that circulating tumor cell (CTC) clusters and polyclonal metastasis of TNBC are driven by aggregation of CD44+ cancer stem cells (CSC) and associated with an unfavorable prognosis, such as low overall survival. However, there is no existing therapeutic that can specifically block CTC or CSC cluster formation.Methods: Using patient-derived xenograft (PDX) models, we established an ex vivo tumor cell clustering assay for a pilot screening of blockade antibodies. After identifying EGFR as a target candidate, we modulated the gene expression and inhibited its kinase activity to determine its functional importance in tumor cell clustering and therapeutic inhibition of lung metastasis. We also examined the molecular regulation network of EGFR and a potential connection to CSC marker CD44 and microRNAs, which regulate CTC clustering.Results: We report here that EGFR inhibition successfully blocks circulating CSC (cCSC) clustering and lung metastasis of TNBC. EGFR enhances CD44-mediated tumor cell aggregation and CD44 stabilizes EGFR. Importantly, blocking EGFR by a novel anti-EGFR monoclonal antibody (clone LA1) effectively blocked cell aggregation in vitro and reduced lung metastasis in vivo. Furthermore, our data demonstrated that the tumor suppressor microRNA-30c serves as another negative regulator of cCSC clustering and lung metastasis by targeting CD44 as well as its downstream effector EGFR.Conclusion: Our studies identify a novel anti-EGFR therapeutic strategy to inhibit cCSC aggregation and therefore abolish cCSC cluster-mediated metastasis of TNBC.

Gossypol Reduces Metastasis and Epithelial-Mesenchymal Transition by Targeting Protease in Human Cervical Cancer.

Metastasis is the most prevalent cause of cancer-associated deaths amongst patients with cervical cancer. Epithelial-mesenchymal transition (EMT) is essential for carcinogenesis, and it confers metastatic properties to cancer cells. Gossypol is a natural polyphenolic compound with anti-inflammation, anti-oxidant, and anticancer activities. In this study, we investigated the antimetastatic and antitumour effects of gossypol on human cervical cancer cells (HeLa and SiHa cells). Gossypol exerted a strong inhibition effect on the migration and invasion of human cervical cancer cells. It reduced the focal adhesion kinase (FAK) pathway-mediated expression of matrix metalloproteinase-2 and urokinase-type plasminogen activator, subsequently inhibiting the invasion of SiHa cells. In addition, gossypol reversed EMT induced by transforming growth factor beta 1 (TGF-[Formula: see text]1) and up-regulated epithelial markers, such as E-cadherin but significantly suppressed Ras homolog family member (Rho)A, RhoB, and p-Samd3. The tail vein injection model showed that gossypol treatment via oral gavage reduced lung metastasis. Gossypol also decreased tumour growth in vivo in the nude mouse xenograft model. All these findings suggest that gossypol suppressed the invasion and migration of human cervical cancer cells by targeting the FAK signaling pathway and reversing TGF-[Formula: see text]1-induced EMT. Hence, gossypol warrants further attention for basic mechanistic studies and drug development.

Controlling cancer-induced inflammation with a nucleic acid scavenger prevents lung metastasis in murine models of breast cancer

Tumor cells release nucleic acid-containing proinflammatory complexes, termed nucleic acid-containing damage-associated molecular patterns (NA DAMPs), passively upon death and actively during stress. NA DAMPs activate pattern recognition receptors on cells in the tumor microenvironment leading to prolonged and intensified inflammation that potentiates metastasis. No strategy exists to control endogenous or therapy-induced inflammation in cancer patients. We discovered that the generation 3.0 polyamidoamine dendrimer (PAMAM-G3) scavenges NA DAMPs and mitigates their proinflammatory effects. In this study, we tested if the nucleic acid scavenger (NAS) PAMAM-G3 reduces lung metastasis in murine models of breast cancer. Our data indicate that PAMAM-G3 treatment decreases cell-free DNA levels and reduces lung metastasis in the experimental intravenous tumor-injection model and the postsurgical tumor-resection model of 4T1 breast cancer. Reduction in lung metastasis is associated with reduction in inflammatory immune cell subsets and proinflammatory cytokine levels in the tumor and the periphery. This study is the first example of NAS-mediated inhibition of metastasis to the lung. The study results provide a strong rationale for inclusion of NAS therapy in women with breast cancer undergoing standard-of-care surgery.

S100A10 Has a Critical Regulatory Function in Mammary Tumor Growth and Metastasis: Insights Using MMTV-PyMT Oncomice and Clinical Patient Sample Analysis.

Simple SummaryThe key challenges that face patients during breast cancer therapy is the metastatic spread and aggressiveness of the disease. Thus, the goal of current breast cancer research is to discover new therapeutic and diagnostic targets that limit the aggressive spread of the cancer. In this study, we investigated the role of protein S100A10 (p11) in breast tumor growth, progression, and metastasis using mouse cancer models and patient tumor sample analysis. We have demonstrated in our previous studies that p11 is critical for the function of a proteolytic enzyme–plasmin, which aids in the digestion of the tissues surrounding the tumor and allows the escape of the cancer cells from the breast tissue to organs such as the lungs and bone. Here, we present evidence that genetic deletion of p11 results in smaller and less aggressive mammary tumors in mice. We also observed that the cancer spread to the lungs is dramatically reduced in the absence of p11 gene in mice. Subsequent analysis of breast cancer patient tissues showed a correlation between higher p11 expression and both poor survival and aggressive cancer.S100A10 (p11) is a plasminogen receptor that regulates cellular plasmin generation by cancer cells. In the current study, we used the MMTV-PyMT mouse breast cancer model, patient tumor microarray, and immunohistochemical (IHC) analysis to investigate the role of p11 in oncogenesis. The genetic deletion of p11 resulted in significantly decreased tumor onset, growth rate, and spontaneous pulmonary metastatic burden in the PyMT/p11-KO (knock-out) mice. This phenotype was accompanied by substantial reduction in Ki67 positivity, macrophage infiltration, decreased vascular density in the primary tumors, and decrease in invasive carcinoma and pulmonary metastasis. Surprisingly, IHC analysis of wild-type MMTV-PyMT mice failed to detect p11 expression in the tumors or metastatic tumor cells and loss of p11 did not decrease plasmin generation in the PyMT tumors and cells. Furthermore, tumor cells expressing p11 displayed dramatically reduced lung metastasis when injected into p11-depleted mice, further strengthening the stromal role of p11 in tumor growth and metastasis. Transcriptome analysis of the PyMT tumors from p11-KO mice showed marked reduction in genes such as Areg, Muc1, and S100a8 involved in breast cancer development, progression, and inflammation. The PyMT/p11-KO tumors displayed a remarkable increase in inflammatory cytokines such as interleukin (Il)-6, Il-10, and interferon (Ifn)-γ. Gene expression profiling and IHC of primary breast cancer samples showed that p11 mRNA and protein levels were significantly higher in tumor tissues compared to normal mammary tissue. P11 mRNA expression was significantly associated with poor patient prognosis and significantly elevated in high grade, triple negative (TN) tumors, and tumors with high proliferative index. This is the first study examining the crucial role of p11 in breast tumor development and metastasis, thus emphasizing its potential as a diagnostic and prognostic biomarker in breast cancer.

Anacardic 6-pentadecyl salicylic acid induces apoptosis in breast cancer tumor cells, immunostimulation in the host and decreases blood toxic effects of taxol in an animal model

Many antineoplastic agents induce myelosuppression and leukopenia as secondary effects in patients. The development of anticancer agents that simultaneously provoke antitumor immune response represents an important therapeutic advance. The administration of 6-pentadecyl salicylic acid (6SA) contributes to the antitumor immunity using 4T1 breast cancer cells in Balb/c female mice, with Taxol as a positive control and in cotreatment with 6SA (6SA + Taxol; CoT).Our results show that 6SA reduces tumor volume and size by inducing caspase-8-mediated apoptosis without reducing tumor infiltrated lymphocytes. Also, 6SA reduced lung metastasis and increased the proportion of immune cells in blood, lymph nodes and bone marrow; more evidently, in the proportion of tumor-infiltrated natural killer (NK) cells and cytotoxic T lymphocytes. Taxol reduces helper and cytotoxic lymphocytes causing systemic immunosuppression and myelosuppression in bone marrow, whereas 6SA does not decrease any immune cell subpopulations in circulating blood and lymph nodes. More importantly, the CoT decreased the Taxol-induced cytotoxicity in circulating T cells and bone marrow. Treatment with 6SA increases the secretion of IL-2, IL-12, GM-CSF, TNF-α and IFN-γ and significantly reduces IL-10 and IL-17 secretion, suggesting that the reduction of regulatory T cells and tumor-associated macrophages contribute to the host control of tumor development.Finally, 6SA has an effective antineoplastic activity against breast cancer cells in an immunocompetent animal, reduces the myelosuppression and leukopenia that Taxol produces, improves the antitumoral immunological microenvironment and increases the overall survival of the animals improving the quality of life of patients with cancer.

Epigenetic Inactivation of α-Internexin Accelerates Microtubule Polymerization in Colorectal Cancer.

DNA methylation contributes to malignant transformation, but little is known about how the methylation drives colorectal cancer evolution at the early stages. Here we identify aberrant INA (α-internexin) gene methylation in colon adenoma and adenocarcinoma by filtering data obtained from a genome-wide screen of methylated genes. The gene encoding INA, a type IV intermediate filament, was frequently hypermethylated in CpG islands located in the promoter region. This hypermethylation preferentially occurred in large tumors and was a prognostic marker for poor overall survival in patients with colorectal cancer. This type of epigenetic alteration silenced INA expression in both adenoma and adenocarcinoma tissues. Gene silencing of INA in colorectal cancer cells increased cell proliferation, migration, and invasion. Restored INA expression blocked migration and invasion in vitro and reduced lung metastasis in vivo. Mechanistically, INA directly inhibited microtubule polymerization in vitro and decreased intracellular microtubule plus-end assembly rates. A peptide array screen surveying the tubulin-binding sites in INA identified a tubulin-binding motif located in the N-terminal head domain that plays a tumor-suppressive role by binding to unpolymerized tubulins and impeding microtubule polymerization. Thus, epigenetic inactivation of INA is an intermediate filament reorganization event that is essential to accelerate microtubule polymerization in the early stages of colorectal cancer. SIGNIFICANCE: This work provides insight into the epigenetic inactivation of INA, a novel identified tumor suppressor, which increases microtubule polymerization during colorectal cancer progression.

Abstract PO-056: Role of the metabolite N-acetylaspartate in breast cancer progression

Abstract Breast cancer (BCa) is a disease that affects over a quarter million women in the US every year. Our laboratory studies reprogrammed energy metabolism in BCa, which is an essential hallmark of the disease. Specifically, we focus on the metabolic rewiring events that occur in BCa, intending to identify cancer-associated metabolic vulnerabilities to treat this aggressive disease. Using a global metabolic profiling approach, we have determined that the metabolite N-acetylaspartate (NAA) is highly accumulated in patients with BCa. Prior studies involving NAA in other cancer types have attributed its accumulation to the upregulation of the enzyme N-acetyl transferase (NAT8L), which synthesizes NAA. However, using matched gene expression data, we found that the accumulation of NAA in these BCa tumors rather coincides with the downregulation of aspartoacylase (ASPA), the enzyme that breaks down NAA. To determine the role of NAA in BCa progression, we generated multiple cell line models containing either NAT8L knockdown, ASPA overexpression, or a combination of the two in the cell line MDA-MB231 in a bid to deplete intracellular NAA. We used these models to test for metastasis by tail vein injection in immunocompromised mice. Consistent with our expectations and other reports, NAT8L knockdown resulted in reduced lung metastasis. Interestingly, however, NAA depletion via ASPA overexpression increased metastasis to the lung in vivo. Furthermore, the combination of ASPA overexpression with the knockdown of NAT8L led to a moderate phenotype between the two independent genetic modifications. Our findings of the metastasis-promoting effect of NAA depletion via ASPA overexpression leads to us believe that it is not just the accumulation of NAA but also its utilization that is important for breast cancer progression. Our future experiments are directed towards determining how NAA utilization causes a pro-tumorigenic phenotype. We would also like to study the current inconsistency between ASPA transcript levels in patient tumors and its observed tumor-promoting function in xenograft experiments. Completion of these studies will potentially define the importance of elevated NAA levels in the context of aggressive BCa. Since NAA is a metabolite that can be detected in serum, our study could also lead to the development of NAA as a non-invasive biomarker for early detection of aggressive BCa, thus providing a bigger window for therapeutic intervention. Citation Format: Akhila Balasubramanian, Weijie Zhang, Franklin Gu, Chandra Ambati, Nagireddy Putluri, Xiang Zhang, Aryan Namboodiri, Stefan Ambs, Arun Sreekumar. Role of the metabolite N-acetylaspartate in breast cancer progression [abstract]. In: Abstracts: AACR Special Virtual Conference on Epigenetics and Metabolism; October 15-16, 2020; 2020 Oct 15-16. Philadelphia (PA): AACR; Cancer Res 2020;80(23 Suppl):Abstract nr PO-056.

Akt isoform-specific effects on thyroid cancer development and progression in a murine thyroid cancer model

The Akt family is comprised of three unique homologous proteins with isoform-specific effects, but isoform-specific in vivo data are limited in follicular thyroid cancer (FTC), a PI3 kinase-driven tumor. Prior studies demonstrated that PI3K/Akt signaling is important in thyroid hormone receptor βPV/PV knock-in (PV) mice that develop metastatic thyroid cancer that most closely resembles FTC. To determine the roles of Akt isoforms in this model we crossed Akt1−/−, Akt2−/−, and Akt3−/− mice with PV mice. Over 12 months, thyroid size was reduced for the Akt null crosses (p < 0.001). Thyroid cancer development and local invasion were delayed in only the PVPV-Akt1 knock out (KO) mice in association with increased apoptosis with no change in proliferation. Primary-cultured PVPV-Akt1KO thyrocytes uniquely displayed a reduced cell motility. In contrast, loss of any Akt isoform reduced lung metastasis while vascular invasion was reduced with Akt1 or 3 loss. Microarray of thyroid RNA displayed incomplete overlap between the Akt KO models. The most upregulated gene was the dendritic cell (DC) marker CD209a only in PVPV-Akt1KO thyroids. Immunohistochemistry demonstrated an increase in CD209a-expressing cells in the PVPV-Akt1KO thyroids. In summary, Akt isoforms exhibit common and differential functions that regulate local and metastatic progression in this model of thyroid cancer.

SHP-2 and PDL1 Inhibition Combined with Radiotherapy Enhances Systemic Antitumor Effects in an Anti-PD1-resistant Model of Non-small-cell-lung Cancer

Immune checkpoint inhibitors such as anti-PD1/PDL1 have emerged as promising therapies for advanced non-small-cell-lung cancer (NSCLC). However, approximately 80% of patients do not respond to immunotherapy given alone because of intrinsic or acquired resistance. Radiotherapy (XRT) can overcome PD1 resistance and improve treatment outcomes, but its efficacy remains suboptimal. The tyrosine phosphatase SHP-2, expressed in some cancers and in immune cells, has been shown to negatively affect antitumor immunity. Our hypothesis was that SHP-2 inhibition in combination with α-PDL1 would enhance immune-mediated responses to XRT and synergistically boost antitumor effects in an anti-PD1-resistant mouse model. We treated 129Sv/Ev mice with anti-PD1-resistant 344SQ NSCLC adenocarcinoma xenografts with oral SHP099 (an SHP-2 inhibitor) combined with XRT and intraperitoneal α-PDL1. Primary tumors were treated with XRT (3 fractions of 12 Gy each), whereas abscopal (out-of-field) tumors were observed but not treated. In vitro experiments include: qRT-PCR, western blot, flow-cytometry, single-cell-sequence and nano-string. Results: o The Results single-cell-sequence of biopsies from advanced NSCLC patients showed that SHP-2 was significantly higher expressed in to anti-PD-1 non-responders (n = 8) compared to responders (n = 8) (P = 0.041). Then, in vitro and in vivo experiments confirmed that SHP-2 was higher expressed in PD-1 resistant NSCLC model comparing to sensitive model in qRT-PCR, western blot and flow-cytometry. o XRT in combination with SHP099 and α-PDL1 promoted local and abscopal responses, reduced lung metastases, and improved mouse survival. o Expression analysis of 770 immune-related genes indicated that triple therapy significantly increased macrophage function (P = 0.008 vs XRT alone, P = 0.028 vs XRT+α-PDL1), T-cell function (P = 0.003 vs control, P = 0.009 vs XRT alone), and decreased cancer progression (P = 0.048 vs control) in abscopal tumor sites. o Flow-cytometry immune profiling indicated that addition of anti-SHP-2 was associated with higher M1/M2 ratio, greater numbers of CD8+ T cells, and fewer regulatory T cells, compared to iRT group (XRT+anti-PD-L1). o The depletion experiment showed that the anti-tumor effects of triple therapy took place mainly through M1 TAMs and CD8+ T cells. This novel triple-combination therapy had strong antitumor effects in this mouse model of anti-PD1-resistant NSCLC, and may be a novel therapeutic approach for anti-PD1- resistant NSCLC in patients.

Upregulated β-arrestin1 predicts poor prognosis and promotes metastasis via AKT/ERK signaling pathway in gastric cancer

Backgroundβ-Arrestins have been found to regulate cell proliferation, invasion and migration; transmit anti-apoptotic survival signals; and affect other characteristics of tumours. However, their role in gastric cancer (GC) is not clear. We investigated the role and mechanism of β-arrestins in the regulation of GC. MethodsWe first examined β-arrestins mRNA levels in 17 pairs of GC tissues by qRT-PCR. We also used immunohistochemistry to further examine the expression of β-arrestins in 60 paraffin-embedded primary GC tissues and 20 normal gastric tissues. Then, the function of β-arrestin1 was investigated in vitro and in vivo. Resultsβ-Arrestin1 was upregulated in GC tissue and was associated with tumour stage, lymph node metastasis, invasion depth and patient sex. High expression of β-arrestin1 expression predicted poor prognosis in GC. β-Arrestin1 promoted GC cell proliferation, migration and invasion, and it suppressed E-cadherin expression and upregulated Vimentin expression via AKT/ERK signalling pathway. The in vivo metastasis assays showed that knockdown of β-arrestin1 reduced lung metastasis and inhibited EMT. ConclusionThe upregulation of β-arrestin1 predicts poor prognosis and promotes metastasis and epithelial-mesenchymal transition in GC through AKT/ERK signalling pathway. This study may provide therapeutic advances for the treatment and early diagnosis of patients with metastatic GC.

Targeting IL-3Rα on tumor-derived endothelial cells blunts metastatic spread of triple-negative breast cancer via extracellular vesicle reprogramming

The lack of approved targeted therapies highlights the need for new treatments for triple-negative breast cancer (TNBC) patients. Interleukin-3 (IL-3) acts as an autocrine factor for tumor–endothelial cells (TEC), and exerts pro-angiogenic paracrine action via extracellular vesicles (EVs). IL-3Rα blockade on TEC changes TEC-EV (anti-IL-3R-EV) microRNA (miR) content and promotes the regression of established vessels. As TEC is the doorway for “drug” entry into tumors, we aimed to assess whether IL-3R blockade on TEC impacts tumor progression via its unique EV cargo. First, the expression of IL-3Rα was evaluated in 27 human TNBC samples. It was noticed that, besides TEC and inflammatory cells, tumor cells from 55.5% of the human TNBC samples expressed IL-3Rα. Using human TNBC cell lines for in vitro studies, we found that, unlike native TEC-EVs (nEVs), anti-IL-3R-EVs increase apoptosis and reduced cell viability and migration. In vivo, anti-IL-3R-EV treatment induced vessel regression in established tumors formed of MDA-MB-231 cells, decreased Vimentin, β-catenin, and TWIST1 expression, almost abolished liver and lung metastases from primary tumors, and reduced lung metastasis generated via the intravenous injection of MDA-MB-231 cells. nEVs depleted of miR-24-3p (antago-miR-24-3p-EVs) were effective as anti-IL-3R-EVs in downregulating TWIST1 and reducing metastatic lesions in vivo. Consistent with network analyses of miR-24-3p gene targeting, anti-IL-3R-EVs and antago-miR-24-3p-EVs upregulate SPRY2 in MDA-MB-231 cells. Finally, SPRY2 silencing prevented anti-IL-3R-EV and antago-miR-24-3p-EV-mediated apoptotic cues.Overall, these data provide the first evidence that IL-3Rα is highly expressed in TNBC cells, TEC, and inflammatory cells, and that IL-3Rα blockade on TEC impacts tumor progression.

Gamabufotalin suppressed osteosarcoma stem cells through the TGF-β/periostin/PI3K/AKT pathway

AimsTo investigate the effect of gamabufotalin (GBT) on metastasis and modulation of stemness features in osteosarcoma, and the molecular mechanisms underlying such effects. MethodsHuman osteosarcoma U2OS/MG-63 cell lines were used in this study. Cell proliferation, migration, and invasion were determined by MTT assay, wound healing assay, and cell invasion assay, respectively. The inhibitive effect of GBT on stemness was assessed by flow cytometry and mammosphere formation. The protein levels of related proteins were detected by western blotting analysis. The effect of GBT on tumorigenicity and metastasis was determined by immunofluorescence staining and immunohistochemistry in vivo experiments. ResultsWe found that GBT suppressed the viability of U2OS/MG-63 cells in a time- and dose-dependent manner. Notably, GBT had no effect on the viability of human fetal osteoblastic (hFOB) 1.19 cells. Moreover, GBT increased the width of wounds, reduced the number of invasive osteosarcoma cells and reversed the epithelial–mesenchymal transition phenotype. Notably, we found that, compared with hFOB1.19 cells, the levels of transforming growth factor-β (TGF-β), periostin, phosphorylated-AKT (p-AKT), and phosphorylated-PI3K (p-PI3K) were higher in spheroids group than in parent cells. In addition, GBT reduced the ratio of CD133+ cells, the size of spheroids and Nanog, as well as the protein levels of SRY-box transcription factor 2 (SOX2), and octamer-binding protein 3/4 (OCT3/4). Our in vivo experiments showed that GBT consistently reduced lung metastasis lesions, the expression levels of matrix metalloproteinase 2 (MMP2), TGF-β, periostin, p-AKT, and p-PI3K (immunohistochemistry staining), as well as that of CD133 in tumor tissues (immunofluorescence analysis). From a mechanistic point of view, exogenous TGF-β/periostin/PI3K/AKT overexpression neutralized the reduction of GBT-decreased invasion/migration and the suppression of stemness properties. ConclusionCollectively, our data demonstrated that GBT inhibited the viability and tumorigenesis capability of osteosarcoma cells by blocking the TGF-β/periostin/PI3K/AKT signaling pathway. Therefore, GBT may represent a promising therapeutic agent for the management of osteosarcoma.