Project description:Background: More than 90% mortality of triple negative breast cancer (TNBC) patients are attributed to cancer metastasis with organotropism. Lung is the frequent site of TNBC metastasis. However, the precise mechanism of lung-specific metastasis of TNBC, particularly the driving factors, is not well understood. Methods: RNA sequencing was performed to identify patterns of gene expression associated with lung metastatic behavior using 4T1-LM3, 231-LM3 and their parental cells (4T1-P, 231-P). Expressions of RGCC, encoding the completion 32 protein response protein, were detected in TNBC cells and tissues by qRT-PCR, western blotting and IHC. Kinase activity assay were performed to evaluated PLK1 kinase activity and the amount of phosphorylated AMP-activated protein kinase α2 (AMPKα2). RGCC-mediated metabolism was determined by UHPLC system. Oxidative phosphorylation was evaluated by JC-1 staining and oxygen consumption rate (OCR) assay. Fatty acid oxidation assay, NADPH and ROS was applied to measure the status of RGCC-mediated fatty acid oxidation. The chemical sensitivity of cells was evaluated by CCK8 assay. Results: RGCC is aberrantly upregulated in pulmonary metastatic cells. High level of RGCC is significantly related with lung metastasis rather than other organ metastases. RGCC can effectively promote the kinase activity of PLK1, and activated PLK1 phosphorylates AMPKα2 to facilitate TNBC lung metastasis. Mechanistically, Survival and colonization of TNBC cells in the lungs by upregulating oxidative phosphorylation of mitochondria to obtain more energy and promoting fatty acid oxidation to produce abundant NADPH and sustains redox homeostasis, thus prevents excessive accumulation of potentially detrimental ROS in metastatic tumor cells, ultimately establishing metastases. Importantly, targeting RGCC in combination with paclitaxel/carboplatin effectively suppresses pulmonary metastases of TNBC in a mouse model. Conclusions: The enhanced RGCC is closely associated with lung-specific metastasis of TNBC. RGCC leads to activation of AMPKα2 and downstream signaling through RGCC-driven PLK1 activity to facilitate TNBC lung metastasis, which may determine a potential value of RGCC-driven OXPHOS and fatty acid oxidation as an important therapeutic target.

Project description:Purpose: Evaluate the anti-tumorigenic efficacy and immunomodulatory effects of chitin as general blocker of immunosuppressive chitinase-like proteins (CLPs) in combination with and without anti-PD-1 immune checkpoint blockade (ICB) in an immunocompetent 4T1- and 66cl4-based intraductal model for triple-negative breast cancer (TNBC). Methods: 4T1 and 66cl4 mammary tumor-bearing female BALB/c mice were either left untreated or were treated with anti-PD-1, chitin or chitin + anti-PD-1 for 2 weeks (w) between 3 and 5 w post-inoculation (p.i.) of the mammary tumor cells. After the 2-w treatment, 4T1 and 66cl4 primary tumors were resected and RNA was isolated from the tissue using in-house developed protocols. Results: Chitin-mediated reduction in innate immunosuppression and increased anti-tumor T-cell immunity in primary tumors of both TNBC models was clearly presented at the genomic level based on RNA-sequencing analysis. More specifically, a selection of 68 and 55 innate immunity-related genes in respectively 4T1 and 66cl4 primary tumors were downregulated upon chitin treatment in combination with or without anti-PD-1. Several of the selected innate immunity genes could be linked to immunosuppressive myeloid cell types, including Ccl2, Cxcl2, Csf1r and Itgam. Genes related to T-cell exhaustion, including Pdcd1, Cd274, Havcr2 and Lag3, were downregulated upon chitin and chitin + anti-PD-1 treatment in 4T1 tumors. Chitin- and chitin + anti-PD-1-treated 66cl4 tumors showed upregulation of genes associated with enhanced T-cell activity, including Cd8a, Cd8b1, Ifng, Il12b and Il18r1. In line with an overall reduced inflammation upon chitin treatment at the protein level, predominantly through myeloid cell reduction, a selected set of 68 and 48 inflammation-related genes were downregulated in chitin- and chitin + anti-PD-1-treated primary tumors of respectively the 4T1- and the 66cl4-based model. Importantly, genes that have been associated with the signaling and pro-tumorigenic activity of a prominent CLP family member, i.e. CHI3L1, were also downregulated in primary tumors following chitin treatment either with or without anti-PD-1, including Usf1 and Rab37 in the 4T1-based model and Lgals3 in the 66cl4-based model. Conclusion: Our findings highlight that chitin, as a general CLP blocker, reduces cancer-associated immunosuppression and enhances anti-tumor immunity as well as ICB responses in complementary ICB-resistant TNBC models, supporting its potential clinical relevance in immunosuppressed TNBC patients.

Project description:Triple negative breast cancer (TNBC) is a heterogeneous disease with limited therapeutic options. Two commonalities of TNBCs are a failure to express receptors for hormone or HER2-targeted therapies and high levels of chromosomal instability (CIN), both of which generate considerable therapeutic challenges. Focusing on the Src Family Kinases (SFKs), we found that treatment of TNBC cells with pan-SFK inhibitors causes abnormal nuclear phenotypes and DNA double strand breaks that are characteristic of excessive CIN, suggesting that SFKs may provide a vulnerability for treating TNBC. While prior clinical studies found that pan-SFK inhibition causes dose-limiting toxicities and minimal efficacy, we asked whether targeting individual family members may provide more specificity and hence, greater impact. Analysis of public datasets revealed that YES1 is one of the most highly expressed SFKs in basal/TNBC breast cancer and that its expression is associated with poor patient outcomes. Genomic and pharmacologic targeting of YES1 in TNBC cells further demonstrated that its sustained activity is essential for normal mitoses, including centrosome composition and bipolar spindle formation, as well as chromosome stability. We then determined if the ability of YES1 inhibition to induce mitotic defects and profound CIN could be leveraged to potentiate the efficacy of taxanes, cornerstone drugs for TNBC that also induce CIN. Combining paclitaxel with the selective YES1 inhibitor, CH695355, synergistically inhibited growth of several TNBC models both in vitro and in vivo. Moreover, YES1 inhibition re-sensitized taxane-resistant TNBC cells to paclitaxel. Elevated tumor expression of YES1 was also associated with worse overall survival of TNBC patients treated with combined taxanes/anthracyclines. Mechanistically, YES1 suppression leads to a loss of FOXM1 expression, a transcription factor whose disruption has also been reported to induce CIN, but has not yet been effectively therapeutically targeted. Together, these studies demonstrate that the SFK family member, YES1, is a novel and essential regulator of genome stability in TNBC. More broadly, these results suggest that YES1 may be a therapeutically targetable vulnerability that could potentiate the efficacy of mitotic inhibitors to induce maladaptive CIN and improve the treatment of TNBC.

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Novel inhibition of central carbon metabolism pathways by Rac and Cdc42 inhibitorMBQ-167 and paclitaxel

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