Immunotherapeutic IL-6R and targeting the MCT-1/IL-6/CXCL7/PD-L1 circuit prevent relapse and metastasis of triple-negative breast cancer.

Rationale: Multiple copies in T-cell malignancy 1 (MCT-1) is a prognostic biomarker for aggressive breast cancers. Overexpressed MCT-1 stimulates the IL-6/IL-6R/gp130/STAT3 axis, which promotes epithelial-to-mesenchymal transition and cancer stemness. Because cancer stemness largely contributes to the tumor metastasis and recurrence, we aimed to identify whether the blockade of MCT-1 and IL-6R can render these effects and to understand the underlying mechanisms that govern the process. Methods: We assessed primary tumor invasion, postsurgical local recurrence and distant metastasis in orthotopic syngeneic mice given the indicated immunotherapy and MCT-1 silencing (shMCT-1). Results: We found that shMCT-1 suppresses the transcriptomes of the inflammatory response and metastatic signaling in TNBC cells and inhibits tumor recurrence, metastasis and mortality in xenograft mice. IL-6R immunotherapy and shMCT-1 combined further decreased intratumoral M2 macrophages and T regulatory cells (Tregs) and avoided postsurgical TNBC expansion. shMCT-1 also enhances IL-6R-based immunotherapy effectively in preventing postsurgical TNBC metastasis, recurrence and mortality. Anti-IL-6R improved helper T, cytotoxic T and natural killer (NK) cells in the lymphatic system and decreased Tregs in the recurrent and metastatic tumors. Combined IL-6R and PD-L1 immunotherapies abridged TNBC cell stemness and M2 macrophage activity to a greater extent than monotherapy. Sequential immunotherapy of PD-L1 and IL-6R demonstrated the best survival outcome and lowest postoperative recurrence and metastasis compared with synchronized therapy, particularly in the shMCT-1 context. Multiple positive feedforward loops of the MCT-1/IL-6/IL-6R/CXCL7/PD-L1 axis were identified in TNBC cells, which boosted metastatic niches and immunosuppressive microenvironments. Clinically, MCT-1high/PD-L1high/CXCL7high and CXCL7high/IL-6high/IL-6Rhigh expression patterns predict worse prognosis and poorer survival of breast cancer patients. Conclusion: Systemic targeting the MCT-1/IL-6/IL-6R/CXCL7/PD-L1 interconnections enhances immune surveillance that inhibits the aggressiveness of TNBC.

SCEL regulates switches between pro-survival and apoptosis of the TNF-α/TNFR1/NF-κB/c-FLIP axis to control lung colonization of triple negative breast cancer.

BACKGROUND: Patients with metastatic triple-negative breast cancer (mTNBC) have a higher probability of developing visceral metastasis within 5 years after the initial diagnosis. Therefore, a deeper understanding of the progression and spread of mTNBC is urgently needed. METHODS: The isobaric tag for relative and absolute quantitation (iTRAQ)-based LC-MS/MS proteomic approach was applied to identify novel membrane-associated proteins in the lung-tropic metastatic cells. Public domain datasets were used to assess the clinical relevance of the candidate proteins. Cell-based and mouse models were used for biochemical and functional characterization of the protein molecule Sciellin (SCEL) identified by iTRAQ to elucidate its role and underlying mechanism in promoting lung colonization of TNBC cells. RESULTS: The iTRAQ-based LC-MS/MS proteomic approach identified a membrane-associated protein SCEL that was overexpressed in the lung-tropic metastatic cells, and its high expression was significantly correlated with the late-stage TNBC and the shorter survival of the patients. Downregulation of SCEL expression significantly impaired the 3D colony-forming ability but not the migration and invasion ability of the lung colonization (LC) cells. Knockdown of SCEL reduced TNF-α-induced activation of the NF-κB/c-FLIP pro-survival and Akt/Erk1/2 growth signaling pathways in the LC cells. Specifically, knockdown of SCEL expression switched TNF-α-mediated cell survival to the caspase 3-dependent apoptosis. Conversely, ectopic expression of SCEL promoted TNF-α-induced activation of NF-κB/c-FLIP pro-survival and Akt/Erk1/2 pro-growth signaling pathway. The result of co-immunoprecipitation (Co-IP) and GST pull-down assay showed that SCEL could interact with TNFR1 to promote its protein stability. The xenograft mouse model experiments revealed that knockdown of SCEL resulted in increase of caspase-3 activity, and decrease of ki67 and TNFR1 expression as well as increase of tumor-associated macrophages in the metastatic lung lesions. Clinically, SCEL expression was found to be positively correlated with TNFR1 in TNBC tissues. Lastly, we showed that blocking TNF-α-mediated cell survival signaling by adalimumab effectively suppressed the lung colonization of the SCEL-positive, but not the SCEL-downregulated LC cells in the tail-vein injection model. CONCLUSIONS: Our findings indicate that SCEL plays an essential role in the metastatic lung colonization of TNBC by promoting the TNF-α/TNFR1/NF-κB/c-FLIP survival and Akt/Erk1/2 proliferation signaling. Thus, SCEL may serve as a biomarker for adalimumab treatment of TNBC patients.

Inhibition of CDCP1 by 8-isopentenylnaringenin synergizes with EGFR inhibitors in lung cancer treatment.

CUB domain-containing protein 1 (CDCP1) contributes to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) resistance by regulating EGFR signaling pathways and is a potential target in lung cancer treatment. This study aims to identify a CDCP1 reducer that synergistically improves TKI treatment. Utilizing a high-throughput drug screening system, a phytoestrogen 8-isopentenylnaringenin (8PN) was identified. Upon 8PN treatment, CDCP1 protein levels and malignant features were reduced. 8PN exposure caused the accumulation of lung cancer cells in G0/G1 phase and increased the proportion of senescent cells. In EGFR TKI-resistant lung cancer cells, the combination of 8PN and TKI synergistically reduced cell malignance, inhibited downstream EGFR pathway signaling, and exerted additive effects on cell death. Moreover, combination therapy effectively reduced tumor growth and enhanced tumor necrosis in tumor xenograft mice models. Mechanistically, 8PN increased interleukin (IL)6 and IL8 expression, induced neutrophil infiltration, and enhanced neutrophil-mediated cytotoxicity to attenuate lung cancer cell growth. In conclusion, 8PN enhances the anticancer efficacy of EGFR TKI on lung cancer and triggers neutrophil-dependent necrosis, highlighting the potential to overcome TKI resistance in lung cancer patients who have EGFR mutation.

rFGF4 alleviates lipopolysaccharide-induced acute lung injury by inhibiting the TLR4/NF-κB signaling pathway.

Acute lung injury (ALI) is a serious and common clinical disease. Despite significant progress in ALI treatment, the morbidity and mortality rates remain high. However, no effective drug has been discovered for ALI. FGF4, a member of the FGF family, plays an important role in the regulation of various physiological and pathological processes. Therefore, in the present study, we aimed to study the protective effects of FGF4 against LPS-induced lung injury in vivo and in vitro. We found that rFGF4 treatment improved the lung W/D weight ratio, the survival rate, immune cell infiltration and protein concentrations in mice with LPS-induced ALI. Histological analysis revealed that rFGF4 significantly attenuated lung tissue injury and cell apoptosis. Furthermore, rFGF4 inhibited the activation of the TLR4/NF-κB signaling pathway and the production of pro-inflammatory mediators in LPS-injured lung tissues, murine alveolar macrophages (MH-S) and murine pulmonary epithelial (MLE-12) cells. The results of cell experiments further verified that rFGF4 inhibited the production of inflammatory mediators in MH-S cells and MLE-12 cells by regulating the TLR4/NF-κB signaling pathway. These results revealed that rFGF4 protected lung tissues and inhibited inflammatory mediators in mice with LPS-induced ALI by inhibiting the TLR4/NF-κB signaling pathway in MH-S and MLE-12 cells.

Hepatocyte leukotriene B4 receptor 1 promotes NAFLD development in obesity.

BACKGROUND AND AIMS: NAFLD is the most prevalent chronic liver disease worldwide and has emerged as a serious public health issue with no approved treatment. The development of NAFLD is strongly associated with hepatic lipid content, and patients with NAFLD have significantly higher rates of hepatic de novo lipogenesis (DNL) than lean individuals. Leukotriene B4 (LTB4), a metabolite of arachidonic acid, is dramatically increased in obesity and plays important role in proinflammatory cytokine production and insulin resistance. But the role of liver LTB4/LTB4 receptor 1 (Ltb4r1) in lipid metabolism is unclear. APPROACH AND RESULTS: Hepatocyte-specific knockout (HKO) of Ltb4r1 improved hepatic steatosis and systemic insulin resistance in both diet-induced and genetically induced obese mice. The mRNA level of key enzymes involved in DNL and fatty acid esterification decreased in Ltb4r1 HKO obese mice. LTB4/Ltb4r1 directly promoted lipogenesis in HepG2 cells and primary hepatocytes. Mechanically, LTB4/Ltb4r1 promoted lipogenesis by activating the cAMP-protein kinase A (PKA)-inositol-requiring enzyme 1α (IRE1α)-spliced X-box-binding protein 1 (XBP1s) axis in hepatocytes, which in turn promoted the expression of lipogenesis genes regulated by XBP1s. In addition, Ltb4r1 suppression through the Ltb4r1 inhibitor or lentivirus-short hairpin RNA delivery alleviated the fatty liver phenotype in obese mice. CONCLUSIONS: LTB4/Ltb4r1 promotes hepatocyte lipogenesis directly by activating PKA-IRE1α-XBP1s to promote lipogenic gene expression. Inhibition of hepatocyte Ltb4r1 improved hepatic steatosis and insulin resistance. Ltb4r1 is a potential therapeutic target for NAFLD.

Periostin promotes ovarian cancer metastasis by enhancing M2 macrophages and cancer-associated fibroblasts via integrin-mediated NF-κB and TGF-ß2 signaling.

BACKGROUND: Ovarian cancer has the highest mortality among gynecological cancers due to late diagnosis and lack of effective targeted therapy. Although the study of interplay between cancer cells with their microenvironment is emerging, how ovarian cancer triggers signaling that coordinates with immune cells to promote metastasis is still elusive. METHODS: Microarray and bioinformatics analysis of low and highly invasive ovarian cancer cell lines were used to reveal periostin (POSTN), a matrix protein with multifunctions in cancer, with elevated expression in the highly invasive cells. Anchorage independent assay, Western blot, RNA interference, confocal analysis and neutralizing antibody treatment were performed to analyze the effects of POSTN on tumor promotion and to explore the underlying mechanism. Chemotaxis, flow cytometry and cytokine array analyses were undertaken to analyze the involvement of POSTN in cancer-associated fibroblast (CAF) and macrophage modulation. Correlations between POSTN expression levels and clinical characteristics were analyzed using the Oncomine, commercial ovarian cancer cDNA and China Medical University Hospital patient cohort. In vivo effect of POSTN on metastasis was studied using a mouse xenograft model. RESULTS: Expression of POSTN was found to be elevated in highly invasive ovarian cancer cells. We observed that POSTN was co-localized with integrin ß3 and integrin ß5, which was important for POSTN-mediated activation of ERK and NF-κB. Ectopic expression of POSTN enhanced whereas knockdown of POSTN decreased cancer cell migration and invasion in vitro, as well as tumor growth and metastasis in vivo. POSTN enhanced integrin/ERK/NF-κB signaling through an autocrine effect on cancer cells to produce macrophage attracting and mobilizing cytokines including MIP-1ß, MCP-1, TNFα and RANTES resulting in increased chemotaxis of THP-1 monocytes and their polarization to M2 macrophages in vitro. In agreement, tumors derived from POSTN-overexpressing SKOV3 harbored more tumor-associated macrophages than the control tumors. POSTN induced TGF-ß2 expression from ovarian cancer cells to promote activation of adipose-derived stromal cells to become CAF-like cells expressing alpha smooth muscle actin and fibroblast activation protein alpha. Consistently, increased CAFs were observed in POSTN overexpressing SKOV3 cells-derived metastatic tumors. In clinical relevance, we found that expression of POSTN was positively correlated with advanced-stage diseases and poor overall survival of patients. CONCLUSIONS: Our study revealed a POSTN-integrin-NF-κB-mediated signaling and its involvement in enhancing M2 macrophages and CAFs, which could potentially participate in promoting tumor growth. Our results suggest that POSTN could be a useful prognosis marker and potential therapeutic target.

MicroRNA-485-5p targets keratin 17 to regulate oral cancer stemness and chemoresistance via the integrin/FAK/Src/ERK/ß-catenin pathway.

BACKGROUND: The development of drug resistance in oral squamous cell carcinoma (OSCC) that frequently leads to recurrence and metastasis after initial treatment remains an unresolved challenge. Presence of cancer stem cells (CSCs) has been increasingly reported to be a critical contributing factor in drug resistance, tumor recurrence and metastasis. Thus, unveiling of mechanisms regulating CSCs and potential targets for developing their inhibitors will be instrumental for improving OSCC therapy. METHODS: siRNA, shRNA and miRNA that specifically target keratin 17 (KRT17) were used for modulation of gene expression and functional analyses. Sphere-formation and invasion/migration assays were utilized to assess cancer cell stemness and epithelial mesenchymal transition (EMT) properties, respectively. Duolink proximity ligation assay (PLA) was used to examine molecular proximity between KRT17 and plectin, which is a large protein that binds cytoskeleton components. Cell proliferation assay was employed to evaluate growth rates and viability of oral cancer cells treated with cisplatin, carboplatin or dasatinib. Xenograft mouse tumor model was used to evaluate the effect of KRT17- knockdown in OSCC cells on tumor growth and drug sensitization. RESULTS: Significantly elevated expression of KRT17 in highly invasive OSCC cell lines and advanced tumor specimens were observed and high KRT17 expression was correlated with poor overall survival. KRT17 gene silencing in OSCC cells attenuated their stemness properties including markedly reduced sphere forming ability and expression of stemness and EMT markers. We identified a novel signaling cascade orchestrated by KRT17 where its association with plectin resulted in activation of integrin ß4/α6, increased phosphorylation of FAK, Src and ERK, as well as stabilization and nuclear translocation of ß-catenin. The activation of this signaling cascade was correlated with enhanced OSCC cancer stemness and elevated expression of CD44 and epidermal growth factor receptor (EGFR). We identified and demonstrated KRT17 to be a direct target of miRNA-485-5p. Ectopic expression of miRNA-485-5p inhibited OSCC sphere formation and caused sensitization of cancer cells towards cisplatin and carboplatin, which could be significantly rescued by KRT17 overexpression. Dasatinib treatment that inhibited KRT17-mediated Src activation also resulted in OSCC drug sensitization. In OSCC xenograft mouse model, KRT17 knockdown significantly inhibited tumor growth, and combinatorial treatment with cisplatin elicited a greater tumor inhibitory effect. Consistently, markedly reduced levels of integrin ß4, active ß-catenin, CD44 and EGFR were observed in the tumors induced by KRT17 knockdown OSCC cells. CONCLUSIONS: A novel miRNA-485-5p/KRT17/integrin/FAK/Src/ERK/ß-catenin signaling pathway is unveiled to modulate OSCC cancer stemness and drug resistance to the common first-line chemotherapeutics. This provides a potential new therapeutic strategy to inhibit OSCC stem cells and counter chemoresistance.

One-Chip Isolation of Drug-Resistant Acute Myeloid Leukemia Cells with CXCR4-Targeted Magnetic Fluorescent Nanoprobes.

Drug resistance and relapse lead to high mortality in acute myeloid leukemia, and studies have shown that CXCR4 overexpression is highly correlated with poor prognosis and drug resistance in leukemia cells. Isolation and detection of AML cells with CXCR4 overexpression will be crucial to the treatment of AML. In this paper, magnetic nanoparticles were firstly prepared successfully by high-temperature thermal decomposition method, and then characterized by TEM, VSM and DLS. Subsequently CXCR4-targeted magnetic fluorescent nanoprobes conjugated with antibody 12G5 were constructed by stepwise coupling. In cell experiments, the obtained probes demonstrated excellent targeting efficacy to CXCR4 overexpressed AML cells HL-60. In addition, HL-60 cells labelled with the magnetic probes can be magnetic isolated successfully in one microfluidics chip, with efficiency of 82.92 ± 7.03%. Overall, this method utilizes the superiority of superparamagnetic nanomaterials and microfluidic technology to achieve the enrichment and capture of drug-resistant cells in a microfluidic chip, providing a new idea for the isolation and detective of drug-resistant acute myeloid leukemia cells.

Strategic synthesis of trimetallic Au@Ag-Pt nanorattles for ultrasensitive colorimetric detection in lateral flow immunoassay.

Achieving metal nanoparticles with high peroxidase activity and visible-light plasmonic property for lateral flow immunoassay has attracted extensive attention in the industry. However, the major challenge lies in establishing a general and robust preparation strategy. In this contribution, we developed a citrate-capped trimetallic Au@Ag-Pt nanorattle by employing seed-mediated growth and galvanic replacement reaction under a convenient condition, which can be translated directly to industrialized production. The rattle-like architecture empowers the Au@Ag-Pt NPs peroxidase-like activity while retaining the plasmonic property with intense color in the visible-light range. According to testing requirements, Au@Ag-Pt NPs-LFIA provides two colorimetric modes: low-sensitivity mode based on the color from their intrinsic plasmonic property and the high-sensitivity mode based on the nanozyme-triggered chromogenic reaction. Human cardiac troponin I (cTnI), one of the most specific markers for cardiac injury, was chosen as the detection model. Mainly, ultrasensitive colorimetric detection of human cTnI was successfully achieved as low as ∼20 pg mL-1. This strategy is robust to guarantee the stability and repeatability of the peroxidase activity without exact control, which can directly dock with the industrialized production of traditional LFIA strips and be readily adapted for on-demand clinical diagnosis.

De-glycosylated membrane PD-L1 in tumor tissues as a biomarker for responsiveness to atezolizumab (Tecentriq) in advanced breast cancer patients.

The atezolizumab (Tecentriq), a humanized antibody against human programmed death ligand 1 (PD-L1), combined with nab-paclitaxel was granted with accelerated approval to treat unresectable locally advanced or metastatic triple-negative breast cancer (TNBC) due to the encouraging positive results of the phase 3 IMpassion130 trial using PD-L1 biomarker from immune cells to stratify patients. However, the post-market study IMpassion131 did not support the original observation, resulting in the voluntary withdrawal of atezolizumab from the indication in breast cancer by Genentech in 2021. Emerging evidence has revealed a high frequency of false negative result using the standard immunohistochemical (IHC) staining due to heavy glycosylation of PD-L1. The removal of glycosylation prevents from the false negative staining, enabling more accurate assessment of PD-L1 levels and improving prediction for response to immune checkpoint therapy. In the present study, the natural and de-glycosylated PD-L1 expression in tumor and immune cells from nine TNBC patients were analyzed by using clone 28-8 monoclonal antibody to correlate with treatment outcome. Our results demonstrate that: (1) Removal of the glycosylation indeed enhances the detection of PD-L1 by IHC staining, (2) The PD-L1 levels on tumor cell surface after removal of the glycosylation correlates well with clinical responses for atezolizumab treatment; (3) The criteria used in the IMpassion130 and IMpassion131 trials which scored the natural PD-L1 in the immune cells failed to correlate with the clinical response. Taken together, tumor cell surface staining of PD-L1 with de-glycosylation has a significant correlation with the clinical response for atezolizumab treatment, suggesting that treatment of atezolizumab may be worthy of further consideration with de-glycosylation procedure as a patient stratification strategy. A larger cohort to validate this important issue is warranted to ensure right patient population who could benefit from the existing FDA-approved drugs.

Novel function of THEMIS2 in the enhancement of cancer stemness and chemoresistance by releasing PTP1B from MET.

Triple negative breast cancer (TNBC) possesses poor prognosis mainly due to lack of effective endocrine or targeted therapies, aggressive nature and high rate of chemoresistance. Cancer stem cells (CSCs) are considered to play critical roles in cancer recurrence and chemoresistance. THEMIS2 was identified as the sole common elevated gene in three triple negative breast cancer (TNBC) and two ovarian CSC lines. We discovered an intrinsic signaling scaffold function of THEMIS2, which acts as a novel regulator of cancer stemness in promoting multiple cancer stemness properties including sphere formation, stemness markers expression, chemoresistance and tumorigenicity with low numbers of cancer cells implantation. For the first time, we demonstrated that THEMIS2 specifically enhanced MET activating phosphorylation by suppressing the association of protein-tyrosine phosphatases 1B (PTP1B) with p-MET and MET, which accounted mainly for THEMIS2-mediated effect on cancer stemness and chemoresistance. Increased THEMIS2 expression was associated with poor survival in TNBC patients and in patients from our breast cancer cohort. We found that non-cytotoxic dosages of cryptotanshinone (CPT) could potently inhibit cancer stemness, chemoresistance and tumorigenicity by suppressing expression of THEMIS2. Notably, stable overexpression of THEMIS2 is associated with enhanced sensitivity toward Capmatinib and CPT treatment. Expression levels of THEMIS2 and p-MET protein were positively correlated in the 465 breast cancer specimens. Our study revealed the novel oncogenic role of THEMIS2 and its underlying mechanism via suppressing PTP1B association with MET and thus leading to its activation. Our findings suggest that THEMIS2 could be a biomarker for MET targeted therapy and also provide a potential clinical application using low dosages of CPT for treatment of THEMIS2 positive TNBC.

Monocytes secrete CXCL7 to promote breast cancer progression.

Certain immune cells and inflammatory cytokines are essential components in the tumor microenvironment to promote breast cancer progression. To identify key immune players in the tumor microenvironment, we applied highly invasive MDA-MB-231 breast cancer cell lines to co-culture with human monocyte THP-1 cells and identified CXCL7 by cytokine array as one of the increasingly secreted cytokines by THP-1 cells. Further investigations indicated that upon co-culturing, breast cancer cells secreted CSF1 to induce expression and release of CXCL7 from monocytes, which in turn acted on cancer cells to promote FAK activation, MMP13 expression, migration, and invasion. In a xenograft mouse model, administration of CXCL7 antibodies significantly reduced abundance of M2 macrophages in tumor microenvironment, as well as decreased tumor growth and distant metastasis. Clinical investigation further suggested that high CXCL7 expression is correlated with breast cancer progression and poor overall survival of patients. Overall, our study unveils an important immune cytokine, CXCL7, which is secreted by tumor infiltrating monocytes, to stimulate cancer cell migration, invasion, and metastasis, contributing to the promotion of breast cancer progression.

LOC550643, a Long Non-coding RNA, Acts as Novel Oncogene in Regulating Breast Cancer Growth and Metastasis.

Metastatic disease is responsible for over 90% of death in patients with breast cancer. Therefore, identifying the molecular mechanisms that regulate metastasis and developing useful therapies are crucial tasks. Long non-coding RNAs (lncRNAs), which are non-coding transcripts with >200 nucleotides, have recently been identified as critical molecules for monitoring cancer progression. This study examined the novel lncRNAs involved in the regulation of tumor progression in breast cancer. This study identified 73 metastasis-related lncRNA candidates from comparison of paired isogenic high and low human metastatic breast cancer cell lines, and their expression levels were verified in clinical tumor samples by using The Cancer Genome Atlas. Among the cell lines, a novel lncRNA, LOC550643, was highly expressed in breast cancer cells. Furthermore, the high expression of LOC550643 was significantly correlated with the poor prognosis of breast cancer patients, especially those with triple-negative breast cancer. Knockdown of LOC550643 inhibited cell proliferation of breast cancer cells by blocking cell cycle progression at S phase. LOC550643 promoted important in vitro metastatic traits such as cell migration and invasion. Furthermore, LOC550643 could inhibit miR-125b-2-3p expression to promote breast cancer cell growth and invasiveness. In addition, by using a xenograft mouse model, we demonstrated that depletion of LOC550643 suppressed the lung metastatic potential of breast cancer cells. Overall, our study shows that LOC550643 plays an important role in breast cancer cell metastasis and growth, and LOC550643 could be a potential diagnosis biomarker and therapeutic target for breast cancer.

Arginine starvation elicits chromatin leakage and cGAS-STING activation via epigenetic silencing of metabolic and DNA-repair genes.

Rationale: One of the most common metabolic defects in cancers is the deficiency in arginine synthesis, which has been exploited therapeutically. Yet, challenges remain, and the mechanisms of arginine-starvation induced killing are largely unclear. Here, we sought to demonstrate the underlying mechanisms by which arginine starvation-induced cell death and to develop a dietary arginine-restriction xenograft model to study the in vivo effects. Methods: Multiple castration-resistant prostate cancer cell lines were treated with arginine starvation followed by comprehensive analysis of microarray, RNA-seq and ChIP-seq were to identify the molecular and epigenetic pathways affected by arginine starvation. Metabolomics and Seahorse Flux analyses were used to determine the metabolic profiles. A dietary arginine-restriction xenograft mouse model was developed to assess the effects of arginine starvation on tumor growth and inflammatory responses. Results: We showed that arginine starvation coordinately and epigenetically suppressed gene expressions, including those involved in oxidative phosphorylation and DNA repair, resulting in DNA damage, chromatin-leakage and cGAS-STING activation, accompanied by the upregulation of type I interferon response. We further demonstrated that arginine starvation-caused depletion of α-ketoglutarate and inactivation of histone demethylases are the underlying causes of epigenetic silencing. Significantly, our dietary arginine-restriction model showed that arginine starvation suppressed prostate cancer growth in vivo, with evidence of enhanced interferon responses and recruitment of immune cells. Conclusions: Arginine-starvation induces tumor cell killing by metabolite depletion and epigenetic silencing of metabolic genes, leading to DNA damage and chromatin leakage. The resulting cGAS-STING activation may further enhance these killing effects.

Nutrient supplements from selected botanicals mediated immune modulation of the tumor microenvironment and antitumor mechanism.

Specific extracts of selected vegetables (SV) have been shown to benefit the survival of stage IIIb/IV non-small cell lung cancer patients in phase I/II studies and is currently in a phase III trial. However, the underlying mechanism of SV-mediated antitumor immune responses has not been elucidated. Our results indicate that SV modulated the NK and adoptive T cell immune responses in antitumor efficacy. Furthermore, antitumor effects of SV were also mediated by innate myeloid cell function, which requires both TLR and ß-glucan signaling in a MyD88/TRIF and Dectin-1-dependent manner, respectively. Additionally, SV treatment reduced granulocytic myeloid-derived suppressor cell (MDSC) infiltration into the tumor and limited monocytic MDSC toward the M2-like functional phenotype. Importantly, SV treatment enhanced antigen-specific immune responses by augmenting the activation of antigen-specific TH1/TH17 cells in secondary lymphoid organs and proliferative response, as well as by reducing the Treg population in the tumor microenvironment, which was driven by SV-primed activated M-MDSC. Our results support the idea that SV can subvert immune-tolerance state in the tumor microenvironment and inhibit tumor growth. The present study suggests that features, such as easy accessibility, favorable clinical efficacy, no detectable side effects and satisfactory safety make SV a feasible, appealing and convincing adjuvant therapy for the treatment of cancer patients and prevent tumor recurrence and/or metastases.

Effects of synthetic glucocorticoids on breast cancer progression.

Glucocorticoids (GCs) are widely prescribed as adjuvant therapy for breast cancer patients. Unlike other steroid hormone receptors, the GC receptor is not considered an oncogene. Research in the past few years has revealed the complexity of GC-mediated signaling, but it remains puzzling whether GCs promote or inhibit tumor progression in different cancer types. Here we evaluated the potential of using a synthetic GC, dexamethasone (DEX), in the treatment of breast cancer. We found that the administration of low-dose DEX suppressed tumor growth and distant metastasis in the MCF-7 and MDA-MB-231 xenograft mouse model, whereas treatment with high-dose DEX enhanced tumor growth and metastasis, respectively. Treatment of breast cancer cells with DEX inhibited cell adhesion, migration, and invasion in a dose-dependent manner. The DEX-mediated inhibition of cell adhesion, migration, and invasion is partly through induction of microRNA-708 and subsequent Rap1B-mediated signaling in MDA-MB-231 cells. On the other hand, in MCF-7 cells, DEX-suppressed cell migration is independent from microRNA-708 mediated signaling. Overall, our data reveal that DEX acts as a double-edged sword during breast-cancer progression and metastasis: Lower concentrations inhibit breast cancer tumor growth and metastasis, whereas higher concentrations may play an undesired role to promote breast cancer progression.

DOCK6 promotes chemo- and radioresistance of gastric cancer by modulating WNT/ß-catenin signaling and cancer stem cell traits.

Gastric cancer (GC) is the third leading cause of cancer-related mortality worldwide and prognosis after potentially curative gastrectomy remains poor. Administration of GC-targeting molecules in combination with adjuvant chemo- or radiotherapy following surgical resection has been proposed as a potentially effective treatment option. Here, we have identified DOCK6, a guanine nucleotide exchange factor (GEF) for Rac1 and CDC42, as an independent biomarker for GC prognosis. Clinical findings indicate the positive correlation of higher DOCK6 expression with tumor size, depth of invasion, lymph node metastasis, vascular invasion, and pathological stage. Furthermore, elevated DOCK6 expression was significantly associated with shorter cumulative survival in both univariate and multivariate analyses. Gene ontology analysis of three independent clinical GC cohorts revealed significant involvement of DOCK6-correlated genes in the WNT/ß-catenin signaling pathway. Ectopic expression of DOCK6 promoted GC cancer stem cell (CSC) characteristics and chemo- or radioresistance concomitantly through Rac1 activation. Conversely, depletion of DOCK6 suppressed CSC phenotypes and progression of GC, further demonstrating the pivotal role of DOCK6 in GC progression. Our results demonstrate a novel mechanistic link between DOCK6, Rac1, and ß-catenin in GCCSC for the first time, supporting the utility of DOCK6 as an independent marker of GC.

Dysregulation of cystathionine γ-lyase promotes prostate cancer progression and metastasis.

Hydrogen sulfide (H2 S), an endogenous signaling gaseous molecule, is involved in various physiological activities, including vessel relaxation, regulation of cellular bioenergetics, inflammation, and angiogenesis. By using xenograft orthotopic implantation of prostate cancer PC3 cells and subsequently comparing bone metastatic with primary tumor-derived cancer cells, we find that H2 S-producing enzyme cystathionine γ-lyase (CTH) is upregulated in bone-metastatic PC3 cells. Clinical data further reveal that the expression of CTH is elevated in late-stage prostate cancer patients, and higher CTH expression correlates with poor survival from The Cancer Genome Atlas (TCGA) prostate cancer RNA-seq datasets. CTH promotes NF-κB nuclear translocation through H2 S-mediated sulfhydration on cysteine-38 of the NF-κB p65 subunit, resulting in increased IL-1ß expression and H2 S-induced cell invasion. Knockdown of CTH in PC3 cells results in the suppression of tumor growth and distant metastasis, while overexpression of CTH in DU145 cells promotes primary tumor growth and lymph node metastasis in the orthotopic implanted xenograft mouse model. Together, our findings provide evidence that CTH generated H2 S promotes prostate cancer progression and metastasis through IL-1ß/NF-κB signaling pathways.

Cancer-Derived VEGF-C Increases Chemokine Production in Lymphatic Endothelial Cells to Promote CXCR2-Dependent Cancer Invasion and MDSC Recruitment.

Breast cancer-derived vascular endothelial growth factor-C (VEGF-C) has been shown to enhance lymphangiogenesis in lymph nodes to accelerate cancer metastasis. However, the remodeling of lymph node microenvironments by VEGF-C remains elusive. By in vivo selection, we established a subline (named as "LC") with strong lymphatic tropism and high VEGF-C expression from the human MDA-MB-231 breast cancer cell line. Co-culture with LC cells or treatment with LC-conditioned medium upregulated the expression of CXC chemokines in lymphatic endothelial cells (LECs), which could be inhibited by pre-incubation with VEGF-C-neutralizing antibodies and VEGFR3 inhibitors. The chemokines produced by LECs enhanced recruitment of myeloid-derived suppressor cells (MDSCs) to tumor-draining and distant lymph nodes in tumor-bearing mice. Treatment with a CXCR2 inhibitor after tumor cell inoculation dramatically decreased the number of MDSCs in lymph nodes, suggesting the importance of the chemokine/CXCR2 signaling axis in MDSC recruitment. In addition, LEC-released chemokines also stimulated the expression of serum amyloid A1 (SAA1) in cancer cells, enhancing their lymphatic invasion by increasing VE-cadherin phosphorylation, junction disruption, and vascular permeability of LECs. Clinical sample validation confirmed that SAA1 expression was associated with increased lymph node metastasis. Collectively, we reveal a novel mechanism by which cancer cell-derived VEGF-C remodels lymphovascular microenvironments by regulating chemokine production in LECs to promote cancer invasion and MDSC recruitment. Our results also suggest that inhibition of CXCR2 is effective in treating lymphatic metastasis.