TRAF3/STAT6 axis regulates macrophage polarization and tumor progression.

Converting tumor-associated macrophages (TAMs) from the M2 to the M1 phenotype is considered an effective strategy for cancer therapy. TRAF3 is known to regulate NF-κB signaling. However, the role of TRAF3 in TAM polarization has not yet been completely elucidated. Here, we found that ablation of TRAF3 increased M1 markers, iNOS, FGR and SLC4A7, while down-regulated M2 markers, CD206, CD36 and ABCC3, expression levels in macrophages. Moreover, TRAF3 deficiency enhanced LPS-induced M1 and abolished IL-4-induced macrophage polarization. Next, quantitative ubiquitomics assays demonstrated that among the quantitative 7618 ubiquitination modification sites on 2598 proteins, ubiquitination modification of IL-4 responding proteins was the most prominently reduced according to enrichment analysis. STAT6, a key factor of IL-4 responding protein, K450 and K129 residue ubiquitination levels were dramatically decreased in TRAF3-deficient macrophages. Ubiquitination assay and luciferase assay demonstrated that TRAF3 promotes STAT6 ubiquitination and transcriptional activity. Site mutation analysis revealed STAT6 K450 site ubiquitination played a vital role in TRAF3-mediated STAT6 activation. Finally, B16 melanoma mouse model demonstrated that myeloid TRAF3 deficiency suppressed tumor growth and lung metastasis in vivo. Taken together, TRAF3 plays a vital role in M2 polarization via regulating STAT6 K450 ubiquitination in macrophages.

TRK-fused gene (TFG) regulates ULK1 stability via TRAF3-mediated ubiquitination and protects macrophages from LPS-induced pyroptosis.

TRK-fused gene (TFG) is known to be involved in protein secretion and plays essential roles in an antiviral innate immune response. However, its function in LPS-induced inflammation and pyroptotic cell death is still unknown. Here, we reported that TFG promotes the stabilization of Unc-51 like autophagy activating kinase (ULK1) and participates in LPS plus nigericin (Ng) induced pyroptotic cell death. Our results showed that TFG-deficient THP-1 macrophages exhibit higher mitochondrial ROS production. LPS/Ng stimulation triggers a much higher level of ROS and induces pyroptotic cell death. ULK1 undergoes a rapid turnover in TFG-deficient THP-1 cells. TFG forms complex with an E3 ligase, tumor necrosis factor receptor-associated factor 3 (TRAF3), and stabilizes ULK1 via disturbing ULK1-TRAF3 interaction. Knockdown of TFG facilitates the interaction of ULK1 with TRAF3 and subsequent K48-linked ULK1 ubiquitination and proteasome degradation. Rescue of ULK1 expression blocks LPS/Ng-induced cell death in TFG-deficient THP-1 macrophages. Taken together, TFG plays an essential role in LPS/Ng-induced pyroptotic cell death via regulating K48-linked ULK1 ubiquitination in macrophages.

Protein Tyrosine Kinase 7 Regulates EGFR/Akt Signaling Pathway and Correlates With Malignant Progression in Triple-Negative Breast Cancer.

PURPOSE: Triple-negative breast cancer (TNBC), the most aggressive subtype of breast cancer, is associated with high invasiveness, high metastatic occurrence and poor prognosis. Protein tyrosine kinase 7 (PTK7) plays an important role in multiple cancers. However, the role of PTK7 in TNBC has not been well addressed. This study was performed to evaluate the role of PTK7 in the progression of TNBC. METHODS: Correlation of PTK7 expression with clinicopathological parameters was assessed using tissue microarray immunohistochemistry (IHC) staining in 280 patients with breast cancer. PTK7 expression in TNBC (MDA-MB-468, MDA-MB-436 and MDA-MB-231) and non-TNBC (MCF7 and SK-BR-3) breast cancer cell lines were examined using immunoblotting assay. PTK7 correlated genes in invasive breast carcinoma were analyzed using cBioPortal breast cancer datasets including 1,904 patients. PTK7 overexpressed or knockdown TNBC cell lines (MDA-MB-468 and MDA-MB-436) were used to analyze the potential roles of PTK7 in TNBC metastasis and tumor progression. A TNBC tumor bearing mouse model was established to further analyze the role of PTK7 in TNBC tumorigenicity in vivo. RESULTS: PTK7 is highly expressed in breast cancer and correlates with worse prognosis and associates with tumor metastasis and progression in TNBC. Co-expression analysis and gain- or loss-of-function of PTK7 in TNBC cell lines revealed that PTK7 participates in EGFR/Akt signaling regulation and associated with extracellular matrix organization and migration genes in breast cancer, including COL1A1, FN1, WNT5B, MMP11, MMP14 and SDC1. Gain- or loss-of-function experiments of PTK7 suggested that PTK7 promotes proliferation and migration in TNBC cell lines. PTK7 knockdown MDA-MB-468 cell bearing mouse model further demonstrated that PTK7-deficiency inhibits TNBC tumor progression in vivo. CONCLUSION: This study identified PTK7 as a potential marker of worse prognosis in TNBC and revealed PTK7 promotes TNBC metastasis and progression via EGFR/Akt signaling pathway.

Overexpression of YB1 C-terminal domain inhibits proliferation, angiogenesis and tumorigenicity in a SK-BR-3 breast cancer xenograft mouse model.

Y-box-binding protein 1 (YB1) is a multifunctional transcription factor with vital roles in proliferation, differentiation and apoptosis. In this study, we have examined the role of its C-terminal domain (YB1 CTD) in proliferation, angiogenesis and tumorigenicity in breast cancer. Breast cancer cell line SK-BR-3 was infected with GFP-tagged YB1 CTD adenovirus expression vector. An 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) proliferation assay showed that YB1 CTD decreased SK-BR-3 cell proliferation, and down-regulated cyclin B1 and up-regulated p21 levels in SK-BR-3 cells. YB1 CTD overexpression changed the cytoskeletal organization and slightly inhibited the migration of SK-BR-3 cells. YB1 CTD also inhibited secreted VEGF expression in SK-BR-3 cells, which decreased SK-BR-3-induced EA.hy926 endothelial cell angiogenesis in vitro. YB1 CTD overexpression attenuated the ability of SK-BR-3 cells to form tumours in nude mice, and decreased in vivo VEGF levels and angiogenesis in the xenografts in SK-BR-3 tumour-bearing mice. Taken together, our findings demonstrate the vital role of YB1 CTD overexpression in inhibiting proliferation, angiogenesis and tumorigenicity of breast cancer cell line SK-BR-3.

Krüppel-like factor 4 induces apoptosis and inhibits tumorigenic progression in SK-BR-3 breast cancer cells.

Krüppel-like factor 4 (KLF4) functions as either a tumor suppressor or an oncogene in different tissues by regulating the expression of various genes. The aim of this study was to reveal the functions of KLF4 in regulating breast cancer apoptosis, proliferation, and tumorigenic progression. KLF4 expression levels in breast cancer tissues and breast cancer cell lines were found to be much lower than those in nontumorous tissues and a nontransformed mammary epithelial cell line. KLF4 was upregulated in the tumor necrosis factor-α-induced SK-BR-3 breast cancer cell apoptotic process. Overexpression of KLF4 promoted SK-BR-3 breast cancer cell apoptosis and suppressed SK-BR-3 cell tumorigenicity in vivo.

[Expressions of immunogenic molecules in low-dose radiotherapy-treated human renal clear cell carcinoma 786-0 cells].

OBJECTIVE: To explore the effects of low-dose radiation on the expression of immunogenic membrane molecules calreticulin (CRT) and MHC-I/II on the surface of human renal clear cell carcinoma 786-0 cells. METHODS: The inhibitory activity of low-dose radiation on cell line 786-0 was examined by CCK-8 assay. And the post-radiation membrane expressions of CRT, MHC-I and MHC-II were measured by flow cytometry while CRT was visualized by immunofluorescence photography. RESULTS: The inhibition rates on the proliferative capacities of four 786-0 cell lines rose with the incremental radiation doses of 0, 6, 12 and 24 Gy. And the CRT expression levels of each experimental group was significantly higher than that of the control group (P < 0.001). Along with incremental doses of irradiation, the average calreticulin fluorescence intensities increased gradually initially and then there was a downward trend. The membrane expressions of MHC-I and MHC-II of each experimental group was significantly higher than those of the control group (P < 0.05). As the irradiation dose increased, the average MHC-I fluorescence intensities increased gradually in a dose-dependent manner. CONCLUSION: The low-dose radiotherapy may up-regulate CRT and MHC class I/II related with the immunogenicity of tumor cells to induce immune response against tumors.

Effective adoptive transfer of haploidentical tumor-specific T cells in B16-melanoma bearing mice.

BACKGROUND: Adoptive transfer of allogeneic tumor-specific T cells often results in severe graft-versus-host disease (GVHD). Here, we sought to maximize graft-versus-tumor and minimize GVHD by using haploidentical T cells in pre-irradiated B16-melanoma bearing mice. METHODS: C57BL/6 mice bearing B16-melanoma tumors were irradiated with 0, 5, or 7 Gy total body irradiation (TBI), or 7 Gy TBI plus bone marrow transplantation. Tumor areas were measured every 3 days to assess the influence of irradiation treatment on tumor regression. B16-melanoma bearing mice were irradiated with 7 Gy TBI; sera and spleens were harvested at days 1, 3, 5, 7, 9, 11, and 13 after irradiation. White blood cell levels were measured and transforming growth factor ß1 (TGF-b1) and interleukin 10 (IL-10) levels in serum were detected using enzyme-linked immunosorbent assay (ELISA) kits. Real-time reverse transcription-polymerase chain reaction (RT-PCR) and flow cytometry were performed to test TGF-b1, IL-10 and Foxp3 mRNA levels and the proportion of CD4+CD25+Foxp3+ T-regulatory cells (Tregs) in spleens. B16-melanoma bearing C57BL/6 mice were irradiated with 7 Gy TBI followed by syngeneic (Syn1/Syn2) or haploidentical (Hap1/Hap2), dendritic cell-induced cytotoxic T lymphocytes (DC-CTLs) treatment, tumor areas and system GVHD were observed every 3 days. Mice were killed 21 days after the DC-CTLs adoptive transfer; histologic analyses of eyes, skin, liver, lungs, and intestine were then performed. RESULTS: Irradiation with 7 Gy TBI on the B16-melanoma-bearing mice did not influence tumor regression compared to the control group; however, it down-regulated the proportion of Tregs in spleens and the TGF-b1 and IL-10 levels in sera and spleens, suggesting inhibition of autoimmunity and intervention of tumor microenvironment. Adoptive transfer of haploidentical DC-CTLs significantly inhibited B16-melanoma growth. GVHD assessment and histology analysis showed no significant difference among the groups. CONCLUSION: Adoptive transfer of haploidentical tumor-specific T cells in irradiation-pretreated B16-melanoma bearing mice preserved antitumor capacity without causing a GVHD response.

[Expression of SM22α and its relationship with lymph node metastasis in breast cancer].

OBJECTIVE: to analyze the relationship between the expression of SM22α and the lymph node (LN) metastasis of breast cancer and to investigate its molecular mechanisms. METHODS: reverse transcription-polymerase chain reaction (RT-PCR) was performed to detect the expression of SM22α in breast cancer tissue and adjacent normal breast tissue. RT-PCR and Western blot were employed to investigate the SM22α mRNA and protein level in tissues of breast fibroadenoma, breast cancer without LN metastasis and breast cancer with LN metastasis. RT-PCR and zymography were used to detect the MMP2 and MMP9 expression and activity and TIMP1 expression level in breast fibroadenoma, breast cancer samples without LN metastasis and those with LN metastasis respectively. RESULTS: the expression level of SM22α mRNA in breast cancer was significantly lower than that in breast fibroadenoma or adjacent normal breast tissue (5.1% ± 2.4% vs 15.1% ± 5.3% vs 30.1% ± 5.1%, P < 0.01). The protein and mRNA expression level of SM22α in breast cancer samples with LN metastasis were significant lower than those of breast cancer without LN metastasis (6.2% ± 3.1% vs 10.1% ± 4.1%, P < 0.01). Both the expression and activity of MMP2 and MMP9 in breast cancer samples with LN metastasis were significant higher than those without LN metastasis (P < 0.01). A strong negative correlation was found between SM22α protein level and MMP2 activity (r = -0.848; n = 27; P < 0.01) or MMP9 activity (r = -0.916; n = 27; P < 0.01) in breast cancer tissue. CONCLUSION: a down-regulation of SM22α in breast cancer is correlated with LN metastasis. SM22α may inhibit the LN metastasis through a negative regulation of MMP2 and MMP9 in breast cancer.