Junfang Qin

Adrenergic receptor β2 activation by stress promotes breast cancer progression through macrophages M2 polarization in tumor microenvironment

Stress and its related hormones epinephrine (E) and norepinephrine (NE) play a crucial role in tumor progression. Macrophages in the tumor microenvironment (TME) polarized to M2 is also a vital pathway for tumor deterioration. Here, we explore the underlying role of macrophages in the effect of stress and E promoting breast cancer growth. It was found that the weight and volume of tumor in tumor bearing mice were increased, and dramatically accompanied with the rising E level after chronic stress using social isolation. What is most noteworthy, the number of M2 macrophages inside tumor was up-regulated with it. The effects of E treatment appear to be directly related to the change of M2 phenotype is reproduced in vitro. Moreover, E receptor ADRβ2 involved in E promoting M2 polarization was comprehended simultaneously. Our results imply psychological stress is influential on specific immune system, more essential for the comprehensive treatment against tumors. [BMB Reports 2015; 48(5): 295-300]

M-CSF cooperating with NFκB induces macrophage transformation from M1 to M2 by upregulating c-Jun.

Increasing evidence suggests tumor-associated macrophages (TAMs) are polarized M2 subtype of macrophage that exerts pro-tumor effects and promote the malignancy of some cancers, but the concrete mechanism is not well defined. Our previous research exhibited that proto-oncogene AP-1 regulated IL-6 expression in macrophages and promoted the formation of M2 macrophages. In this study, we investigate whether extra-cellular stimulus M-CSF help this process or nuclear factor NFκB has a synergistic role in the activation state of polarized M2 subtype of macrophage. RAW 264.7 macrophage and 4T1 mouse breast cancer cells were co-cultured to reconstruct tumor microenvironment. Being co-cultured with 4T1 or its supernatant, the expression of c-Jun, the member of AP-1 family, has a dramatically increase both on the level of gene and on the protein in RAW 264.7 macrophages, but the expression of c-Fos does not increase neither on the level of gene nor on the protein. After co-cultured with 4T1, RAW 264.7 has a higher consumption of M-CSF than RAW 264.7 macrophages alone. With the stimulation of M-CSF, the mRNA of c-Jun increased significantly, but decreased remarkably after adding the anti-M-CSF. And at the same time, p50, the member of NFκB family, has a similar tendency to c-Jun. WB results suggest that with the stimulation of M-CSF, p-Jun in nuclear increases heavily but decreases after the neutralizing antibody added. Coimmunoprecipitation and immunoblotting techniques confirmed that c-Jun and p50 NFκB coprecipitated, and c-Jun protein expression is properly enhanced with rM-CSF effect. In conclusion, M-CSF induces macrophage transformation by upregulating c-Jun with a certain synergy of NFκB. Our study may present a novel therapeutic strategy against cancer.

PTEN inhibits macrophage polarization from M1 to M2 through CCL2 and VEGF-A reduction and NHERF-1 synergism

PTEN has been studied in several tumor models as a tumor suppressor. In this study, we explored the role of PTEN in the inhibition state of polarized M2 subtype of macrophage in tumor microenvironment (TME) and the underlying mechanisms. To elucidate the potential effect in TME, RAW 264.7 macrophages and 4T1 mouse breast cancer cells were co-cultured to reconstruct tumor microenvironment. After PTEN was down-regulated with shRNA, the expression of CCL2 and VEGF-A, which are definited to promote the formation of M2 macrophages, have a dramatically increase on the level of both gene and protein in co-cultured RAW 264.7 macrophages. And at the same time, NHERF-1 (Na+/H+ exchanger regulating factor-1), another tumor suppressor has a similar tendency to PTEN. Q-PCR and WB results suggested that PTEN and NHERF-1 were consistent with one another no matter at mRNA or protein level when exposed to the same stimulus. Coimmunoprecipitation and immunofluorescence techniques confirmed that PTEN and NHERF-1 were coprecipitated, and NHERF-1 protein expression was properly reduced with rCCL2 effect. In addition, cell immunofluorescence images revealed a profound transferance, in co-cultured RAW 264.7 macrophages, an up-regulation of NHERF-1 could promote the PTEN marked expression on the cell membrane, and this form for the interaction was not negligible. These observations illustrate PTEN with a certain synergy of NHERF-1, as well as down-regulation of CCL2 suppressing M2 macrophage transformation pathway. The results suggest that the activation of PTEN and NHERF-1 may impede the evolution of macrophages beyond the M1 into M2 phenotype in tumor microenvironment.

hTERT rs2736098 genetic variants and susceptibility of hepatocellular carcinoma in the Chinese population:a case-control study

BACKGROUND The human telomerase reverse transcriptase (hTERT) gene encodes the catalytic subunit of telomerase, which mediates pleiotropic effects, including the regulation of senescence and proliferation and plays an important role in carcinogenesis. This study attempts to clarify the genetic predisposition to hepatocellular carcinoma (HCC), focusing on the hTERT gene rs2736098 polymorphism. METHOD Four hundred patients with HCC and 400 non-cancer controls were genotyped to elucidate the potential association between hTERT rs2736098 polymorphism and HCC risks. RESULTS Compared with the controls, the patients with HCC had a lower frequency of G/G genotype (33.3% vs 44.3%, P=0.001) and a higher frequency of G/A (51.5% vs 39.5%, P=0.001). Allele genotypic frequencies in the patients differed from those of the controls (P=0.040). The data of this study rs2736098[A] allele contributed significantly to HCC risk in female patients (OR=1.78, 95% CI, 1.17-2.72, P=0.007), patients with HCV infection (OR=2.89, 95% CI, 1.08-7.70, P=0.031), non-drinker patients (OR=1.32, 95% CI, 1.06-1.65, P=0.015), and patients not affected by HBV (OR=1.77, 95% CI, 1.30-2.40, P<0.001). CONCLUSIONS rs2736098[A] may be an independent hereditary parameter in HCC, but some risk factors would cover up the association by more powerful hepatocarcinogenesis. These results are important guidance for further studies in detecting HCC-associated single nucleotide polymorphisms.

Autophagy suppresses isoprenaline-induced M2 macrophage polarization via the ROS/ERK and mTOR signaling pathway

Abstract The objective of this study was to examine the effect of autophagy on stress‐induced M2 macrophage polarization in the tumor microenvironment of breast cancer and to determine whether the underlying mechanism was related to the reactive oxygen species (ROS)/ERK and mTOR pathway. In vitro, we found that the basal autophagy level in mouse RAW 264.7 macrophages decreased with the incubation of tumor cell culture supernatant. Similarly, the polarization of RAW 264.7 to M2 macrophages was inhibited by the autophagy inducer rapamycin and increased by the autophagy inhibitor 3‐MA or by siBeclin1. In addition, we found that not only was M2 molecule expression down‐regulated but intracellular ROS generation was also blocked by autophagy induction. In vivo, we observed that mice that received an isoprenaline injection as a stress agent exhibited augmented implanted breast tumor growth, lung metastasis, intratumoral mRNA expression of M2 molecules and serum ROS generation. In contrast, the intratumoral expression of LC3‐II and Beclin1 was decreased. In addition, we observed that isoprenaline induced the up‐regulation of the intratumoral expression of phosphorylated mTOR, phosphorylated ERK1/2, phosphorylated Tyr705‐STAT3 and HIF‐1&agr;, whereas rapamycin induced an opposite effect on the same molecules and could abolish the effects of isoprenaline. These results suggest that autophagy might suppress M2 macrophage polarization induced by isoprenaline via the ROS/ERK and mTOR signaling pathway. Our findings provide a theoretical basis for why high levels of stress hormones accelerate the progression of breast cancer, and autophagy may play a role in determining the outcomes of cancer therapy. Graphical abstract Figure. No caption available. HighlightsAn interplay between stress and autophagy is investigated in macrophage polarization.Autophagy inhibits M2 polarization in the TME and then suppresses cell migration.Iso enhances TAM transformation to the M2 phenotype and cancer progression in Balb/c mice.ROS/ERK and mTOR signaling mediates the inhibition of autophagy on Iso‐induced M2 polarization.

The influence of miR-34a expression on stemness and cytotoxic susceptibility of breast cancer stem cells.

ABSTRACT In this study, we investigate the effect of miR-34a expression and biological characteristics of breast cancer stem cells (BCSCs). The mammospheres were formed from murine breast cancer cell line 4T1 and regarded as murine BCSCs. Identification of stemness molecules and cloning experiments validate the biological characteristics of BCSCs we have established. We showed that miR-34a, as a tumor suppressor, could separately reduce the stemness of BCSCs and activate the cytotoxic susceptibility of BCSCs to natural killer (NK) cells in vitro via down regulating the expression of Notch1 signaling molecules. Moreover, miR-34a could completely restrain established mice breast tumor xenografts in vivo in the NOD/SCID mice that have functional NK cells at a normal level, whereas it was less effective in NOD/SCID/ CD122/IL-2Rβ mice that do not have functional NK cells. We conclude that miR-34a is a crucial, dual tumor suppressor and BCSCs-targeting immunotherapeutic agent and has shown efficacy in the treatment of murine breast cancer. The results also suggest that impaired NK cells could contribute to the resistance to therapies.

IDO1 impairs NK cell cytotoxicity by decreasing NKG2D/NKG2DLs via promoting miR-18a

HIGHLIGHTSmiR‐18a directly down‐regulated both the expression of NKG2D in NK cells and Mult‐1(NKG2DL) in cancer cells.IDO1/ miR‐18a/ NKG2D/NKG2DL regulatory axis in the regulation of NK cell function.IDO1 inhibitors or miR‐18a interference on NK cells activation might inhibit cancer development. Background Indoleamine‐2,3‐dioxygenase 1 (IDO1) is an important enzyme for altering the tumour microenvironment and assisting tumour cells to escape the immune system. Results In this study, a significant reduction in NK cell cytotoxicity that was associated with a high expression of IDO1 in a reconstructed tumour microenvironment was observed. In a co‐culture system of tumour cell culture supernatant (TSN) and murine NK cell, IDO1 was substantially increased, while NKG2D was markedly downregulated in NK cells. Based on computational predictions, miR‐18a, which has two definite binding sites consisting of the 3′UTR of NKG2D and the 3′UTR of NKG2D ligand (Mult‐1), was suspected to be a negative regulator of which its conjoined. As expected, the IDO1 could promote the expression of miR‐18a and promote the downregulation effect of miR‐18a on NKG2D and NKG2DL, and INCB024360 (INCB) could reverse the result. For digging the mechanism deeper, we authenticated IDO1 promoted the combination of miR‐18a and AGO2 after argonaute 2 (AGO2) co‐immunoprecipitation, which then degraded Mult‐1 mRNA and inhibited the translation of it, further destructing NK cell cytotoxicity. Conclusion Our findings revealed a new regulatory axis, IDO1/miR‐18a/NKG2D/NKG2DL, in the regulation of NK cell function. This may provide insight into the mechanism of the priming effect of IDO1 inhibitors and miR‐18a interference, then elicit possible new methods of cancer treatment.

miR-21a negatively modulates tumor suppressor genes PTEN and miR-200c and further promotes the transformation of M2 macrophages

miR‐21a is well‐known to inhibit PTEN expression. We have previously shown that PTEN suppressed the transformation of M2 macrophages in the tumor microenvironment. Therefore, we hypothesized that miR‐21a could influence M2 macrophage transformation by regulating PTEN expression. In this study, we identified how miR‐21a reduced the expression of both PTEN mRNA and protein in murine macrophage cell lines and primary macrophages. Moreover, opposite effects were identified upon the use of a miR‐21a inhibitor. Using a cytokine array, we identified the cytokines closely associated with miR‐21a‐mediated macrophage transformation to the M2 phenotype. miR‐21a mimics could also enhance the migratory ability of murine breast cancer 4T1 cells, the growth of breast cancer in vivo and CD206 intratumor expression. In addition, quantitative PCR (qPCR) and methylation‐specific PCR analysis showed that miR‐21a enhanced miR‐200c methylation and then decreased miR‐200c and PTEN expression. These effects could be reversed by treatment with 5′‐Aza, a DNA‐demethylating agent. MiR‐200c was predicted to target the PTEN 3′UTR, but qPCR illustrated the miR‐200c mimic that increased PTEN expression, and 5′‐Aza could enhance its effect. The above results indicate that miR‐21a negatively modulates two tumor suppressor genes, miR‐200c and PTEN, thereby promoting M2 macrophage transformation. This demonstrates that miR‐21a represents a novel target for improving the overall tumor microenvironment.

Downregulation of MCT4 for lactate exchange promotes the cytotoxicity of NK cells in breast carcinoma

Monocarboxylate transporter‐4 (MCT4), a monocarboxylic acid transporter, demonstrates significantly increased expression in the majority of malignancies. We performed an experiment using BALB/C mice, and our results showed that ShMCT4 transfection or the pharmaceutic inhibition of MCT4 with 7acc1 strengthens the activity of NK cells. The results of a calcein assay revealed that the cytotoxicity of NK cells was strengthened via inhibition of MCT4. In addition, ELISA testing showed that the content of perforin and CD107a was increased, and PCR amplification and immunoblotting revealed that the expression of NKG2D and H60 was upregulated after the inhibition of MCT4. Further, we observed an elevated pH value, decreased extracellular lactate flow, and attenuated tumor growth. Therefore, we concluded that the inhibition of MCT4 enhanced the cytotoxicity of NK cells by blocking lactate flux and reversing the acidified tumor microenvironment. In addition to these findings, we also discovered that MCT4 depletion may have a pronounced impact on autophagy, which was surmised by observing that the inhibition of autophagy (3MA) pulled the enhanced cytotoxicity of NK cells downwards. Together, these data suggest that the key effect of MCT4 depletion on NK cells probably utilizes inductive autophagy as a compensatory metabolic mechanism to minimize the acidic extracellular microenvironment associated with lactate export in tumors.