Tumor-activated neutrophils promote metastasis in breast cancer via the G-CSF-RLN2-MMP-9 axis.

The immune component of the tumor microenvironment is essential for the regulation of cancer progression. In breast cancer (BC), a patient's tumor mass is frequently infiltrated by neutrophils (tumor-associated neutrophils, TANs). Our study addressed the role of TANs and their mechanism of action in BC. Using quantitative IHC, ROC, and Cox analysis, we demonstrated that a high density of TANs infiltrating the tumor parenchyma was predictive of poor prognosis and of decreased progression-free survival of patients with BC, who underwent surgical tumor removal without previous neoadjuvant chemotherapy, in 3 different cohorts: training, validation, and independent cohorts. Conditioned medium from human BC cell lines prolonged the lifespan of healthy donor neutrophils ex vivo. Neutrophils activated by the supernatants of BC lines demonstrated an increased ability to stimulate proliferation, migration, and invasive activity of BC cells. Cytokines involved in this process were identified using antibody arrays. The relationship between these cytokines and the density of TANs was validated by ELISA and IHC in fresh BC surgical samples. It was determined that tumor-derived G-CSF significantly extended the lifespan and increased the metastasis-promoting activities of neutrophils via the PI3K-AKT and NF-κB pathways. Simultaneously, TAN-derived RLN2 promoted the migratory abilities of MCF7 cells via PI3K-AKT-MMP-9. Analysis of tumor tissues from 20 patients with BC identified a positive correlation between the density of TANs and the activation of the G-CSF-RLN2-MMP-9 axis. Finally, our data demonstrated that TANs in human BC have detrimental effects, supporting malignant cell invasion and migration.

Multifaceted roles of CCL20 (C-C motif chemokine ligand 20): mechanisms and communication networks in breast cancer progression.

Emerging studies have demonstrated notable roles of CCL20 in breast cancer progression. Based on these findings, CCL20 has become a potential therapeutic target for cancer immunotherapy. Accordingly, studies utilizing monoclonal antibodies to target CCL20 are currently being experimented. However, the existence of cytokine network in the tumor microenvironment collectively regulates tumor progression. Hence, a deeper understanding of the role of CCL20 and the underlying signaling pathways regulating the functions of CCL20 may provide a novel strategy for therapeutic interventions. This review provides the current knowledge on how CCL20 interacts with breast cancer cells to influence tumor progression via immunosuppression, angiogenesis, epithelial to mesenchymal transition, migration/invasion and chemoresistance. As a possible candidate biomarker, we also reviewed signal pathways and other factors in the tumor microenvironment regulating the tumor-promoting functions of CCL20.These new insights may be useful to design new potent and selective CCL20 inhibitors against breast cancer in the future.

Tumor-associated neutrophils activated by tumor-derived CCL20 (C-C motif chemokine ligand 20) promote T cell immunosuppression via programmed death-ligand 1 (PD-L1) in breast cancer.

Breast cancer is the leading cause of cancer-related death among women despite the significant improvement in diagnosis and treatment. Tumor-associated neutrophils have been shown to suppress antitumor functions of the host. However, how breast cancer tumor microenvironment influences the phenotype and functions of neutrophils to potentiate T cell immunosuppression is unknown. Herein, neutrophils isolated from peripheral blood of healthy donors were treated with supernatants from breast cancer cell lines or recombinant human CCL20. PD-L1 expression on neutrophils was then evaluated by immunofluorescence and flow cytometry. Neutrophils and Jurkat T cells were cocultured to evaluate the effect of tumor-associated neutrophils on T cell functions. Finally, immunohistochemical staining was performed to evaluate the clinical relevance of neutrophils infiltrating breast tumor tissues. Tumor-derived CCL20 activated and upregulated PD-L1 expression on neutrophils. A significant positive correlation was found between CCL20 and CD66b+ neutrophils in tumor tissues. Through in vitro experiment, tumor-associated neutrophils (TANs) effectively suppressed T cell immunity which was reversed upon PD-L1 blockade.Moreover, a high density of TANs was associated with short disease free survival in breast cancer patients. Furthermore, receiver operating curve showed that the density of TANs could accurately predict disease-free survival in breast cancer patients. Our findings suggest that targeting TANs via CCL20 immunosuppressive pathway may be a novel therapeutic strategy for breast cancer treatment.

Lung Adenocarcinoma Cells Promote Self-Migration and Self-Invasion by Activating Neutrophils to Upregulate Notch3 Expression of Cancer Cells.

Background: Invasion and migration of cancer cells play a key role in lung cancer progression and metastasis. Tumor-associated neutrophils (TANs) are related to poor prognosis in many types of cancer. However, the role of TANs in lung cancer is controversial. In this study, we investigated the effect of TANs on the invasion and migration of lung adenocarcinoma. Methods: Immunohistochemistry was performed to detect the density of infiltrating TANs and the expression of Notch3 in 100 lung adenocarcinoma tissues. Flow cytometry was used to observe the viability of neutrophils, which were isolated from healthy peripheral blood and then exposed to the supernatant of cultured lung adenocarcinoma cell lines. After treating with tumor-associated neutrophils culture supernatant, NeuCS (supernatant of cultured neutrophils), tumor cells culture supernatant, Medium (serum-free medium), respectively, the migration and invasion of the lung cancer cells before and after transfected by si-Notch3 were detected by transwell assay and wound healing assay. Kaplan-Meier plotter (http://kmplot.com/analysis/index.php?p) was used to analyze the prognostic role of the density of TANs on lung adenocarcinoma and TIMER ((http://cistrome.dfci.harvard.edu/TIMER/) was used to detect the expression of Notch3 on lung adenocarcinoma. Results: The infiltration of TANs was observed in the parenchyma and stroma of the lung adenocarcinoma, the density of TANs was positively related to the TNM stage and negatively related to the differentiation and prognosis. Notch3 expression of cancer cells was negatively related to the tumor differentiation and prognosis. Compared to quiescent neutrophils, the viability of TCCS-activated neutrophils was enhanced. Both migration and invasion of A549 and PC9 cells were significantly promoted by TANs, while after knocking down Notch3, the migration and invasion of the cancer cells were not affected by TANs. Bioinformatics analysis showed that the density of TANs and the expression of Notch3 were related to the poor prognosis. Conclusion: The results indicated that lung adenocarcinoma cells promote self-invasion and self-migration by activating neutrophils to upregulate the Notch3 expression of cancer cells. The density of infiltrating TANs may be a novel marker for the poor prognosis of lung adenocarcinoma. Targeting TANs might be a potential therapeutic strategy for lung cancer treatment.

ARPC2 promotes breast cancer proliferation and metastasis.

Actin-related protein 2/3 complex (ARPC2) is an actin­binding component involved in the regulation of actin polymerization. It mediates the formation of branched actin networks and contacts the mother actin filament. Migration and invasion are key processes which enable tumor cells to infiltrate blood vessels or lymphatic vessels, and the actin pathway plays a very important role. Given that ARPC2 is critical to this progression, the present study focused on ARPC2 activity in breast cancer (BrCa) cell invasion and migration. Limited data are available on the expression and role of ARPC2 proteins in breast carcinomas. We screened the Oncomine database for messenger RNAs (mRNAs) that are upregulated in BrCa and found that ARPC2 was one of the most consistently involved mRNAs in BrCa. The analysis of immunohistochemical data revealed that ARPC2 expression was higher in breast cancerous tissues than in adjacent non­cancerous tissues. In addition, ARPC2 was highly associated with the tumor stage, nodal metastasis, and overall survival of patients with BrCa. We performed siRNA­ARPC2 transfection to investigate the effect of ARPC2 on the proliferation, migration, invasion and arrest of BrCa cells. It was revealed that ectopic ARPC2 expression significantly upregulated N­cadherin, vimentin, ZEB1, MMP­9 and MMP­3 expression and also activated the TGF­ß pathway to contribute to epithelial­mesenchymal transition (EMT). These results collectively indicated that ARPC2 promoted the tumorigenesis of breast carcinoma and the initiation of EMT. Therefore, ARPC2 was revealed to be a potential therapeutic target in patients with BrCa.

LMO4 mediates trastuzumab resistance in HER2 positive breast cancer cells.

Breast cancer is the leading cause of cancer-related mortality in women worldwide. Trastuzumab (Herceptin) is an effective antibody drug for HER2 positive breast cancer; de novo or acquired trastuzumab resistance retarded the use of trastuzumab for at least 70% of HER2 positive breast cancers. In this study, we reported LMO4 (a member of LIM-only proteins) promoted trastuzumab resistance in human breast cancer cells. Over-expression of LMO4 was observed in acquired trastuzumab resistance breast cancer cells SKBR3 HR and BT474 HR. Depletion of LMO4 partly abolished the trastuzumab resistance of SKBR3 HR and BT474 HR cells. Forced expression of LMO4 significantly increased trastuzumab resistance of HER2 positive breast cancer cells both in vitro and in vivo. BCL-2 was regulated by LMO4 and mediated the promoting role of LMO4 in trastuzumab resistance of HER2 positive breast cancer cells. High level of LMO4 was associated with worse clinicopathological parameters (including tumor size and histological grade) and lower survival rate in HER2 positive breast cancer patients. LMO4 therefore could be used as a target to develop diagnostic and therapeutic methods for human HER2 positive breast cancer.

Decreased digit ratio (2D:4D) and gastric cancer in Chinese men.

BACKGROUND: The development of finger length is influenced by the level of hormones during pregnancy in the womb. The relative length of 2nd to 4th digit (2D:4D) is considered as a putative marker for prenatal hormone exposure and may represent an individual susceptibility to certain diseases, particularly those hormone-related cancers (e.g., gastric cancer). AIMS: The aim of this study is to investigate whether there is a possible relationship between 2D:4D ratio and gastric cancer (GCA) in Chinese men. METHODS: 94 male patients with GCA and 91 controls were chosen to participate in this study. Photographs of both hands were collected and then the lengths of second and fourth digits of both hands were measured. Left hand, right hand, mean hand, and right minus left hand (∆R-L) 2D:4D ratios were analyzed and compared. RESULTS: In GCA group, 2D:4D ratios were significantly lower (right hand: p<0.01; left hand, mean hand: p<0.001) than controls. No association was observed between 2D:4D ratio and tumor staging (neither in tumor size (T) nor in lymph node involvement (N) or distant metastases (M)). There was also no correlation between 2D:4D ratio and age of onset. CONCLUSIONS: Decreased 2D:4D ratio may be an indicator for forecasting the susceptibility to develop GCA.

Digit ratio (2D:4D) in a Chinese population with schizophrenia.

BACKGROUND: Prenatal gonadal hormones may play a major role in pathogenesis of schizophrenia. It has been reported that second to fourth digit ratio (2D:4D) was influenced by the levels of exposure to prenatal testosterone and estrogen. So, 2D:4D may help to predict the disease susceptibility to schizophrenia. AIMS: The aim of this study was to investigate the relationship between the digit ratio (2D:4D) and schizophrenia in Chinese population. METHODS: We recruited 178 schizophrenics (males: 76; females: 102) and 365 controls (males: 218; females: 147) in this study. Photocopies of both hands were collected and left hand, right hand, mean hand and left minus right hand (DL-R) 2D:4D were analyzed. RESULTS: The right and mean hand 2D:4D ratios were significantly higher in schizophrenics compared to that of controls in both males and females. The left hand 2D:4D ratio in female schizophrenics was also significantly higher than in controls. Compared to controls, the DL-R 2D:4D in male schizophrenics was obviously higher. There was a weakly (but not significantly) negative correlation between the mean hand 2D:4D ratio and the age of onset. CONCLUSIONS: The 2D:4D ratio may correlate with the schizophrenia in Chinese population, and it may be an indicator of schizophrenia.

[Correlation of the second to fourth digit ratio with the body mass index of infertile men].

OBJECTIVE: To investigate the relationship between the second to the fourth digit ratio (2D:4D) and body mass index (BMI) in infertile men of the Han ethnic group in Ningxia. METHODS: Using anthropometry, we calculated the mean ratio of 2D:4D and BMI of 197 infertile men and 148 normal healthy male controls, followed by analysis of their relationship. RESULTS: The BMI was correlated positively with the 2D:4D ratio of the left hand in the infertile men (P < 0.05) and in the patients with a higher 2D:4D ratio of the left hand (P < 0.05), but negatively with the 2D:4D ratio of the righ/left (Dr-1) (left: P < 0.01; Dr-l: P < 0.05). The mean 2D: 4D ratio and BMI were both lower in the normal control than in the infertile men, with statistically significant differences in BMI (P < 0.05) and the 2D:4D ratio of the left hand (P < 0.05). CONCLUSION: There is a correlation between the 2D:4D ratio and BMI in infertile men.

Dermatoglyphs in Coronary Artery Disease Among Ningxia Population of North China.

BACKGROUND: Coronary artery disease (CAD) is an enormous health problem in the world. Dermatoglyphs are cutaneous ridges on the fingers, palms, and soles, formed by genetic regulation and control during early intrauterine life. The Dermatoglyphic traits do not change significantly as the growth of the age. They may be the phenotypic characters of individual genes and represent the predisposition to certain diseases. AIMS AND OBJECTIVES: The study was carried out to document characteristic dermatoglyphic patterns in coronary artery disease which could be useful in early diagnosis of the disease. MATERIALS AND METHODS: Dermatoglyphic study of 258 male (129 coronary artery disease cases and 129 normal subjects) of Ningxia China were studied in the present cross-sectional study. It involved the digital patterns, ATD angles, A-B ridge counts on the hands. Chi-square test, t-test were used for the statistical analysis in this study. RESULTS: The overall frequency of whorls was higher followed by loop and arch in both two groups. It was observed that there was significant difference of digital frequency of whorls and ulnar loops in patients in both hands as compared to controls (p≤0.01). The mean value of finger ridge counts, total ridge counts were similar between two groups. The A-B ridge counts were significantly higher in coronary artery disease compared with controls on the right palm (p≤0.01). However, the mean ATD angle values were significantly higher in cases than those of in normal on both hands (p<0.05). CONCLUSION: Abnormally high A-B ridge count, ATD angles and the frequency of whorls are characteristic dermatoglyphic patterns of coronary artery disease. Dermatoglyphics may have an important role in early diagnosis of coronary artery disease in future.