Yunjie Zeng

miR-142-5p and miR-130a-3p are regulated by IL-4 and IL-13 and control profibrogenic macrophage program

Macrophages play a pivotal role in tissue fibrogenesis, which underlies the pathogenesis of many end-stage chronic inflammatory diseases. MicroRNAs are key regulators of immune cell functions, but their roles in macrophages fibrogenesis have not been characterized. Here we show that IL-4 and IL-13 induce miR-142-5p and downregulate miR-130a-3p in macrophages; these changes sustain the profibrogenic effect of macrophages. In vitro, miR-142-5p mimic prolongs STAT6 phosphorylation by targeting its negative regulator, SOCS1. Blocking miR-130a relieves its inhibition of PPARγ, which coordinates STAT6 signalling. In vivo, inhibiting miR-142-5p and increasing miR-130a-3p expression with locked nucleic acid-modified oligonucleotides inhibits CCL4-induced liver fibrosis and bleomycin-induced lung fibrosis in mice. Furthermore, macrophages from the tissue samples of patients with liver cirrhosis and idiopathic pulmonary fibrosis display increased miR-142-5p and decreased miR-130a-3p expression. Therefore, miR-142-5p and miR-130a-3p regulate macrophage profibrogenic gene expression in chronic inflammation.

miR-21 Induces Myofibroblast Differentiation and Promotes the Malignant Progression of Breast Phyllodes Tumors

Phyllodes tumors of breast, even histologically diagnosed as benign, can recur locally and have metastatic potential. Histologic markers only have limited value in predicting the clinical behavior of phyllodes tumors. It remains unknown what drives the malignant progression of phyllodes tumors. We found that the expression of myofibroblast markers, α-smooth muscle actin (α-SMA), fibroblast activation protein (FAP), and stromal cell-derived factor-1 (SDF-1), is progressively increased in the malignant progression of phyllodes tumors. Microarray showed that miR-21 was one of the most significantly upregulated microRNAs in malignant phyllodes tumors compared with benign phyllodes tumors. In addition, increased miR-21 expression was primarily localized to α-SMA-positive myofibroblasts. More importantly, α-SMA and miR-21 are independent predictors of recurrence and metastasis, with their predictive value of recurrence better than histologic grading. Furthermore, miR-21 mimics promoted, whereas miR-21 antisense oligos inhibited, the expression of α-SMA, FAP, and SDF-1, as well as the proliferation and invasion of primary stromal cells of phyllodes tumors. The ability of miR-21 to induce myofibroblast differentiation was mediated by its regulation on Smad7 and PTEN, which regulate the migration and proliferation, respectively. In breast phyllodes tumor xenografts, miR-21 accelerated tumor growth, induced myofibroblast differentiation, and promoted metastasis. This study suggests an important role of myofibroblast differentiation in the malignant progression of phyllodes tumors that is driven by increased miR-21.

CCL18 from tumor-associated macrophages promotes angiogenesis in breast cancer

The infiltration of tumor-associated macrophages (TAMs) is associated with extensive angiogenesis, which contributes to a poor prognosis in breast cancer. However, anti-angiogenic therapy with VEGF-specific monotherapy has been unsuccessful in treating breast cancer, and the molecular mechanisms associated with chemoresistance remain unclear. Here, we investigated whether CCL18, a chemokine produced by TAMs, can stimulate angiogenesis in breast cancer, as well as the underlying mechanisms. Double immunohistochemical staining for CCL18 and CD34/CD31/vWF was performed in 80 breast cancer samples to study the correlation between CCL18+ TAMs and microvascular density (MVD). Cocultures of TAMs with human umbilical vein endothelial cells (HUVECs) were used to model the inflammatory microenvironment, and CCL18-induced angiogenesis was evaluated both in vitro and in vivo. We demonstrated that CCL18+ TAM infiltration positively associated with MVD in breast cancer samples, which was correlated with tumor metastasis and poor prognosis. We confirmed, both in vitro and in vivo, that CCL18 and VEGF synergistically promoted endothelial cell migration and angiogenesis. Conversely, blocking CCL18 or VEGF with neutralizing antibodies synergistically inhibited the promigratory effects of TAMs. Silencing PITPNM3, a putative CCL18 receptor, on the surface of HUVECs abrogated CCL18-mediated promigration and the enhancement of HUVEC tube formation, independently of VEGFR signaling. Moreover, CCL18 exposure induced the endothelial-mesenchymal transformation and activated ERK and Akt/GSK-3β/Snail signaling in HUVECs, thereby contributing to its pro-angiogenic effects. In conclusion, our findings suggest that CCL18 released from TAMs promotes angiogenesis and tumor progression in breast cancer; thus, CCL18 may serve as a novel target for anti-angiogenic therapies.

Ubc9 expression predicts chemoresistance in breast cancer.

Ubiquitin-conjugating enzyme 9 (Ubc9), the sole conjugating enzyme for sumoylation, regulates protein function and plays an important role in tumorigenesis. Whether Ubc9 is involved in the chemoresistance of breast cancer remains unknown. In this study, we aimed to evaluate the contribution of Ubc9 in the chemoresistance of breast cancer. Immunohistochemistry (IHC) was used to examine the expression level of Ubc9. Chi-square test, Wilcoxon test, and one-way ANOVA were applied to analyze the relationship between Ubc9 expression, clinicopathologic features, and clinical response to neoadjuvant chemotherapy. The significance of variables for survival was analyzed by the Cox proportional hazards model in a multivariate analysis. Kaplan-Meier survival curves were plotted and log-rank test was performed. The proportion of Ubc9-positive cells was higher in invasive ductal carcinoma than in normal breast tissues [(48.48 ± 17.94)% vs. (5.82 ± 2.80)%, P < 0.001]. High Ubc9 expression was associated with poor differentiation (χ2 = 6.538, P = 0.038), larger tumor size (χ2 = 4.701, P = 0.030), advanced clinical stage (χ2 = 4.651, P = 0.031), lymph node metastasis (χ2 = 9.913, P = 0.010), basal-like phenotype (χ2 = 8.660, P = 0.034), and poor clinical response to neoadjuvant chemotherapy (χ2 = 11.09, P = 0.001). The expected 6-year cumulative disease-free survival rate was 87.32% in patients with low Ubc9 expression compared to 68.78% in those with high Ubc9 expression (χ2 = 4.289, P = 0.038). These data indicate that high Ubc9 expression correlates with poor response to chemotherapy and poor clinical prognosis.

Comparison of ER/PR and HER2 statuses in primary and paired liver metastatic sites of breast carcinoma in patients with or without treatment

PurposeThe aim of this study was to determine whether estrogen receptor (ER)/progesterone receptor (PR) and human epidermal growth factor receptor type 2 (HER2) statuses between primary tumors and paired liver metastatic localizations of breast carcinoma were modified by treatment or during the natural metastatic process.MethodsER, PR, and HER2 expressions were analyzed on paired tissue specimens taken from the primary and the liver metastatic tumors in breast cancer patients. The first group included 46 women who presented with T1–T4, N0–N3, M0 breast carcinoma when first diagnosed and were treated by neoadjuvant therapy or directly underwent surgery, then received postoperative treatment and developed liver metastasis several months/years later. The second group included 12 patients with liver metastatic breast carcinoma when first diagnosed for breast cancer. HER2 status was determined by immunohistochemistry as well as fluorescence in situ hybridization.ResultsAmong the 46 patients in the first group, the ER/PR and HER2 statuses (when considered as a whole histological subtype) were changed between primary tumor and liver metastatic lesions in 12 patients (26.1%). While ER and PR status were modified in 14 (30.4%) and 25 (54.3%) patients, respectively, there were only 5 (10.9%) cases showed a discrepancy in the HER2 status. In the second group, the ER/PR and HER2 statuses (when considered as a whole subtype) were consistent between primary and liver metastatic tumor in 10 of 12 (83.3%) patients. ER, PR, and HER2 statuses were modified in 0 of 12 (0%), 4 of 12 (33.3%), and 1 of 12 (8.3%) cases, respectively.ConclusionsER/PR and HER2 statuses between primary and liver metastatic lesions of breast carcinoma can be modified after treatment but are stable in most cases during the natural metastatic process.

Blocking the recruitment of naive CD4 + T cells reverses immunosuppression in breast cancer

The origin of tumor-infiltrating Tregs, critical mediators of tumor immunosuppression, is unclear. Here, we show that tumor-infiltrating naive CD4+ T cells and Tregs in human breast cancer have overlapping TCR repertoires, while hardly overlap with circulating Tregs, suggesting that intratumoral Tregs mainly develop from naive T cells in situ rather than from recruited Tregs. Furthermore, the abundance of naive CD4+ T cells and Tregs is closely correlated, both indicating poor prognosis for breast cancer patients. Naive CD4+ T cells adhere to tumor slices in proportion to the abundance of CCL18-producing macrophages. Moreover, adoptively transferred human naive CD4+ T cells infiltrate human breast cancer orthotopic xenografts in a CCL18-dependent manner. In human breast cancer xenografts in humanized mice, blocking the recruitment of naive CD4+ T cells into tumor by knocking down the expression of PITPNM3, a CCL18 receptor, significantly reduces intratumoral Tregs and inhibits tumor progression. These findings suggest that breast tumor-infiltrating Tregs arise from chemotaxis of circulating naive CD4+ T cells that differentiate into Tregs in situ. Inhibiting naive CD4+ T cell recruitment into tumors by interfering with PITPNM3 recognition of CCL18 may be an attractive strategy for anticancer immunotherapy.

Validation and comparison of models to predict non-sentinel lymph node metastasis in breast cancer patients.

Several models for predicting the risk of non‐sentinel lymph node (NSLN) metastasis in breast cancer patients with positive sentinel lymph nodes (SLNs) have been developed. The purpose of this study was to validate and compare these models in Chinese patients. A total of 159 breast cancer patients with positive SLNs treated at our institution were included. Among them, 81 (50.9%) patients had at least one NSLN involvement. The Cambridge, Mou, Mayo, Tenon, MDA, Memorial Sloan‐Kettering Cancer Center (MSKCC), Ljubljana, SNUH, Turkish, Louisville, Stanford, and Saidi models were evaluated and compared using receiver operating characteristic (ROC) curves, calibration plots, and false negative (FN) rates. The Cambridge and Mou models outperformed the others, both with area under the ROC curves (AUCs) of 0.73. The Mayo, Tenon, MDA, MSKCC, Turkish, Ljubljana, SNUH, and Louisville models had AUCs of 0.68, 0.66, 0.66, 0.64, 0.63, 0.62, 0.61, and 0.60, respectively. The Stanford and Saidi models did not present any discriminative capabilities, with AUCs of 0.54 and 0.50, respectively. The Cambridge, MSKCC, and Mayo models were well calibrated. With adjusted thresholds, the Mayo model outperformed the others by classifying the highest proportion of patients (20%) into the low‐risk group. Our study revealed that the Cambridge and Mou models performed well in Chinese patients. The ROC curves, calibration plots, and FN rates should be used together for the accurate evaluation of prediction models. Selection of these models should be based on the clinicopathological features of the targeted population. The models specifically designed for patients with micrometastases or macrometastases of SLNs are needed in the future. (Cancer Sci 2012; 103: 274–281)

A comparison of survival outcomes and side effects of toremifene or tamoxifen therapy in premenopausal estrogen and progesterone receptor positive breast cancer patients: a retrospective cohort study.

BackgroundIn premenopausal women, endocrine adjuvant therapy for breast cancer primarily consists of tamoxifen alone or with ovarian suppressive strategies. Toremifene is a chlorinated derivative of tamoxifen, but with a superior risk-benefit profile. In this retrospective study, we sought to establish the role of toremifene as an endocrine therapy for premenopausal patients with estrogen and/or progesterone receptor positive breast cancer besides tamoxifen.MethodsPatients with early invasive breast cancer were selected from the breast tumor registries at the Sun Yat-Sen Memorial Hospital (China). Premenopausal patients with endocrine responsive breast cancer who underwent standard therapy and adjuvant therapy with toremifene or tamoxifen were considered eligible. Patients with breast sarcoma, carcinosarcoma, concurrent contralateral primary breast cancer, or with distant metastases at diagnosis, or those who had not undergone surgery and endocrine therapy were ineligible. Overall survival and recurrence-free survival were the primary outcomes measured. Toxicity data was also collected and compared between the two groups.ResultsOf the 810 patients reviewed, 452 patients were analyzed in the study: 240 received tamoxifen and 212 received toremifene. The median and mean follow up times were 50.8 and 57.3 months, respectively. Toremifene and tamoxifen yielded similar overall survival values, with 5-year overall survival rates of 100% and 98.4%, respectively (p = 0.087). However, recurrence-free survival was significantly better in the toremifene group than in the tamoxifen group (p = 0.022). Multivariate analysis showed that recurrence-free survival improved independently with toremifene (HR = 0.385, 95% CI = 0.154-0.961; p = 0.041). Toxicity was similar in the two treatment groups with no women experiencing severe complications, other than hot flashes, which was more frequent in the toremifene patients (p = 0.049). No patients developed endometrial cancer.ConclusionToremifene may be a valid and safe alternative to tamoxifen in premenopausal women with endocrine-responsive breast cancer.

CCL18/PITPNM3 enhances migration, invasion, and EMT through the NF-κB signaling pathway in hepatocellular carcinoma

Chemokine ligand 18 (CCL18) has been associated with hepatocellular carcinoma (HCC) metastasis. Here, we demonstrated a novel mechanism through which CCL18 enhances cell migration, invasion, and epithelial–mesenchymal transition (EMT) in HCC. (1) Using immunohistochemistry, we analyzed the expression of PITPNM3, a molecule that correlated with CCL18 signaling, in 149 HCC tissue specimens. The results showed that PITPNM3 expression is highly associated with tumor metastasis and differentiation; (2) in vitro experiments showed that CCL18 enhances cell migration, invasion, and EMT in PITPNM3(+) HCC cells but not in PITPNM3(-) cells. Silencing of PITPNM3 by short interfering RNA (siRNA) inhibited the induction of cell migration, invasion, and EMT by CCL18; (3) Cell migration, invasion, and EMT induced by CCL18 accompanied with the phosphorylation of IKK and IKBα as well as p65 nuclear translocation in PITPNM3(+) HCC cells, but not in the cells that PITPNM3 is silenced with siRNA, implying that the activation of NF-κB signaling is involved in the action of CCL18/PITPNM3. These results suggest that CCL18 enhances HCC cell migration, invasion, and EMT through the expression of PITPNM3 and the activation of the NF-κB signaling pathway.

HER-2 positive breast cancer is associated with an increased risk of positive cavity margins after initial lumpectomy

BackgroundThe effect of breast cancer subtype on margin status after lumpectomy remains unclear. This study aims to determine whether approximated breast cancer subtype is associated with positive margins after lumpectomy, which could be used to determine if there is an increased risk of developing local recurrence (LR) following breast-conserving surgery.MethodsWe studied 1,032 consecutive patients with invasive cancer who received lumpectomies and cavity margin (CM) assessments from January 2003 to November 2012. The following data were collected: patient age, cT stage, pT stage, grade, status of CM, lymph node status, menopausal status, ER, PR, HER-2, and Ki67, as well as the presence of extensive intraductal component (EIC) and lymphovascular invasion (LVI). A χ2 test was used to compare categorical baseline characteristics. Univariate and multivariate logistic regression analyses were performed to evaluate associations between pathologic features of CM status. Kaplan-Meier actuarial cumulative rates of LR (ipsilateral in-breast) were calculated.ResultsA total of 7,884 pieces of marginal tissue were collected from 1,032 patients, and 209 patients had positive CMs. Of the patients tested, 52.3% had luminal A subtype, 14.9% were luminal B, 12.8% were luminal-HER-2, 8.1% were HER-2 enriched, and 11.8% were triple negative. Univariate analysis showed that EIC (P <0.001), LVI (P = 0.026), pN stage (N1 vs. N0: P = 0.018; N3 vs. N0: P <0.001), and luminal B (P = 0.001) and HER-2 (P <0.001) subtypes were associated with positive CMs. Multivariable analysis indicated that only EIC (P <0.001), pN stage (P = 0.003), and HER-2 subtype (P <0.001) were significantly correlated with positive CMs. On multivariable analysis, HER-2 subtype was an independent prognostic factor in LR (P = 0.031).ConclusionsThe HER-2 subtype was the predictive factor most associated with positive CMs and an independent prognostic factor for LR. This result suggests that the increased risk of LR in HER-2 breast cancer is due to an increased microscopic invasive tumor burden, which is indicated by margin status after lumpectomy.