Does the Dose of Standard Adjuvant Chemotherapy Affect the Triple-negative Breast Cancer Benefit from Extended Capecitabine Metronomic Therapy? An Exploratory Analysis of the SYSUCC-001 Trial.

Purpose: Results from studies of extended capecitabine after the standard adjuvant chemotherapy in early stage triple-negative breast cancer (TNBC) were inconsistent, and only low-dose capecitabine from the SYSUCC-001 trial improved disease-free survival (DFS). Adjustment of the conventional adjuvant chemotherapy doses affect the prognosis and may affect the efficacy of subsequent treatments. This study investigated whether the survival benefit of the SYSUCC-001 trial was affected by dose adjustment of the standard adjuvant chemotherapy or not. Patients and Methods: We reviewed the adjuvant chemotherapy regimens before the extended capecitabine in the SYSUCC-001 trial. Patients were classified into "consistent" (standard acceptable dose) and "inconsistent" (doses lower than acceptable dose) dose based on the minimum acceptable dose range in the landmark clinical trials. Cox proportional hazards model was used to investigate the impact of dose on the survival outcomes. Results: All 434 patients in SYSUCC-001 trial were enrolled in this study. Most of patients administered the anthracycline-taxane regimen accounted for 88.94%. Among patients in the "inconsistent" dose, 60.8% and 47% received lower doses of anthracycline and taxane separately. In the observation group, the "inconsistent" dose of anthracycline and taxane did not affect DFS compared with the "consistent" dose. Moreover, in the capecitabine group, the "inconsistent" anthracycline dose did not affect DFS compared with the "consistent" dose. However, patients with "consistent" taxane doses benefited significantly from extended capecitabine (P=0.014). The sufficient dose of adjuvant taxane had a positive effect of extended capecitabine (hazard ratio [HR] 2.04; 95% confidence interval [CI] 1.02 to 4.06). Conclusion: This study found the dose reduction of adjuvant taxane might negatively impact the efficacy of capecitabine. Therefore, the reduction of anthracycline dose over paclitaxel should be given priority during conventional adjuvant chemotherapy, if patients need dose reduction and plan for extended capecitabine.

Effect of Capecitabine Maintenance Therapy Using Lower Dosage and Higher Frequency vs Observation on Disease-Free Survival Among Patients With Early-Stage Triple-Negative Breast Cancer Who Had Received Standard Treatment: The SYSUCC-001 Randomized Clinical Trial.

Importance: Among all subtypes of breast cancer, triple-negative breast cancer has a relatively high relapse rate and poor outcome after standard treatment. Effective strategies to reduce the risk of relapse and death are needed. Objective: To evaluate the efficacy and adverse effects of low-dose capecitabine maintenance after standard adjuvant chemotherapy in early-stage triple-negative breast cancer. Design, Setting, and Participants: Randomized clinical trial conducted at 13 academic centers and clinical sites in China from April 2010 to December 2016 and final date of follow-up was April 30, 2020. Patients (n = 443) had early-stage triple-negative breast cancer and had completed standard adjuvant chemotherapy. Interventions: Eligible patients were randomized 1:1 to receive capecitabine (n = 222) at a dose of 650 mg/m2 twice a day by mouth for 1 year without interruption or to observation (n = 221) after completion of standard adjuvant chemotherapy. Main Outcomes and Measures: The primary end point was disease-free survival. Secondary end points included distant disease-free survival, overall survival, locoregional recurrence-free survival, and adverse events. Results: Among 443 women who were randomized, 434 were included in the full analysis set (mean [SD] age, 46 [9.9] years; T1/T2 stage, 93.1%; node-negative, 61.8%) (98.0% completed the trial). After a median follow-up of 61 months (interquartile range, 44-82), 94 events were observed, including 38 events (37 recurrences and 32 deaths) in the capecitabine group and 56 events (56 recurrences and 40 deaths) in the observation group. The estimated 5-year disease-free survival was 82.8% in the capecitabine group and 73.0% in the observation group (hazard ratio [HR] for risk of recurrence or death, 0.64 [95% CI, 0.42-0.95]; P = .03). In the capecitabine group vs the observation group, the estimated 5-year distant disease-free survival was 85.8% vs 75.8% (HR for risk of distant metastasis or death, 0.60 [95% CI, 0.38-0.92]; P = .02), the estimated 5-year overall survival was 85.5% vs 81.3% (HR for risk of death, 0.75 [95% CI, 0.47-1.19]; P = .22), and the estimated 5-year locoregional recurrence-free survival was 85.0% vs 80.8% (HR for risk of locoregional recurrence or death, 0.72 [95% CI, 0.46-1.13]; P = .15). The most common capecitabine-related adverse event was hand-foot syndrome (45.2%), with 7.7% of patients experiencing a grade 3 event. Conclusions and Relevance: Among women with early-stage triple-negative breast cancer who received standard adjuvant treatment, low-dose capecitabine maintenance therapy for 1 year, compared with observation, resulted in significantly improved 5-year disease-free survival. Trial Registration: ClinicalTrials.gov Identifier: NCT01112826.

Regulation of Hippo/YAP signaling and Esophageal Squamous Carcinoma progression by an E3 ubiquitin ligase PARK2.

Objective: Esophageal squamous cell carcinoma (ESCC) is one of the most commonly diagnosed cancer types in China. Recent genomic sequencing analysis indicated the over-activation of Hippo/YAP signaling might play important roles for the carcinogenic process and progression for ESCC patients. However, little is known about the molecular mechanisms that controls Hippo signaling activity in ESCC. Our previous studies indicated that PLCE1-an important risk factor for ESCC-linked to ESCC progression through snail signaling, during this period, we found PARK2 was an important downstream target of PLCE1-snail axis. PARK2 was decreased in ESCC human samples, and correlated with good prognosis in ESCC patients. Further research showed that PARK2 could inhibit YAP, which functions as key downstream effectors of the Hippo pathway. Here, we aim to reveal the molecular mechanisms of PARK2 modulated Hippo pathway in ESCC. Methods: To evaluate the function of PARK2 in ESCC, we used a tissue microarray (TMA) of 223 human ESCC patients and immunohistochemistry to analyze the correlation between PARK2 expression and clinicopathologic variables. Depletion of endogenous PARK2 and YAP from ESCC cells using CRISPR/Cas9 technologies. Flow cytometry and EdU cell proliferation assay were used to detect proliferation of ESCC cells. Nude mice subcutaneous injection and Ki-67 staining were used to evaluate tumor growth in vivo. Migration and invasion assays were performed. In addition, lung metastasis models in mice were used to validate the function of PARK2 in vivo. Identification of PARK2 involved in hippo pathway was achieved by expression microarray screening, double immunofluorescence staining and co-immunoprecipitation assays. The RNA-seq analysis results were validated through quantitative real-time PCR (qRT-PCR) analysis. The protein half-life of YAP was analyzed by Cycloheximide assay, and the TEAD activity was detected by Luciferase reporter assays. Results: Clinical sample of ESCC revealed that low PARK2 expression correlated with late tumor stage (P < 0.001), poor differentiation (P < 0.04), lymph node (P < 0.001) and distant metastasis (P = 0.0087). Multivariate Cox proportional regression analysis further revealed that PARK2 expression (P = 0.032) is an independent prognostic factor for the overall survival of ESCC patients. Besides, the immunohistochemistry results showed that PARK2 negatively correlated with YAP protein level (P < 0.001). PARK2 depletion promotes ESCC progression both through Hippo/YAP axis, while PARK2 overexpression suppresses ESCC tumor progression by Hippo signaling. Co-IP and ubiquitination assays revealed that PARK2 could interact with YAP in the cytosol and promotes YAP K48-linked ubiquitination at K90 sites. Conclusion: Clinical sample analysis and mechanistic study have validated PARK2 as a tumor suppressor for ESCC. Multivariate Cox proportional regression analysis further revealed that PARK2 is an independent prognostic factor for the overall survival of ESCC patients. Cellular and molecular mechanisms in this study showed that PARK2 associated with YAP protein in the cytosol, promoted YAP ubiquitination and proteasome-dependent degradation in ESCC cells. Therefore, as a novel modulator for Hippo signaling, modulation of PARK2 activity or gene expression level could be an appealing strategy to treat esophageal.

RACO-1 modulates Hippo signalling in oesophageal squamous cell carcinoma.

Oesophageal cancer is one of the most lethal malignancies worldwide, whereas the 5-year survival is less than 20%. Although the detailed carcinogenic mechanisms are not totally clear, recent genomic sequencing data showed dysregulation of Hippo signalling could be a critical factor for oesophageal squamous cell carcinoma (ESCC) progression. Therefore, understanding of the molecular mechanisms that control Hippo signalling activity is of great importance to improve ESCC diagnostics and therapeutics. Our current study revealed RACO-1 as an inhibitory protein for YAP/TEAD axis. Depletion of RACO-1 increases the protein level of YAP and expression of YAP/TEAD target gene. Besides, RACO-1 silencing could promote ESCC cell invasion and migration, which effect could be rescued by YAP depletion in ESCC cells. Immunoprecipitation showed that RACO-1 associated with YAP and promote ubiquitination and degradation of YAP at k48 poly-ubiquitination site. Our research discovered a new regulator of Hippo signalling via modulating YAP stability. RACO-1 could be a promising factor, which serves cancer diagnostics and therapeutics in ESCC patients.

SHARPIN Inhibits Esophageal Squamous Cell Carcinoma Progression by Modulating Hippo Signaling.

Esophageal cancer is one of the leading malignancies worldwide, while around sixty percent of newly diagnosed cases are in China. In recent years, genome-wide sequencing studies and cancer biology studies show that Hippo signaling functions a critical role in esophageal squamous cell carcinoma (ESCC) progression, which could be a promising therapeutic targets in ESCC treatment. However, the detailed mechanisms of Hippo signaling dys-regulation in ESCC remain not clear. Here we identify SHARPIN protein as an endogenous inhibitor for YAP protein. SHARPIN depletion significantly decreases cell migration and invasion capacity in ESCC, which effects could be rescued by further YAP depletion. Depletion SHARPIN increases YAP protein level and YAP/TEAD target genes, such as CTGF and CYR61 in ESCC. Immuno-precipitation assay shows that SHARPIN associates with YAP, promoting YAP degradation possibly via inducing YAP K48-dependent poly-ubiquitination. Our study reveals a novel post-translational mechanism in modulating Hippo signaling in ESCC. Overexpression or activation of SHARPIN could be a promising strategy to target Hippo signaling for ESCC patients.

RNF168 facilitates proliferation and invasion of esophageal carcinoma, possibly via stabilizing STAT1.

Oesophageal cancer ranks as one of the most common malignancy in China and worldwide. Although genome-wide association studies and molecular biology studies aim to elucidate the driver molecules in oesophageal cancer progression, the detailed mechanisms remain to be identified. Interestingly, RNF168 (RING finger protein 168) shows a high frequency of gene amplification in oesophageal cancer from TCGA database. Here, we report an important function for RNF168 protein in supporting oesophageal cancer growth and invasion by stabilizing STAT1 protein. RNF168 gene is amplified in oesophageal cancer samples, which tends to correlate with poor prognosis. Depletion RNF168 causes decreased cell proliferation and invasion in oesophageal cancer cells. Through unbiased RNA sequencing in RNF168 depleted oesophageal cancer cell, we identifies JAK-STAT pathway is dramatically decreased. Depletion RNF168 reduced JAK-STAT target genes, such as IRF1, IRF9 and IFITM1. Immuno-precipitation reveals that RNF168 associates with STAT1 in the nucleus, stabilizing STAT1 protein and inhibiting its poly-ubiquitination and degradation. Our study provides a novel mechanism that RNF168 promoting JAK-STAT signalling in supporting oesophageal cancer progression. It could be a promising strategy to target RNF168 for oesophageal cancer treatment.

Circulating CD8+ T-cell repertoires reveal the biological characteristics of tumors and clinical responses to chemotherapy in breast cancer patients.

PURPOSE: CD8+ T cells are primarily cytotoxic cells that provide immunological protection against malignant cells. Considerable evidence suggests that the T-cell repertoire is closely associated with the host immune response and the development of cancer. In this study, we explored the characteristics of the circulating CD8+ T-cell repertoire and their potential value in predicting the clinical response of breast cancer patients to chemotherapy. EXPERIMENTAL DESIGN: We applied a high-throughput TCR ß-chain sequencing method to characterize the CD8+ T-cell repertoire of the peripheral blood from 26 breast cancer patients. In addition, changes in the circulating CD8+ T-cell repertoire during chemotherapy were analyzed. RESULTS: We found that the HEC ratios of the CD8+ T-cell repertoires from HER2+ breast cancer patients were significantly higher than those of HER2- patients, suggesting that the HER2 protein is released into circulation where it is targeted by CD8+ T cells. Several Vß and CDR3 motifs preferentially used in HER2+ patients were identified. Besides, we found that the circulating CD8+ T-cell repertoires evolved during chemotherapy and correlated with patient clinical responses to chemotherapy. Increased CD8+ T-cell repertoire heterogeneity during chemotherapy was associated with a better clinical response. CONCLUSIONS: Although functional studies of clonally expanded CD8+ T-cell populations are clearly required, our results suggest that the circulating CD8+ T-cell repertoire reflects the characteristics of the tumor-associated biomolecules released into the blood and correlates with the clinical responses of the patients to chemotherapy which might assist in making treatment decisions.

Antityrosinase and antioxidant properties of mung bean seed proanthocyanidins: Novel insights into the inhibitory mechanism.

This study investigated the structure, antioxidant activity, antityrosinase activity and mechanism of proanthocyanidins from mung bean seed [Vigna radiata (L.) Wilczek]. The structural composition were characterized by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), electrospray ionization-full-mass spectrometry (ESI-Full-MS), and high-pressure liquid chromatography-electrospray ionization-mass spectrometry (HPLC-ESI-MS) techniques. The mung bean seed proanthocyanidins were composed of procyanidins, prodelphinidins, and their rhamnosides. According to enzyme kinetic analysis, these compounds were potent, reversible, and mixed-type inhibitors of tyrosinase. They inhibited the enzyme activity by interacting with enzyme as well as substrates. The results of molecular docking showed that the interaction between mung bean seed proanthocyanidins and tyrosinase was driven by hydrogen bond, hydrophobic and electrostatic interactions. In addition, mung bean seed proanthocyanidins were demonstrated as powerful antioxidants. Therefore, this study confirmed a novel tyrosinase inhibitor and would lay a scientific foundation for their utilization in pharmaceutical and food industries.

Different Modulatory Effects of IL-17, IL-22, and IL-23 on Osteoblast Differentiation.

OBJECTIVES: To examine the expressions of IL-17, IL-22, and IL-23 receptors in four osteoblast models and the effects of IL-17, IL-22, and IL-23 on osteoblasts. METHODS: Gene expression levels of receptors, alkaline phosphatase (ALP), osteocalcin (OCN), and Runt-related transcription factor 2 (Runx-2), were evaluated by RT-PCR and real-time RT-PCR. Proliferative responses and cell cycle analysis were detected by a CCK-8 assay and flow cytometry, respectively. ALP activity and ALP mass were detected by an ALP activity assay and ALP staining, respectively. RESULTS: In primary osteoblasts, only the IL-17 receptor was expressed. In C2C12, MC3T3-E1, and Saos-2 cells, the genes of IL-17, IL-22, and IL-23 receptors were not detectable. None of IL-17, IL-22, and IL-23 had an obvious effect on the proliferation of primary osteoblasts, but IL-17 exhibited an inhibitory effect on the gene expression of ALP, OCN, and Runx-2. The ALP activity and ALP mass of primary osteoblasts were downregulated by IL-17 treatment in a dose-dependent manner, and IL-17 failed to inhibit BMP-2-induced phosphorylation of Smad. CONCLUSION: Primary osteoblasts constitutively express IL-17 receptors, but none of C2C12 cells, MC3T3-E1 cells, and Saos-2 cells express any receptors for IL-17, IL-22, and IL-23. IL-17 inhibits BMP-2-induced osteoblast differentiation via the BMP/Smad-independent pathway.

Anti-aging pharmacology in cutaneous wound healing: effects of metformin, resveratrol, and rapamycin by local application.

Cutaneous wounds are among the most common soft tissue injuries and are particularly hard to heal in aging. Caloric restriction (CR) is well documented to extend longevity; pharmacologically, profound rejuvenative effects of CR mimetics have been uncovered, especially metformin (MET), resveratrol (RSV), and rapamycin (RAPA). However, locally applied impacts and functional differences of these agents on wound healing remain to be established. Here, we discovered that chronic topical administration of MET and RSV, but not RAPA, accelerated wound healing with improved epidermis, hair follicles, and collagen deposition in young rodents, and MET exerted more profound effects. Furthermore, locally applied MET and RSV improved vascularization of the wound beds, which were attributed to stimulation of adenosine monophosphate-activated protein kinase (AMPK) pathway, the key mediator of wound healing. Notably, in aged skin, AMPK pathway was inhibited, correlated with impaired vasculature and reduced healing ability. As therapeutic approaches, local treatments of MET and RSV prevented age-related AMPK suppression and angiogenic inhibition in wound beds. Moreover, in aged rats, rejuvenative effects of topically applied MET and RSV on cell viability of wound beds were confirmed, of which MET showed more prominent anti-aging effects. We further verified that only MET promoted wound healing and cutaneous integrity in aged skin. These findings clarified differential effects of CR-based anti-aging pharmacology in wound healing, identified critical angiogenic and rejuvenative mechanisms through AMPK pathway in both young and aged skin, and unraveled chronic local application of MET as the optimal and promising regenerative agent in treating cutaneous wound defects.

[Effects of Modified Ruji Recipe in Preventing Relapse and Metastasis of Breast Cancer Patients with Negative Hormone Receptor].

Objective To observe the effect of Ruji Recipe (RR) (treated by syndrome typing) in preventing the relapse and metastasis of invasive ductal breast cancer patients with negative hormone receptor (HR) after surgery and chemotherapy. Methods Using a prospective, cohort method, 136 pa- tients with stage I - III C HR negative invasive ductal breast cancer were equally assigned to the treat- ment group (treated by RR in syndrome typing way) and the control group (routine follow-ups). Disease free survival (DFS) , overall survival (OS) , relapse and metastasis were observed in the two groups. Re- sults All patients were followed-up for 15 to 57 months, with the median follow-up of 44 months. The median DFS and OS had not reached. The 1. 0, 1. 5, 2. -0, and 3. 0 years DFS were 94.1 % (64/68) , 86. 4 % (51/59), 81. 8% (45/55), and 72. 0% (36/50) in the treatment group. They were 77. 9% (53/68), 67.2% (45)67), 60. 6% (40)66), and 54. 5% (36/66) in the control group. Significant difference existed in 1. 0, 1. 5, and 2. 0 years DFS between the two groups (X² = 7.403, 6.426, 6.459; P =0. 012, 0.013, 0. 016). No statistical difference existed in 1. 0, 1. 5, 2. 0, and 3. 0 years OS between the two groups (P >0. 05). Among triple negative breast cancer (TNBC) patients, 1. 0, 1. 5, 2. 0, and 3. 0 years DFS were 97. 0% (32/33), 92. 9% (26/28), 92.6% (2527), and 84. 6% (22/26) in the treatment group, 81. 5% (2227), 66. 7% (1827), 61. 5% (16/26), and 57. 7% (15/26) in the control group. Of them, significant difference existed in 1. 5, 2. 0, and 3. 0 years DFS between the two groups (X² =5. 893, 7. 293, 4. 591 ; P = 0. 015, 0. 007, 0. 032). At the end of follow-ups, relapse and metastasis occurred in 15 patients, local recur- rence in 2 patients, single organ metastasis in 6 patients, and multiple organs metastasis in 7 patients of the treatment group. The relapse and metastasis occurred in 30 patients , local recurrence in 2 patients , single organ metastasis in 12 patients, and multiple organs metastasis in 16 patients of the treatment group. Conclusions RR ( by syndrome typing) could improve DFS and delay progression of invasive ductal breast cancer patients with negative HR in the first 2 years after surgery. It also had certain value for relapse and metastasis of TNBC patients within 2 years.

[Expression of RANKL/OPG in male mandible of different ages].

PURPOSE: To investigate the relationship between male osteoporosis and the expression of receptor activator of NF-κB ligand(RANKL) and osteoprotegerin(OPG)-mRNA in male mandible at different ages. METHODS: Between May 2008 and January 2009, bone tissues of the mandible were collected as the experimental material from 46 patients suffering from jaw facial deformity and extraction. The patients with periodontist, systemic disorder and smoking as well as drinking were excluded.They were divided into three groups: young group, whose age was 10-29 years old ;middle age group, whose age was 30-59 years old; aged group, whose age was 60-89 years old. The expression of RANKL mRNA and OPG mRNA was examined by real-time PCR. The data was analyzed using ANOVA followed by least significant difference (LSD) test with SPSS16.0 software package. RESULTS: (1)Compared with the young group, RANKL mRNA level in mandible was 2.1-fold and 5.3-fold higher in the middle age and aged groups, respectively, whereas OPG mRNA level was 3.3-fold and 4.8-fold higher in middle age and aged groups, respectively. RANKL and OPG were positively correlated with age.(2)The ratio of RANKL/OPG in middle age group was lower than that of young group and old group,respectively. CONCLUSIONS: (1)The expression of RANKL and OPG increases with age remarkably.(2)Bone formation of the mandible is activated in middle aged group. The formation is over absorption.(3)Bone formation of the mandible in aged group is at low level. Bone absorption exceeds bone formation.

[Effect of clpE gene deletion on virulence of Streptococcus pneumoniae].

OBJECTIVE: To study the effect of clpE gene deletion on the virulence of Streptococcus pneumoniae. METHODS: The clpE-deficient strain was constructed by LFH-PCR and identified by PCR and sequencing. The impact of clpE mutant on the virulence of S. pneumoniae was evaluated in a mouse model. In addition,we also studied the effect of clpE mutant on adherence and invasion of host cells. Real time RT-PCR was used to measure the mRNA expression levels of autolysin A, pneumococcal surface adhesion A, pneumolysin, pneumococcal surface protein A and neuraminidase. RESULTS: The clpE gene was replaced completely by erm cassette. Mice virulence experiments showed that the median lethal time of the wide-type was 54 h, whereas that of clpE mutant was 21d (P < 0.01). Cell culture infection experiments indicated that adherence and invasion of clpE mutant were strongly reduced (P < 0.05). The expression of virulent factors in clpE mutant was lower than that of the wild-type (P < 0.05). CONCLUSION: ClpE is involved in virulence by modulating the expressions of virulence factors.