Identification of Differentially Expressed Hub Genes Associated With Immune Cell Recruitment in Claudin-Low Breast Cancer

Breast cancer (BCa) is the most common malignancy in women and claudin-low breast cancer (CL-BCa) is a newly identified BCa subtype characterized by low expression of claudin 3&4&7. However, the hub genes associated with the recruitment of immune cells into CL-BCa were rarely described. This study aimed at exploring the differentially expressed hub genes associated with tumor-infiltrating immune cells in CL-BCa by a multi-approach bioinformatics analysis. The top 200 genes associated with CL-BCa were screened in the METABRIC dataset; the PPI network was constructed using STRING and Cytoscape; tumor-infiltrating immune cells were analyzed by TIMER 2.0; and the correlation of feature cytokines and claudins on survival was examined in METABRIC and TCGA datasets. Consequently, we found that the fraction of tumor-infiltrating immune cells, especially CD8+T cells and macrophages, increased in the CL-BCa. Differentially expressed cytokines (CCL5, CCL19, CXCL9 and CXCL10) were related to the overall survival, and their expression levels were also examined both in tumor tissues of CL-BCa patients by IHC and in typical CL-BCa cell lines by qPCR. Moreover, the BCa patients with low expression of these differentially expressed claudins (CLDN8, CLDN11 and CLDN19) showed a worse overall survival. This study sheds light on molecular features of CL-BCa on immune microenvironments and contributes to identification of prognosis biomarkers for the CL-BCa patients

Triptolide Inhibits the Proliferation of Prostate Cancer Cells and Down-Regulates SUMO-Specific Protease 1 Expression

Recently, traditional Chinese medicine and medicinal herbs have attracted more attentions worldwide for its anti-tumor efficacy. Celastrol and Triptolide, two active components extracted from the Chinese herb Tripterygium wilfordii Hook F (known as Lei Gong Teng or Thunder of God Vine), have shown anti-tumor effects. Celastrol was identified as a natural 26 s proteasome inhibitor which promotes cell apoptosis and inhibits tumor growth. The effect and mechanism of Triptolide on prostate cancer (PCa) is not well studied. Here we demonstrated that Triptolide, more potent than Celastrol, inhibited cell growth and induced cell death in LNCaP and PC-3 cell lines. Triptolide also significantly inhibited the xenografted PC-3 tumor growth in nude mice. Moreover, Triptolide induced PCa cell apoptosis through caspases activation and PARP cleavage. Unbalance between SUMOylation and deSUMOylation was reported to play an important role in PCa progression. SUMO-specific protease 1 (SENP1) was thought to be a potential marker and therapeutical target of PCa. Importantly, we observed that Triptolide down-regulated SENP1 expression in both mRNA and protein levels in dose-dependent and time-dependent manners, resulting in an enhanced cellular SUMOylation in PCa cells. Meanwhile, Triptolide decreased AR and c-Jun expression at similar manners, and suppressed AR and c-Jun transcription activity. Furthermore, knockdown or ectopic SENP1, c-Jun and AR expression in PCa cells inhibited the Triptolide anti-PCa effects. Taken together, our data suggest that Triptolide is a natural compound with potential therapeutic value for PCa. Its anti-tumor activity may be attributed to mechanisms involving down-regulation of SENP1 that restores SUMOylation and deSUMOyaltion balance and negative regulation of AR and c-Jun expression that inhibits the AR and c-Jun mediated transcription in PCa

DataSheet_1_Identification of Differentially Expressed Hub Genes Associated With Immune Cell Recruitment in Claudin-Low Breast Cancer.pdf

Breast cancer (BCa) is the most common malignancy in women and claudin-low breast cancer (CL-BCa) is a newly identified BCa subtype characterized by low expression of claudin 3&4&7. However, the hub genes associated with the recruitment of immune cells into CL-BCa were rarely described. This study aimed at exploring the differentially expressed hub genes associated with tumor-infiltrating immune cells in CL-BCa by a multi-approach bioinformatics analysis. The top 200 genes associated with CL-BCa were screened in the METABRIC dataset; the PPI network was constructed using STRING and Cytoscape; tumor-infiltrating immune cells were analyzed by TIMER 2.0; and the correlation of feature cytokines and claudins on survival was examined in METABRIC and TCGA datasets. Consequently, we found that the fraction of tumor-infiltrating immune cells, especially CD8+T cells and macrophages, increased in the CL-BCa. Differentially expressed cytokines (CCL5, CCL19, CXCL9 and CXCL10) were related to the overall survival, and their expression levels were also examined both in tumor tissues of CL-BCa patients by IHC and in typical CL-BCa cell lines by qPCR. Moreover, the BCa patients with low expression of these differentially expressed claudins (CLDN8, CLDN11 and CLDN19) showed a worse overall survival. This study sheds light on molecular features of CL-BCa on immune microenvironments and contributes to identification of prognosis biomarkers for the CL-BCa patients.</p

Triptolide down-regulated SENP1 expression in PCa cells.

<p>(A) Triptolide decreased SENP1 mRNA level in both LNCaP and PC-3 cells in a dose-dependent manner. Cells were treated with indicated doses of Triptolide or Celastrol for 24 h before analysis. qRT-PCR were performed using the specific primers for SENP1 and β-actin (used as an internal control). (B) Triptolide decreased SENP1 mRNA level in both LNCaP and PC-3 cells in a time-dependent manner. Cells were treated with 0.1 µM Triptolide or Celastrol for indicated times, SENP1 and β-actin mRNA levels were determined by qRT-PCR. (C) Effect of Triptolide on SENP1 mRNA level in PCa cells. IC₅₀ was shown as calculated by Prism 5.04. (D) and (E) Triptolide decreased SENP1 protein level in LNCaP (D) and PC-3 (E) in a dose-dependent manner. Cells were treated with indicated doses of Triptolide or Celastrol for 24 h before Western blot analysis. α-tubulin was used as a loading control. (F) Triptolide decreased SENP1 protein level in PCa cells in a time-dependent manner. PC-3 cells were treated with 1 µM Triptolide for indicated time before Western blot analysis. β-actin was used as a loading control. (G) Triptolide enhanced cellular SUMOylation in PC-3 cells. PC-3 cells were treated with 1 µM Triptolide or Celastrol for 24 h before Western blot analysis using SUMO-1 antibody. NEM was used as a positive control for a SUMO protease inhibitor.</p

Hypothetic mechanisms of anti-PCa effects of Triptolide.

<p>Triptolide suppresses SENP1, AR and c-Jun expression, induces restoration of SUMOylation/deSUMOyaltion balance, inhibitions of the AR and c-Jun mediated transcription, which may all contribute to the anti-PCa effect of Triptolide. Other genes or proteins may involve in mechanism of Triptolide effect (see Discussion for detail).</p

Down-regulation or over-expression of SENP1, c-Jun or AR inhibited Triptolide anti-PCa efficacy.

<p>(A) and (B) Down-regulation of SENP1, c-Jun or AR expression increased cell viability upon Triptolide treatment. LNCaP and PC-3 cells were transfected with specific siRNA against SENP1, c-Jun and AR. 24 h after transfection, the transfected cells were treated with 100 nM triptolide or vehicle control for 48 h, then the viable cell number was counted in each treatment. (A) Effect of knockdown SENP1, c-Jun and AR on Triptolide efficacy in LNCaP cell. Left chart shows the viable cell number in each treatment. Right chart shows the viability ratio. (B) Effect of knockdown SENP1 and c-Jun on Triptolide efficacy in PC-3 cell. * indicates P<0.05 (compared to control). (C) Down-regulation of SENP1, c-Jun and AR protein levels by siRNA in (A) and (B). (D) and (E) Over-expression of SENP1, c-Jun and AR rescued cell viability upon Triptolide treatment. PCa cells were transfected or cotransfected with 800 ng SENP1, c-Jun and AR expression plasmids DNA as indicated. EGFP protein was used as a negative control and the Triptolide binding protein XPB was used as a positive control. Empty vector was used as blank control and to keep the total amount of plasmids DNA equal in each well. 48 h after transfection, cells were treated with Triptolide for another 48 h and viable cell number were counted. (D) Effect of ectopic expression or coexpression of SENP1, c-Jun and AR on Triptolide efficacy in LNCaP cells. (E) Effect of ectopic expression or coexpression of SENP1 and c-Jun on Triptolide efficacy in PC-3 cells. * indicates P<0.05 (compared to control). (F) Ectopic expression or coexpression of SENP1, c-Jun and AR in PCa cells evaluated by Western blot using anti-Flag and anti-HA antibodies.</p

Triptolide and Celastrol induce apoptosis in PCa cells <i>in vitro</i>.

<p>(A) and (B) Fluorescence microscopy observation of Triptolide- and Celastrol- induced apoptosis in LNCaP (A) and PC-3 (B) cells. After treatment with 1 µM Celastrol or Triptolide for 24 h, LNCaP and PC-3 cells were incubated with AV-FITC (green) and PI (red). Representative bright field and fluorescent images are shown. (C) Flow cytometric detection of apoptosis in LNCaP and PC-3 cells treated as above. Percentage of intact cells (AV−/PI−), early apoptotic cells (AV+/PI−), late apoptotic/necrotic cells (AV+/PI+) and necrotic cells (AV−/PI+) are presented. (D) and (E) Western blot analysis of caspase-3 protein in Triptolide- or Celastrol-treated LNCaP (D) and PC-3 (E) cells. Cells were treated with indicated concentrations of Triptolide or Celastrol for 24 h and subjected to analysis. Uncleaved caspase-3 and cleaved products are indicated. α-tubulin was used as a loading control. (F) and (G) Western blot analysis of PARP and caspase-3 proteins in Triptolide- or Celastrol-treated LNCaP (F) and PC-3 (G) cells. Cells were treated with 1 µM Triptolide or Celastrol for desired times. Uncleaved PARP and caspase-3 and their cleaved products are indicated. α-tubulin was used as a loading control.</p

Triptolide and Celastrol inhibited cell growth and induced cell death in PCa cell lines.

<p>(A) and (B) The chemical structure of Triptolide (A) and Celastrol (B), respectively. (C), (D), (E) and (F) PCa cells were treated with indicated doses of Triptolide or Celastrol, viable cell numbers were counted every day for 6 days. All assays were performed in triplicate, and data shown are mean ± SD of three independent experiments. * indicates P<0.05 (compared to control). (C) Effect of Triptolide on LNCaP cells. (D) Effect of Celastrol on LNCaP cells. (E) Effect of Triptolide on PC-3 cells. (F) Effect of Celastrol on PC-3 cells. (G) and (H) PCa Cells were treated with indicated concentrations of Triptolide or Celastrol and cells viability was determined by MTT assay. All assays were performed in triplicate, and data shown are mean ± SD of four independent experiments. (G) Effect of Triptolide and Celastrol on LNCaP cells. (H) Effect of Triptolide and Celastrol on PC-3 cells.</p

Triptolide suppressed PC-3 tumor progression in mouse xenograft model.

<p>PC-3 cells (2×10⁶ per mouse) were injected into 5-week-old nude mice. When solid tumors grew to about 100 mm³, the mice were treated intraperitonneally with either a vehicle control or Triptolide at 0.4 mg/kg daily for 15 days. Tumor sizes and body weight were measured every three days. (A) Effect of Triptolide on xenograft tumor volume. (B) Effect of Triptolide on nude mice body weight. (C) Effect of Triptolide on xenograft tumor weight. (D) Image of xenograft tumor treated with or without Triptolide.* indicates P<0.05 (compared to control).</p