Univariate and multivariate analysis for prognostic value of p-ERK1/2 expression in breast cancer patients.

<p>a: lymph node; b: negative; c: positive.</p

High levels of p-ERK1/2 correlated with loss of Bik and overexpression of Bcl-Xl and Mcl-1.

<p>High levels of p-ERK1/2 correlated with loss of Bik and overexpression of Bcl-Xl and Mcl-1.</p

BikDDA had a longer half-life than BikDD.

<p>A. 293 cells were transfected for 16h with same amount of pUK21- BikDD or pUK21-BikDDA and treated with protein biosynthesis inhibitor cycloheximide (CHX), proteasome inhibitor MG132 or Mek1/2 inhibitor UO126. B. Histogram and line charts showed the gray scale quantitative analysis for western blotting using Gel-pro software. The relative levels before CHX treatment were set as 100%. These results demonstrated that BikDDA degradation rate was lower than BikDD.</p

BikDDA eliminated TNBC cells more powerfully than BikDD.

<p>A. S124A and previous mutant sites T33D and S35D didn’t locate in the pro-apoptotic region of Bik. B. MDA-MB-231 cells were transfected for 28h with the same amount of pUK21, pUK21-BikDD or pUK21-BikDDA. Apoptotic cells were monitored by Annexin V/PI staining and flow-cytometry analysis. The right-lower or right upper quadrant of each plot showed early apoptotic or late apoptotic cells. C. Histogram directly showed the enhanced apoptosis-inducing activity of BikDDA. D. The cytotoxicity of BikDD and BikDDA were analyzed by Cell counting kit and MTT assay (setting at 100% in vector group). E. Western blotting was performed to confirm the prolonged half-life of BikDDA in 231 cells. F. MEK1/2 inhibitor UO126 and selective ERK1/2 inhibitor FR180204 increased the stability of BikDD in 231 cells.</p

Higher p-ERK1/2 correlated with 5-FU resistance and BIKDDA could enhance the cytotoxicity of 5-FU.

<p>A. Kaplan-Meier curve of disease-free survival showed the decreased disease-free survival time of high p-ERK1/2 group who received adjuvant 5-FU based chemotherapy than the low. B and C. High levels of p-ERK1/2 correlated with 5-FU resistance (patients with metastasis and/or recurrence after adjuvant 5-FU based chemotherapy). After stratified by lymph node metastasis the correlation also exhibited statistics significance in metastasis positive cohort. D. 435 and 231 cells were transfected with vector control or BikDDA in combination with 25 ug/ml 5-FU or not, 24h later cell viability was analyzed by MTT assay (vector transfected only setting as 100%). These data showed BIKDDA can enhance the cytotoxicity of 5-FU.</p

BikDDA, a Mutant of Bik with Longer Half-Life Expression Protein, Can Be a Novel Therapeutic Gene for Triple-Negative Breast Cancer

<div><p>Our previous studies showed that BikDD, a constitutively active mutant form of Bik, exhibited powerful antitumor effects in preclinical pancreatic, lung and breast cancer models. Howerver, the antitumor activity of BikDD in triple-negative breast cancer (TNBC) is unknown. Here we show that aberrant expression of p-ERK1/2 was a meaningful molecular phenotype in TNBC patients, and can be an obstacle for treatment because of the converse correlation with Bik. A novel mutant, BikDDA, in which Ser¹²⁴ was changed to Alanine to block BikDD phosphorylation by p-ERK1/2 prevented subsequent ubiquitin-proteasome degradation. BikDDA showed a prolonged half-life and enhanced pro-apoptotic ability in TNBC cells compared with BikDD. Moreover, aberrant expression of p-ERK1/2 was associated with 5-fluorouracil resistance in breast cancer patients and BikDDA enhanced the therapeutic effects of 5-fluorouracil in vitro.</p></div

p-ERK1/2 as a meaningful biomarker in triple-negative breast cancer.

<p>A and B. Kaplan-Meier curves showed overall survival (OS) and disease-free survival (DFS) according to p-ERK1/2 expression. C. Aberrant p-ERK1/2 expression but absent expression of Bik were exhibited in the same triple-negative breast cancer patients’ samples. D. Co-overexpression of p-ERK1/2 and Bcl-Xl or Mcl-1 in the same breast cancer patients’ samples. E. The distribution of p-ERK1/2, Bik, Bcl-Xl and Mcl-1 status categorized by subgroup. F. The relative expression levels of Bik and p-ERK1/2 in eight breast cancer patients (five TNBC and three NTNBC) by western blotting. Semi-quantitative analysis for Bik and p-ERK1/2 expression: β-actin and total ERK1/2 as control respectively.</p

Association between p-ERK1/2 expression and clinicopathological parameters in 114 patients with breast cancer treated by surgery.

<p>a: Non-Triple-negative Breast cancer; b: Triple-negative Breast cancer; c: lymph node.</p

Supplemental Information from PARP Inhibitor Upregulates PD-L1 Expression and Enhances Cancer-Associated Immunosuppression

Supplementary Figure S1. PARPi upregulates PD-L1 in a dose-dependent manner in MD-MB-231 cells. Supplementary Figure S2. Downregulation of BRCA1 or BRCA2 has no effect on PARPi-induced PD-L1 upregulation. Supplementary Figure S3. PARPi inactivates GSK3β. Supplementary Figure S4. PARPi blocks cell proliferation of MDA-MB-231 cells but not activated PBMCs. Supplementary Figure S5. Toxicity assessment of treatment on BALB/c mice.</p

Supplementary Movie S1 from PARP Inhibitor Upregulates PD-L1 Expression and Enhances Cancer-Associated Immunosuppression

PARPi treatment inhibits cell proliferation of MDA-MB-231 cells. Time-lapse video of 231 (Nuc-RFP) cells without olaparib (control) taken using the IncuCyte Zoom microscope.</p