HDAC Inhibitors as Epigenetic Regulators

More importantly, we provide evidence showing that it is possibly a pervasive regulatory mechanism commonly occurring in different cancer types. implicated in human cancer due to its frequent loss in various cancer types. Methods: Expression of FBP1 and PD-L1 was analyzed in various cancer cell lines. Tin(IV) mesoporphyrin IX dichloride Western blot and RT-qPCR were performed to determine whether FBP1 regulates PD-L1 expression. Co-immunoprecipitation and glutathione S-transferase (GST) pulldown assay were employed to define the underlying regulatory mechanisms. Immunohistochemistry was conducted to determine the correlation between FBP1 and PD-L1 expression in a cohort of patients. A cancer syngeneic mouse model was utilized to examine how FBP1 affects tumor immunity. Results: We Tin(IV) mesoporphyrin IX dichloride demonstrated that in a manner independent of Rabbit Polyclonal to RNF138 its enzymatic activity FBP1 downregulates the expression of PD-L1 in various cell lines of different cancer types including pancreatic and prostate cancer. We further showed that this regulation occurs at the transcriptional level and is mediated by FBP1 inhibition of signal transducer and activator of transcription-3 (STAT3)-dependent PD-L1 transcription. Moreover, FBP1 and PD-L1 protein expression were negatively correlated in pancreatic ductal adenocarcinoma (PDAC) specimens from a cohort of patients. Most importantly, we demonstrated that decreased FBP1 expression promotes tumor growth and resistance to immune checkpoint blockade therapy in mice. Conclusions: Our findings reveal a new tumor suppressor function of FBP1 in inhibiting PD-L1 expression and enhancing cancer immunity. They also suggest that FBP1-deficient human cancers could be therapeutically targeted by PD-1/PD-L1-based immune checkpoint blockade therapy. gene 7-11. These transduction pathways can be activated by pro-inflammatory cytokines such as IFN-, TNF-, IL-1 or due to loss or inactivation of tumor suppressor genes such as and PTEN-CaP8 murine prostate cancer cells (5 ) infected with lentivirus expressing control or Fbp1-specific shRNAs was injected subcutaneously into the right flank of mice. The volume of allografts was measured every other day until the tumor volume reached 300 mm3 and calculated by the formula (L W2 0.5). At the end of measurement, mice were euthanized and tumors were isolated and weighed. Flow cytometry analysis PANC-1 and MIA PaCa-2 cells infected with shRNA were harvested and washed with 1 PBS. Cells were fixed with 4% paraformaldehyde for 15 minutes. Cells were incubated with ice-cold 100% methanol for 30 minutes on ice followed by wash with 1 PBS. Cells were washed with 1 PBS one more time and incubated with antibody or isotype IgG for 1 hour at room temperature. Cells were incubated with secondary antibody conjugated with Alexa Fluor (Thermo Fisher Scientific) for 1 hour at room temperature followed by wash with 1 PBS. After washed three times with 1 PBS, cells were resuspended with 1 PBS and analyzed using flow cytometer. For the preparation of flow cytometry analysis of mouse tissue Tin(IV) mesoporphyrin IX dichloride samples, tumors were cut into small pieces and digested with 2 mg/ mL collagenase (Sigma Aldrich) in DMEM for 1 hour at 37 . Cells were filtered through 70 m nylon strainer and resuspended in red blood cell lysis buffer (Biolegend) for 3 minutes at room temperature. Cells were suspended in 1 PBS with 2% BSA and co-stained with antibodies. After incubated with antibody for 30 minutes, cells were washed with 1 PBS and analyzed with flow cytometer. Statistical analysis Statistical analysis were carried out by one-sided or two-sided paired Student’s t-test for single comparison and one-way ANOVA with a post-hoc test for multiple comparisons, and values 0.05 was considered statistically significant. All the values are expressed as the means SD. Results FBP1 negatively regulates PD-L1 expression in multiple cell lines of different cancer types It has been shown previously that FBP1 is frequently lost in many types of human cancers including renal carcinoma, basal-like breast cancer, hepatocellular carcinoma and pancreatic cancer and that loss of FBP1 promotes cancer progression, metabolic reprogramming and drug resistance 28, 31, 33, 34. Given that PD-L1 is a key immune checkpoint molecule and it is often deregulated in human cancers 3-5, 15, 35, we sought to determine whether FBP1 expression influence cancer immunity by regulating PD-L1 expression in cancer.