Fig 2: PFKP is a direct onco-target of YY1 in prostate cancer. (A, B) Volcano plots of the RNA-seq profiles highlight PFKP among the most altered transcripts upon YY1 depletion in 22Rv1 (A) and C4-2 (B) cells. (C) Correlation between the expression levels of PFKP and YY1 among prostate cancer patients using the StandUp2Cancer (SU2C) dataset (43). (D, E) Box plots showing the PFKP expression levels among patient samples from the indicated cohort reported by Tomlins et al (30) (D) or Varambally et al (42) (E). (F) Immunoblotting of endogenous YY1 and PFKP post-KD of YY1, relative to mock, in 22Rv1 (left) and C4-2 (right) cells. (G) Immunoblotting of exogenous YY1 (using HA antibody) and endogenous PFKP post-transduction of HA-tagged YY1WT or YY1S365D into 22Rv1 (left) or C4-2 (right) cells with stable YY1 KD (by shYY1#94, which targets 3'-UTR of YY1). (H) IGV views of the YY1 ChIP-seq profile at the proximal promoter of PFKP. (I, J) Relative transcription activities from a luciferase reporter that carries the PFKP promoter (right panel), either –2008 to +7 (I) or –575 to +37 bp (J) from transcription start site, after its co-transduction with HA-tagged YY1WT or YY1S365D into 22Rv1 cells. Left panels show the reporter results using the cells transduced with the empty luciferase reporter. Y-axis shows the averaged fold-change in the reporter signals after normalization to those of internal luciferase control and then to mock samples (the first sample); n = 3 independent experiments; ** P< 0.01. (K) Scheme shows the used mutation (mut) of putative YY1-binding sites within the PFKP promoter. Empty box denotes the mutated YY1-binding site. (L, M) Luciferase activity from a PFKP promoter reporter, which carries either WT or the indicated mutation of YY1-binding sites (refer to K), after its co-transduction with WT YY1 cDNA into 22Rv1 (L) or C4-2 cells (M). Y-axis shows the averaged fold-change in the reporter signals after normalization to those of internal luciferase controls and then to empty vector-transfected cells (n = 3 independent experiments). * P < 0.05; ** P < 0.01; *** P < 0.001; NS, not significant.

Fig 3: PFKP is critically involved in prostate tumorigenesis in vitro and in vivo. (A, B) Assays for 22Rv1 (A; insert, PFKP blotting) or C4-2 (B) cell proliferation after PFKP depletion (sh#75 or sh#77), compared to transduction of a scramble shRNA (shScramble). *P < 0.05; **P < 0.01; ***P < 0.001. (C, D) Soft agar-based growth of 22Rv1 (C; left) or C4-2 cells (C; right) and quantification of colony number (D) after PFKP depletion, compared to mock. *P < 0.05; **P < 0.01; ***P < 0.001. (E, F) Quantitation of cell cycle phases (E) and immunoblotting of the indicated apoptotic markers (F; n = 2 replicated samples) after PFKP depletion, compared to mock, in 22Rv1 cells. (G, H) Assays for cell proliferation after transduction of empty vector (blue) or an exogenous PFKP (red) into the YY1-depleted 22Rv1 (G) or C4-2 cells (H); cells without YY1 depletion were used as control (shEV; gray). ** P < 0.01. (I–K) Summary of xenografted tumor sizes (I) after subcutaneous transplantation of 22Rv1 cells, which were stably transduced with shEV (black) or shPFKP (red), into castrated NSG mice (n = 6 per group). ** P< 0.005, *** P< 0.0005. The images of tumor xenografts isolated at the end of study and immunoblotting of PFKP in these samples are shown in J and K, respectively. (L) Model illustrating the requirement of YY1 and bromodomain-containing coactivators for potentiation of the expression of genes related to energy metabolism in advanced prostate tumor.

Fig 4: YY1 potentiates prostate tumor cell glycolysis partly via PFKP. (A, B) Measurement of extra cellular acidification rate (ECAR) in 22Rv1 (A) or C4-2 cells (B) after YY1 KD, compared to mock (shEV). The scheme showing the injection of compounds during the assays is presented in the left panel. Data quantifications are shown in the right panel as mean ± SEM. ** P< 0.01, *** P < 0.001. (C–E) Immunoblotting for exogenous HA-YY1 (C; anti-HA immunoblot), as well as ECAR measurement using the 22Rv1 (D) or C4-2 (E) cells post-transduction of the indicated HA-YY1, either WT or with its N-terminal transactivation domain deleted (?1–100aa). *P < 0.05; **P < 0.01; NS, not significant. (F) ECAR measurement using the 22Rv1 cells post-KD of BRD2 (sh#8), compared to scramble control. * P< 0.05; ** P < 0.01; *** P < 0.001. (G) ECAR measurement using 22Rv1 cells, which were transduced with empty vector (mock) or WT YY1, followed by cell treatment with either DMSO or 500nM of JQ1 for 8 h. * P< 0.05; ** P < 0.01; *** P < 0.001. (H, I) Immunoblotting for endogenous YY1 and exogenous HA-PFKP (H) and ECAR measurement (I) after transduction of empty vector (mock) or HA-PFKP (H, lane 3 versus 2), into the YY1-depleted 22Rv1 cells (lanes 2–3). The shEV-transduced cells (lane 1) serve as control. * P< 0.05; ** P < 0.01; NS, not significant. (J, K) ECAR measurement using the 22Rv1 (J) or C4-2 (K) cells post-KD of PFKP (sh#75 or sh#77), compared to mock (shEV). * P< 0.05; ** P < 0.01; *** P < 0.001. (L, M) Immunoblotting for endogenous PFKP and exogenous HA-YY1 (L) and ECAR measurements (M) after transduction of vector (mock; L, lanes 1 and 3) or HA-tagged YY1 (L, lanes 2 and 4) into 22Rv1 cells with stable expression of either shEV (lanes 1–2) or YY1-targeting shRNA (lanes 3–4). * P< 0.05; ** P < 0.01; NS, not significant.

Fig 5: PTEN knockdown by siRNAs promotes glucose consumption and lactate production. (A) The siRNA sequences used in this experiment. (B) DU-145 and 22Rv1 cells were transfected with 40 nM control siRNA and indicated concentrations of two different siRNAs targeting PTEN (PTEN siRNA-1 and PTEN siRNA-2) for 48 h, after which samples were collected for quantitative real-time PCR and (C) Western blotting with antibodies against PTEN and ß-actin. (D) DU-145 and 22Rv1 cells were transfected with 40 nM control siRNA and PTEN siRNAs (PTEN siRNA-1 and PTEN siRNA-2) for 48 h, after which samples were collected for Western blotting with antibodies against PTEN, phospho-Akt (Ser473), Akt and ß-actin. (E) Glucose consumption and lactate production was assessed in DU-145 cells and 22Rv1 cells which were transfected with 40 nM control siRNA and PTEN siRNAs (PTEN siRNA-1 and PTEN siRNA-2) for 48 h. (F) The expression of catalytic enzymes in glycometabolism was detected by western blotting in DU-145 cells and 22Rv1 cells which were transfected with 40 nM control siRNA and PTEN siRNAs (PTEN siRNA-1 and PTEN siRNA-2) for 48 h. (G) DU-145 and 22Rv1 cells which were infected with lentivirus containing PTEN shRNA or scramble shRNA, then cell viability was analyzed by MTT assay. HK1, hexokinase 1; HK2, hexokinase 2; PKM1/2, pyruvate kinase isozymes M1/M2; PKM2, pyruvate kinase isozyme M2; LDHA, lactate dehydrogenase A; PDH, pyruvate dehydrogenase; PFKP, phosphofructokinase platelet type; PC, pyruvate carboxylase; PTEN, gene of phosphate and tension homology deleted on chromosome ten. *P<0.05, **P<0.01 compared with the cells transfected with control siRNA.