PKM2 promotes metastasis by recruiting myeloid-derived suppressor cells and indicates poor prognosis for hepatocellular carcinoma

Abstract

Pyruvate kinase M2 (PKM2) is a member of the pyruvate kinase family. Recent work has defined the "non-metabolic" functions of PKM2. However, the role of PKM2 in HCC remains unclear. To investigate the role of PKM2 in tumor growth, invasion and the prognosis of hepatocellular carcinoma (HCC), PKM2 expression was measured in HCC cell lines and tissues using qRT-PCR, western blot, and immunofluorescence assays. In in vitro experiments, PKM2 was knocked down using a short hairpin RNA lentivirus vector, and tumor cell behavior and the downstream signaling pathways and chemokine were analyzed. For the analysis of in vivo tumor growth, intratumoral and peritumoral lymphocyte infiltration were examined in nude mice. The prognostic value of PKM2 was analyzed by immunohistochemistry in two cohorts including 721 HCC patients. Together, our data obtained from cell lines, tumorigenicity studies, and primary HCC samples illustrate an oncogenic role for PKM2 in tumors. Moreover, PKM2 may serve as a novel prognostic indicator for HCC patients after curative resection, targeted therapy aimed at PKM2 may represent an effective treatment approach for HCC.

Figure 1. Expression of PKM2 in human cancers and HCC tissues/cell lines

(A) Microarray data analyses of PKM2 expression in human cancers are shown. PKM2 mRNA levels in human normal/cancer tissues (breast, lung, and colon) are plotted. The Student t test was conducted using the Oncomine software. The boxes represent the 25th through 75th percentiles. The horizontal lines represent the medians. The whiskers represent the 10th and 90th percentiles, and the asterisks represent the end of the ranges. (B) Quantitative RT-PCR confirmed different PKM2 mRNA expression in HCC cell lines. Data shown as mean (± SD) from three independent experiments. (C) Expression of PKM2 in normal human liver epithelial cells (L02) and 7 HCC cell lines was examined using Western blotting; β-actin was used as a loading control. (D) Quantitative RT-PCR analysis of PKM2 levels in HCC tissues and corresponding peritumoral tissue. β-actin was used as a control. Error bars indicate standard deviation (SD) (n = 48). (E) Fluorescence microscopic analysis for PKM2 expression. *P < 0.05. **P < 0.01.

Figure 2. Effect of PKM2 gene suppression on HCCLM3 HCC cell lines

(A) PKM2 expression in HCCLM3 was modified by vshRNA interference and was verified by qRT-PCR and western blot. (B) Cell proliferation was detected by CCK-8 assay. (C) The apoptosis of cancer cells were analyzed by flow cytometry for annexin-V/PI assay. (D) Cell cycles of cancer cells were analyzed by flow cytometry for PI staining. (E) The migration and invasion of cancer cells was measured by transwell assays. The data represent the mean ± SD of three different experiments. *P < 0.05. **P < 0.01. ***P < 0.001.

Figure 3. PKM2 promotes HCC progression in a xenograft nude mice model

(A) Liver bearing tumors formed by implanted cells with different expression levels of PKM2. Weights and tumor volume of the tumors in livers of nude mice were measured. Error bar indicates standard deviation (n = 7). Error bar indicates standard deviation (n = 7). (B) H&E stained images of metastatic clusters in lungs from all groups with magnification of the selected areas. Scale bar, 40×, 200 μm. 200×, 50 μm. All grades of metastatic clusters in lungs of all xenograft nude mice groups. **P < 0.01. ***P < 0.001.

Figure 4. PKM2 induces MDSC infiltration in a xenograft nude mice model

(A) Flow cytometry analysis with Gr-1, CD11b, F4/80, L6yC in different group. The percentages of Gr-1⁺CD11b⁺, F4/80⁺CD11b⁺, and L6yC⁺ CD11b⁺ cells were calculated. (B) Representative images from tumor serial sections stained with PKM2, Gr-1, and α-SMA by immunofluorescence. Scale bar, 100×, 100 μm. (C) Representative images from tumor serial sections stained with PKM2, Gr-1, α-SMA, and F4/80 by immunohistochemistry. (D) Representative images from lung serial sections stained with Gr-1 by immunohistochemistry. (E) PKM2 induces phosphorylation and activates inflammatory cytokine production in HCC cell lines. Scale bar, 200×, 50 μm. 400×, 25 μm. *P < 0.05.

Figure 5. PKM2 expression and prognostic value in HCC tissue (training cohort, n = 367)

(A) Representative photomicrographs of tumor tissues of the different staining patterns are presented here and are graded from 0 to +++, images of representative staining are shown. Scale bar, 200×, 50 μm. 400×, 25 μm. (B, C) Kaplan-Meier analysis of OS and TTR for PKM2 expression. (D) Prognostic role of PKM2 in Edmonson I-II, Edmonson III-IV, Single tumor, AF P < 20 ng/mL, and TNM stage I subgroups.