Pim1 supports human colorectal cancer growth during glucose deprivation by enhancing the Warburg effect

Abstract

Cancer cells metabolize glucose mainly by glycolysis and are well adapted to metabolic stress. Pim1 is an oncogene that promotes colorectal cancer (CRC) growth and metastasis, and its expression is positively correlated with CRC progression. However, the mechanism underlying Pim1 overexpression during CRC progression and the role of Pim1 in CRC metabolism remains unclear. In the present study, we discovered that Pim1 expression was significantly upregulated in response to glucose deprivation-induced metabolic stress by AMP-activated protein kinase signaling. Pim1 promoted CRC cell proliferation in vitro and tumorigenicity in vivo. Clinical observations showed that Pim1 expression was higher in CRC tissues than in adjacent normal tissues. Pim1 overexpression in CRC tissues not only predicted CRC prognosis in patients but also showed a positive relationship with ¹⁸ F-fluorodeoxyglucose uptake. Further in vitro experiments showed that Pim1 promoted the Warburg effect and that Pim1 expression was positively correlated with hexokinase 2 and lactate dehydrogenase A expression. Pim1-silenced cells were more vulnerable to glucose starvation, and Pim1-induced tumor proliferation or tolerance to glucose starvation was attenuated by blocking the Warburg effect. In conclusion, glucose deprivation is one of the mechanisms that leads to elevated Pim1 expression in CRC, and Pim1 upregulation ensures CRC growth in response to glucose deprivation by facilitating the Warburg effect in a compensatory way.

Keywords: AMPK; Pim1; Warburg effect; colorectal cancer; glucose deprivation.

Figure 1

Pim1 is highly expressed in colorectal cancer (CRC) tissues and predicts poor prognosis. A, Representative immunohistochemistry for Pim1 in normal colorectal epithelial and CRC tissues indicating cytoplasmic staining, nuclear staining or both. B, mRNA level of Pim1 in 72 CRC tissues and adjacent normal tissues detected by qRT‐PCR. C, Representative western blots showing Pim1 protein level. D,E, Kaplan‐Meier analysis of overall survival (OS) and disease‐free survival (DFS) for 296 CRC patients. Patients with positive Pim1 expression had poorer (D) OS and (E) DFS than patients with negative Pim1 expression

Figure 2

Pim1 is upregulated by glucose deprivation through AMP‐activated protein kinase (AMPK). A,B, Colorectal cancer cells were cultured in medium with 0 or 25 mmol/L glucose for 24 h or rescued in complete medium with 25 mmol/L glucose after incubation with glucose‐free medium for another 24 h and then subjected to (A) qRT‐PCR or (B) western blot analysis. C, Pim1 expression was detected after HCT116 cells were treated with 100 μm A769662. D, Pim1 expression was detected after HCT116 cells were treated with 20 μmol/L Compound C in 25 or 0 mmol/L glucose‐containing medium for 24 h

Figure 3

Efficiency of overexpression and knockdown of Pim1 in colorectal cancer (CRC). Baseline (A) mRNA and (B) protein expression of Pim1 in 8 CRC cell lines. C, mRNA and (D) protein levels of Pim1 detected by qRT‐PCR or western blotting, respectively, in RKO and LOVO cells transfected with siRNAs and in HT29 cells transfected with pc3.1‐Pim1

Figure 4

Pim1 promotes colorectal cancer cell proliferation in vitro and in vivo. A, CCK8 assays showed proliferative capacity of indicated cells. B, Colony formation assays showed clonogenicity of indicated cells. C, Nude mouse xenograft models showing that tumors formed by RKO‐shPim1 cells had lower growth rates and weight than tumors formed by RKO‐shNC cells (*P < .05). Representative images of tumors in nude mice (lower). D, Representative images of immunohistochemistry staining for Ki‐67 and Pim1 (upper) and tumors formed by RKO‐shPim1 cells showed lower Ki‐67 index (lower). Sc, scramble. * Indicates P < .05

Figure 5

Pim1 facilitates the Warburg effect in colorectal cancer cells. A, Glucose uptake and lactate production in indicated cells 24 h after transfection (*P < .05). B, Knockdown of Pim1 decreased hexokinase 2 (HK2) and lactate dehydrogenase A (LDHA) mRNA levels (*P < .05). C, Pim1 overexpression facilitated the Warburg effect in HT29 cells and (D) increased HK2 and LDHA mRNA levels (*P < .05). E, Knockdown of Pim1 decreased HK2 and LDHA protein levels. F, Pim1 overexpression increased HK2 and LDHA protein levels. G, Pim1 H score based on immunohistochemistry was positively correlated with maximum standard uptake value (SUVmax). H, Relationship of Pim1 with HK2 and LDHA was analyzed using GEPIA (http://gepia.cancer-pku.cn/). Sc, scramble

Figure 6

Pim1 ensures colorectal cancer cell survival during glucose deprivation. A, RKO and (B) LOVO transfected with Pim1 siRNAs or scramble siRNA (Sc) were cultured in glucose‐free medium for different numbers of days as indicated, and then subjected to CCK8 assays (*P < .05). C, Pim1 overexpression increased HT29 cell survival under glucose deprivation conditions, but was then partially abolished by 2‐deoxyglucose (2‐DG, 5 mmol/L). D, 2‐DG attenuated Pim1‐induced tumor proliferation when indicated cells were cultured in medium containing glucose (*P < .05)

Figure 7

Pim1 facilitates the Warburg effect in colorectal cancer (CRC) cells. Diagram summarizing the role of Pim1 in metabolic regulation in CRC. AMP‐activated protein kinase (AMPK) is activated by phosphorylation under glucose deprivation, thus upregulating Pim1 expression. Pim1 increases the expression of several glycolytic enzymes, including hexokinase 2 (HK2) and lactate dehydrogenase A (LDHA). Furthermore, the Warburg effect is enhanced in a compensatory way to promote CRC cell survival and proliferation. These findings explain why Pim1 expression increases with the progression of CRC and how Pim1 promotes tumor malignancy