Hexokinase-2 is a key therapeutic target in brain metastasis derived from breast cancer.

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The prevalence of brain metastasis from breast cancer is increasing. Current treatment options are limited and have no impact on survival. We are evaluating several potential therapeutic targets identified from cDNA microarray analysis of surgically resected human brain metastases. One of these, hexokinase II (HK-2), was validated using quantitative RT-PCR, and showed an approximately 2-fold up-regulation (p=0.01) in brain metastases (n=8) compared to unmatched primary invasive ductal breast carcinomas (n=8). Since HK-2 is not normally found in brain, this suggests that this isoenzyme may be a selective target for metastatic disease in this organ. HK-2 is up-regulated in many tumors, and belongs to a family of four hexokinases which play a vital role in the generation of ATP as they catalyze the first step of the glycolytic pathway converting glucose to glucose-6-phosphate. Production of this substrate also drives the pentose phosphate pathway providing many components required for cell growth. In addition, through mitochondrial binding, HK-2 inhibits apoptosis. We therefore hypothesized that HK-2 would play a key role in cell growth and survival in brain metastasis derived from breast cancer, and that its level of expression would affect overall survival. To test this hypothesis we examined survival curves in patients that were divided into one of two groups based on the degree of HK-2 expression in brain metastases, as determined by immunohistochemistry. Median survival for patients (n=94) with a high level of HK-2 was 9.6 months post-surgery as compared to a median survival of 17.5 months in patients (n=19) with a low level of HK-2 (p=0.0275 by two-tailed log rank test). To further validate HK-2 as a therapeutic target and evaluate the functional role of HK-2 in brain metastases, we are using a human breast cancer cell subline that is selectively metastatic to the brain (MDA-MB-231BR). These cells were transduced using VSV-g pseudotyped lentiviral particles carrying a shRNA targeting either HK-2 or a non-target control (Sigma-Aldrich). Knockdown of HK-2 was verified by Western blotting and QT-PCR. Cell growth is measured using 5-bromo-2'-deoxy-uridine labeling. Knockdown of HK-2 expression by 95 % inhibited tumor cell proliferation by 40 % (p=0.0012 by two-tailed t test) under normal growth conditions at five days. These data suggest that over-expression of HK-2 is associated with poorer overall patient survival because it plays a key role in the proliferation and long-term survival of metastatic cells in brain. Thus, HK-2 is likely an important therapeutic targetin brain metastasis derived from breast cancer.