The results of this study show that diffuse gastric carcinoma overexpress HK2, GLS1 and FASN proteins as assessed by immunohistochemistry. These three enzymes are key for glycolysis, glutaminolysis and the de novo synthesis of fatty acids pathways which are commonly overactivated in gastric cancer. Interestingly, the overexpression of HK2, the combined expression of HK2/FASN and HK2/GLS/FASN were related with shorter PFS whereas HK2/GLS/FASN with OS. Nonetheless, enzyme over-expression had no independent prognostic significance in the multivariate analysis for either PFS or OS. This work is important because studies had evaluated in primary gastric carcinomas, the expression at the protein or messenger level individually, but not simultaneously. This has potential therapeutic relevance because may suggest the combined use of inhibitors of these pathways could be more effective.
Hexokinases catalyze the conversion of glucose to glucose-6-phosphate. There are four HK isoforms (1–4), but only the HK2 isoform is related to the Warburg effect. Moreover, it translocates from cytoplasm to the outer mitochondrial membrane to complex with the voltage-dependent anion channel (VDAC) that inhibits mitochondrial-induced apoptosis (Krasnov et al. 2013). So far, only three studies have evaluated the expression of HK2 in gastric adenocarcinoma. Rho et al. (2007) reported that HK2 overexpressed in 16.7% (43 out of 257) of gastric cancers, it was positively correlated with Bcl-2 expression, and its overexpression portended shorter survival. Another study reported HK2 overexpression in 21.3% (40 out of 188) gastric tumor samples and was an independent adverse prognostic factor for survival (Qiu et al. 2011). A third study reported overexpression in only 7 (4.1%) out of 152 patients and had no correlation with clinicopathological characteristics or prognosis (Hur et al. 2013). The enzyme FASN functions as a central regulator of lipid metabolism and plays a critical role in the growth and survival of tumors including gastric carcinoma (Fhu and Ali, 2020). In a study analyzing 90 patients, the positivity rate observed was 28.9%. The patients with higher expression showed a decreased 3-year OS (Hou et al. 2012). A second study reported overexpression of FASN by RT-PCR and western blot in 12 and 3 gastric cancer patients as compared to normal adjacent tissue. These results were confirmed in 167 tumors by IHC. The overall, positivity rate was 50.8%, and its overexpression predicted a shorter survival time in those with intense expression (Duan et al., 2016). Finally, Xian et al. (2015) reported that 90% (54 out of 60 patients) had FASN expression which correlated with shorter survival in the multivariate analysis. There is only a single study on GLS1 expression in primary gastric cancers. By proteomics, Jian et al. reported that GLS1 is overexpressed 1.68-fold as compared to normal tissue and data were validated by confirming its overexpression by IHC in 75.6% of primary tumors compared with expression of 19.1% in adjacent cancer tissues. Moreover, GLS1 expression was associated with larger tumor size and lymph node metastases. Its prognostic significance was not reported (Jiang et al. 2019).
Our results showing the over-expression of these enzymes somehow agree with previous studies, though our results in terms of percentage of expression can hardly be compared. A number of variables including patient populations, different antibodies, techniques for routine processing of biopsies and most importantly different semi-quantitative evaluation scores could explain the wide variability in results, though it is clear that these enzymes are indeed overexpressed on gastric carcinomas. A number of preclinical studies have shown the genetic depletion or pharmacological inhibition of each of these three enzymes induce antitumor effects in gastric cancer cell lines. Depleting HK2 expression via miR-181b overexpression represses cell proliferation and migration in NCI-N87 and MGC80-3 gastric carcinoma cells (Li et al. 2016). Likewise, Licochalcone A, an herbal Chinese medicine, suppresses the expression of HK2 and induce antitumor effects in vitro and in vivo which is associated with glycolysis inhibition. The forced overexpression of HK2 reverses the induction of apoptosis of Licochalcone A (Wu et al. 2017). Both, oleanolic acid and the glycolysis inhibitor 2-deoxy-d-glucose, inhibit antitumor effects in vitro via glycolysis inhibition in human MKN-45 and SGC-7901 gastric cancer cells (Li et al. 2019). Regarding fatty acid synthesis, knocking down FASN in the SGC-7901 gastric cancer cell line suppressed cell proliferation and reversed EMT via regulation of AMP-activated protein kinase/mTOR and protein kinase B/mTOR signaling (Sun et al. 2018), while in MNK-45 and AGS cells siFASN transfection reduced anoikis, migration and invasion rates via p-ERK1/2/Bcl-xL pathway (Yu et al. 2021). GLS1 is an overexpressed enzyme in tumors with high rates of glutaminolysis. A study in gastric cancer cell lines show that its genetic or pharmacological depletion induces antitumor effects in vitro and in vivo (Kitayama et al. 2017). We have previously demonstrated in the gastric cancer cell line AGS that the combined use of lonidamine, DON and orlistat inhibitors of HK2, GLS and FASN respectively exert profound antitumor effects in vitro and that there are effective and well tolerated in mouse model of colon cancer (Cervantes-Madrid et al. 2017; Cervantes-Madrid and Dueñas-González, 2015).
Altogether, our results demonstrating that key enzymes of glycolysis, glutaminolysis and de novo fatty acid synthesis are overexpressed in diffused gastric carcinomas should encourage studies to support the use of these pathway inhibitors for gastric cancer. Of major relevance is the finding that around 10% of patients simultaneously overexpress either HK2/FASN or HK2/GLS1/FASN suggesting that some tumors preferently rely on single or combined pathways. The findings lend support to that gastric carcinoma may reprogram and shifth metabolic pathways upon inhibition. For instances, the combined attack upon glycolysis and FA synthesis with knockout of the glycolytic enzyme PGM1 enhances the effect of orlistat, a FASN inhibitor in gastric cancer cells (Cao et al. 2021). Our study has some limitations. The number of patients is limited, we used a semiquantitative score for rating expression, we studied only diffuse gastric carcinomas and do not provide mechanistics insights on how enzyme overexpression could determine shorter survival. In conclusion, in our knowledge, this is the first study to demonstrate that HK2, GLS1 and FASN are concurrently overexpressed which provide support for further studies evaluating the antitumor effects of inhibitors of glycolysis, glutaminolysis and de novo synthesis of fatty acids for metabolic therapy of gastric cancer.