Differences in expression of metabolism-related markers between cancer cells and stromal cells according to the molecular subtypes of breast cancer

Abstract

Alteration of energy metabolism of cancer cells is described by Warburg effect, a phenomenon that tumor cells obtain energy by glycolysis rather than by oxidative phosphorylation. The ‘reverse Warburg effect’ that human breast cancer cells instruct the neighboring stromal fibroblasts to provide energy by aerobic glycolysis is suggested. Breast cancer shows great heterogeneity in tumor and stromal morphology, and it is categorized into molecular subtypes identified by analyzing the gene expresseion profile; luminal A, luminal B, HER-2 type, and basal-like type. The aim of this study is to evaluate the difference of metabolic interaction between cancer cells and stromal cells according to the molecular subtype of breast cancer by investigating the markers related to glycolysis, mitochondrial status, and autophagy status, and to analyze the relationship between the expression of these metabolic markers and clinic-pathological parameters. Cell culture of six types of human breast cancer cell lines (MCF-7, MDA-MB-361, MDA-MB-453, MDA-MB-435S, MDA-MB-231 & MDA-MB-486), co-culture with fibroblasts with inhibition study by siRNA and Western blot analysis for metabolic markers (glycolysis; Glut-1, CAIX, mitochondrial dysfunction; GC1qR, BNIP3, and autophagy; beclin1, LC3A, LC3B) were performed. Tissue microarray from 740 cases of breast cancer samples which underwent mastectomy due to invasive breast cancer from 2002 to 2005 were constructed for immunohistochemical and FISH studies of markers related to molecular classification (ER, PR, HER2, Ki67) and metabolism-related markers (glycolysis; Glut-1, CAIX, MCT4, mitochondrial dysfunction; BNIP3, and autophagy; beclin1, LC3A, LC3B, p62), followed by statistical analysis. Breast cancer phenotypes were classified as luminal A type (ER or/and PR positive and HER-2 negative and Ki-67 LI <14%), Luminal B type [HER-2 negative] (ER or/and PR positive and HER-2 negative and Ki-67 LI ≥14%), Luminal B type [HER-2 positive] (ER or/and PR positive and HER-2 overexpressed or/and amplified), HER-2 type (ER and PR negative and HER-2 overexpressed or/and amplified), TNBC type (ER, PR, and HER-2 negative). The metabolic subtypes were defined as Warburg type (tumor: glycolysis type, stroma: non-glycolysis type), reverse Warburg type (tumor: non-glycolysis type, stroma: glycolysis type), mixed type (tumor: glycolysis type, stroma: glycolysis type), null type (tumor: non-glycolysis type, stroma: non-glycolysis type). In cell line study, the expression levels of metabolic markers [autophagy-related markers (beclin-1, LC3A, LC3B), mitophagy marker (BNIP3), and glycolysis-related markers (CAIX, GLUT-1)] were higher in stromal cells than in tumor cells in MCF-7, whereas tumor cells show higher expression levels of metabolic markers [autophagy-related markers (beclin-1, LC3B), mitophagy marker (GC1qR, BNIP3), and glycolysis-related markers (CAIX, GLUT-1)] than stromal cells in HER2 type and TNBC type. GLUT-1 and LC3B inhibition studies showed reduction in the cancer cell proliferation rate; in luminal type, the reduction rate of cancer cell proliferation was greater in stromal inhibition than in tumoral inhibition, in TNBC type, it was greater in tumoral inhibition than in stromal inhibition. Human breast cancer tissues were classified into 298 (40.3%) cases of luminal A type, 166 (22.4%) cases of luminal B type, 69 (9.3%) cases of HER-2 type, and 207 (28.0%) cases of TNBC type. The clinicopathologic features and the expression levels of metabolism-related proteins are different according to these phenotypes. Tissues were composed of 298 Warburg type (40.3%), 54 reverse Warburg type (7.3%), 62 mixed type (8.4%), and 326 null type (44.0%). TNBC consisted dominant portion of Warburg and mixed types, and luminal A constituted mainly of reverse Warburg and null types (P < 0.001). The mixed type had a higher histologic grade, higher rate of ER negativity, higher rate of PR negativity, higher Ki-67 index, higher rate of activated tumor autophagy status, whereas the null type showed lower histologic grade, higher rate of ER positivity, higher rate of PR positivity, lower Ki-67 index and higher rate of non- activated tumor autophagy status (P≤0.001). Breast cancer is heterogeneous in its metabolic status and the expression levels of metabolism-related markers are different according to molecular subtypes of breast cancer. The metabolic phenotypes of breast cancer have correlations with molecular subtypes along with biology of breast cancer.