The effects of restricted glycolysis on stem-cell like characteristics of breast cancer cells

Abstract

Altered glycolysis is a characteristic of many cancers, and can also be associated with changes in stem cell-like cancer (SCLC) cell populations. We therefore set out to directly examine the effect of glycolysis on SCLC cell phenotype, using a model where glycolysis is stably reduced by adapting the cells to a sugar source other than glucose. Restricting glycolysis using this approach consistently resulted in cells with increased oncogenic potential; including an increase in SCLC cells, proliferation in 3D matrigel, invasiveness, chemoresistance, and altered global gene expression. Tumorigenicity in vivo was also markedly increased. SCLC cells exhibited increased dependence upon alternate metabolic pathways. They also became c-KIT dependent, indicating that their apparent state of maturation is regulated by glycolysis. Single-cell mRNA sequencing identified altered networks of metabolic-, stem- and signaling- gene expression within SCLC-enriched populations in response to glycolytic restriction. Therefore, reduced glycolysis, which may occur in niches within tumors where glucose availability is limiting, can promote tumor aggressiveness by increasing SCLC cell populations, but can also introduce novel, potentially exploitable, vulnerabilities in SCLC cells.

Stem cell-like breast cancer cells with acquired resistance to metformin are sensitive to inhibitors of NADH-dependent CtBP dimerization

“…3D matrigel cells growth, cell invasion and colony growth were performed as previously described (14). For all assays metformin (2 mM) was present in the medium for the metformin-adapted cells.…”

“…Briefly, cells were incubated with ALDEFLUOR substrate (BAAA, BODIPYaminoacetaldehyde. Stem Cell Technologies, UK) to define the ALDEFLUOR-positive cells, and a specific inhibitor of ALDH1, diethylaminobenzaldehyde (DEAB), was Immunoblotting was performed as previously described (14). Primary antibodies used were αV5 (clone E10/V4RR) (Thermo Fisher, UK), R41 polyclonal CtBP (30) and rabbit polyclonal anti-β-actin antibody (Sigma, St Louis, MO).…”

“…Primary antibodies used were αV5 (clone E10/V4RR) (Thermo Fisher, UK), R41 polyclonal CtBP (30) and rabbit polyclonal anti-β-actin antibody (Sigma, St Louis, MO). Reverse transcription-quantitative PCR (RT-qPCR) was performed as previously described (14). Primers and probes were Roche universal probe library assays (Roche, Germany), ABCG2: TGGCTTAGACTCAAGCACAGC/ TCGTCCCTGCTTAGACATCC/#56; CTNNB1: GCTTTCAGTTGAGCTGACCA/CAAGTCCAAGATCAGCAGTCTC/#21; DVL1: AAGAACGTGCTCAGCAACC/AGCTTGGCATTGTCATCAAA/#63; BCL-2: GTACCTGAACCGGCATCTG/GGGGCCATATAGTTCCACAA/#75; SLC7A5: GTGGAAAAACAAGCCCAAGT/GCATGAGCTTCTGACACAGG/#25; SAA2: ACCTCTCCCTGGCAGACA/AGAAGCTTCGGCTGCTGAC /#68; BMI1: CCATTGAATTCTTTGACCAGAA/CTGCTGGGCATCGTAAGTATC/#63; SAA1: GCAGCTCAGCTACAGCACA/AGAAGCTTCGGCTGCTGAC /#68; CD49f /ITGA6: ATTCTCATGCGAGCCTTCAT/GGAAACACAGTCACTCGAACC/#74; CXCL1: TCCTGCATCCCCCATAGTTA/CTTCAGGAACAGCCACCAGT/#52; ID1: CCAGAACCGCAAGGTGAG/GGTCCCTGATGTAGTCGATGA/#39; ID3: CATCTCCAACGACAAAAGGAG/CTTCCGGCAGGAGAGGTT/#59; S100A4: CGCTTCTTCTTTCTTGGTTTG/GAGTACTTGTGGAAGGTGGACA/#81.…”

“…mRNA expression in metforminadapted cells is shown relative to the expression in non-adapted cells. (14), 3 x 10 4 cells of either non-adapted or metformin-adapted MDA-MB-231 IND CtBP DD , which had each been cultured in the absence or presence of 1 μg / mL doxycycline for 24 hours were injected in the mammary gland of NOD/SCID mice on both flanks in three mice per group. Doxycycline (200 μg/mL) and/or metformin (1.75 mg/mL to achieve an estimated mg/kg/day) was included in the drinking water according to the treatment regimen, commencing 5 days prior to injection.…”

“…Even within tumours derived from the same tissue, such as breast cancers, SCLC cells can be either highly glycolytic or, conversely, dependent on mitochondrial oxidative phosphorylation (OXPHOS) (4,12,13). This heterogeneity may be due to cells adapting to particular environmental and / or experimental conditions (5,14). It does mean, however, that highly valuable adjuncts for the use of therapies targeting glycolytic metabolism would be strategies to restrict the SCLC cells into a state of glycolytic dependency, as well as biomarkers of cells that are in this state.…”