TBC1 domain-containing proteins are frequently involved in triple-negative breast cancers in connection with the induction of a glycolytic phenotype

Metabolic plasticity is a hallmark of cancer, and metabolic alterations represent a promising therapeutic target. Since cellular metabolism is controlled by membrane traffic at multiple levels, we investigated the involvement of TBC1 domain-containing proteins (TBC1Ds) in the regulation of cancer metabolism. These proteins are characterized by the presence of a RAB-GAP domain, the TBC1 domain, and typically function as attenuators of RABs, the master switches of membrane traffic. However, a number of TBC1Ds harbor mutations in their catalytic residues, predicting biological functions different from direct regulation of RAB activities. Herein, we report that several genes encoding for TBC1Ds are expressed at higher levels in triple-negative breast cancers (TNBC) vs. other subtypes of breast cancers (BC), and predict prognosis. Orthogonal transcriptomics/metabolomics analysis revealed that the expression of prognostic TBC1Ds correlates with elevated glycolytic metabolism in BC cell lines. In-depth investigations of the three top hits from the previous analyses (TBC1D31, TBC1D22B and TBC1D7) revealed that their elevated expression is causal in determining a glycolytic phenotype in TNBC cell lines. We further showed that the impact of TBC1D7 on glycolytic metabolism of BC cells is independent of its known participation in the TSC1/TSC2 complex and consequent downregulation of mTORC1 activity. Since TBC1D7 behaves as an independent prognostic biomarker in TNBC, it could be used to distinguish good prognosis patients who could be spared aggressive therapy from those with a poor prognosis who might benefit from anti-glycolytic targeted therapies. Together, our results highlight how TBC1Ds connect disease aggressiveness with metabolic alterations in TNBC. Given the high level of heterogeneity among this BC subtype, TBC1Ds could represent important tools in predicting prognosis and guiding therapy decision-making. Overall design: High expression of TBC1D7 correlates with worse prognosis in multivariate analysis within the Triple Negative subtype (TNBC) of breast cancer patients. To investigate the underlying molecular mechanism, we selected TNBC cell lines carrying high or low amount of the TBC1D7 protein. From an initial screening, we identified three lines with these characteristics, MDA-MB-468 (used also in the screening in Fig. 3A) and MDA-MB-231 are high expressors, while Hs578T is a low expressing cell line (Fig. 7A). Silencing of TBC1D7 (Fig. 7A) significantly decreased the intracellular levels of L-lactate in the high expressors (MDA-MB-468 and MDA-MB-231, for a rescue experiment, see Fig. 7C), while it did not affect the low-expressing cell line Hs578T (Fig. 7B). The reduction in L-lactate production was mirrored by a reduction in glucose uptake, which is also a hallmark of glycolytic metabolism (Fig. 7C). Thus, in model TNBC cell lines, high levels of TBC1D7 expression are needed to maintain active glycolysis. To gain insights into the glycolytic function of TBC1D7 we performed RNAseq analysis from MDA-MB-468 cells in which we downregulated the expression of TBC1D7. This was achieved by transient transfection of a pool of 4 siRNA oligos for TBC1D7 (siTBC1D7) or non-silencing control oligos (siCTRL)