Oncogenic or tumor suppressive signaling often causes imbalances in posttranslational modifications (PTMs) in human cancer. The receptor tyrosine kinase HER2 is an oncogene amplified or overexpressed in approximately 25% of breast cancers and is associated with tumor aggressiveness and poor prognosis. A recent study demonstrated that HER2 is translocated to mitochondria in a kinase activity-dependent manner to alter metabolism and promote drug resistance in breast cancer cells. Consistent with these findings, our recent study showed that oncogenic fibroblast growth factor receptor (FGFR) 1, which is also localized in mitochondria, directly tyrosine phosphorylates mitochondrial pyruvate dehydrogenase kinase (PDHK) 1, enhancing its enzyme activity to promote tumor growth in mice. Taken together, these results strongly suggest that mitochondrial localization of oncogenic tyrosine kinases, including HER2, regulate mitochondrial metabolic pathways that contribute to tumor growth. However, which mitochondrial metabolic enzymes are regulated by HER2 in breast cancer is totally unknown. Thus, we performed phospho-proteomic analyses of mitochondria- enriched fractions from HER2+ BT474 breast cancer cells and found that mitochondrial carnitine palmitoyltransferase (CPT) 1A is tyrosine phosphorylated at Y514. Further biochemical analyses showed that CPT1A Y514 phosphorylation by HER2 promotes its carnitine palmitoyltransferase (CPTase) activity. Interestingly, we recently found that CPT1A has an additional enzymatic activity as a lysine succinyltransferase (LSTase) to succinylate lysine residues of substrate proteins in vitro and in vivo .To our knowledge, CPT1A is a first and only LSTase in mammalian cells. Importantly, mutation of CPT1A Gly710 (G710E) selectively inactivated CPTase activity but not LSTase activity. Similar to CPT1A WT, CPT1A G710E increased total lysine succinylation in cells without affecting intracellular succinyl-CoA levels. These findings suggest the unprecedented role of CPT1A as LSTase that can regulate lysine succinylation-dependent signal transduction in cells independent of its canonical CPTase activity that facilitates mitochondrial fatty acid oxidation (FAO) pathway. Notably, however, how the LSTase activity of CPT1A is regulated and whether LSTase activity is important for tumorigenesis is not known. Previous studies reported that multiple types of cancers including melanoma, prostate, breast, and ovarian cancers showed elevated expression of CPT1A and knockdown of CPT1A with si/shRNAs decreased cancer cell proliferation. We also found that high CPT1A mRNA expression in HER2+ breast cancer patients strongly correlates with reduced survival while no correlation between CPT1A mRNA expression and reduced patient survival was observed in other types of breast tumors. In addition, we found that knockdown of CPT1A, as well as inhibition of HER2 by lapatinib, decreased CPT1A-subsrate motif LVxxK succinylation and suppressed BT474 cell proliferation. These results suggest a link between CPT1A LSTase activity and HER2 signaling in regulation of breast cancer cell proliferation. We thus hypothesize that HER2-mediated phosphorylation of CPT1A at Y514 may promote its LSTase activity in addition to the canonical CPTase activity to reprogram metabolism, thereby increasing tumor proliferation. To address this, we propose in Aim 1 to examine how HER2-dependent CPT1A Y514 phosphorylation affects its dual enzymatic activities.
In Aim 2, we will examine whether either or both of the novel LSTase and canonical CPTase activities of CPT1A alter breast cancer metabolism.
In Aim 3, we will examine whether the LSTase activity of CPT1A promotes breast cancer cell proliferation and tumor growth via CPT1A Y514 phosphorylation. Overall, the proposed studies will shed new mechanistic insight into the role of the dual enzymatic activities (LSTase and CPTase activities) of CPT1A in HER2+ breast tumorigenesis and provide the comprehensive understanding of how two distinct PTMs (tyrosine phosphorylation and lysine succinylation) are coordinated to alter metabolism through the signaling hub protein CPT1A to promote tumor growth.
We propose in this application to study the interplay of two distinct PTMs, tyrosine phosphorylation and lysine succinylation, in regulation of cancer metabolism and tumor growth. The proposed studies will shed new mechanistic insight on how HER2 impacts on breast cancer metabolism and tumor growth by regulating the dual enzymatic activities of CPT1A (the novel lysine succinyltransferase and canonical carnitine palmitoyltransferase activities) via tyrosine phosphorylation.
|Kurmi, Kiran; Hitosugi, Sadae; Yu, Jia et al. (2018) Tyrosine Phosphorylation of Mitochondrial Creatine Kinase 1 Enhances a Druggable Tumor Energy Shuttle Pathway. Cell Metab 28:833-847.e8|
|Wiese, Elizabeth K; Hitosugi, Taro (2018) Tyrosine Kinase Signaling in Cancer Metabolism: PKM2 Paradox in the Warburg Effect. Front Cell Dev Biol 6:79|