Scientists in the U.S. have found that a dietary supplement added to sports drinks can effectively reduce the growth of human drug-resistant HER2-positive breast cancer tumors in mice. The studies, headed by Mayo Clinic Pharmacologist Taro Hitosugi, Ph.D., found that the supplement, cyclocreatine targets mitochondrial creatine kinase 1 (MtCK1), which is involved in a mitochondrial metabolic pathway that is upregulated in HER2-positive breast tumors. Tumor proliferation was reduced in cyclocreatine-treated mice with human trastuzumab-resistant HER2+ xenografts. Combining cyclocreatine with the HER2 kinase inhibitor lapatinib resulted in even greater antiproliferative effects in the mouse xenograft models than either treatment alone.

“Mitochondrial creatine kinase 1 may be a new drug target for the treatment of HER2 positive breast cancer,” says Matthew Goetz, M.D., director of the Mayo Clinic Breast Cancer research program, and co-author of the team’s published paper in Cell Metabolism. “Future clinical trials will be necessary to determine the effectiveness of this drug for HER2 positive breast cancer resistant to standard therapies.” The rodent studies are published in a paper entitled, “Tyrosine Phosphorylation of Mitochondrial Creatine Kinase 1 Enhances a Druggable Tumor Energy Shuttle Pathway.

Many key metabolic processes take place in the mitochondria, and the concept that activated oncogenes may upregulate metabolic pathways to match the energy and biosynthesis requirements of rapidly dividing cancer cells has led to the development of a number of clinical-stage drug candidates that target mitochondrial metabolism.  One of the potential drawbacks of these drugs is toxicity to normal cells, which also rely on mitochondrial metabolism.” Therefore, identifying tumor-specific mitochondrial metabolic alterations could aid in the design of drugs that can selectively target cancer cells while sparing normal tissue,” the authors write.

The HER2 receptor tyrosine kinase functions as an 'on' or 'off' switch in cellular functions. Representing a key driver of breast cancer, HER2 is overexpressed in about a quarter of all breast cancers,” says Taro Hitosugi, Ph.D., a pharmacologist at Mayo Clinic and corresponding author of the team’s published paper. “While drugs such as trastuzumab improved outcomes for some patients with HER2 positive breast cancer, some tumors are or may become resistant to this drug.”

Recent studies have also suggested that the HER2 – a member of the epidermal growth factor receptor (EGFR) family – alters mitochondrial metabolism, but what hasn’t been understood is which mitochondrial protein or proteins represent the HER2 target, how HER2 signals to mitochondria, and how the resulting changes in mitochondrial metabolism link to cancer cell proliferation.

One potential link in this mechanism is MtCK1, a mitochondrial protein involved in the phosphocreatine (PCr) energy shuttle, a pathway by that helps to maintain energy reserves in metabolically active cells. 

The Mayo Clinic team’s studies have now shown that HER2 signalling induces phosphorylation of MtCK1 tyrosine 153 by the tyrosine kinase ABL, which leads to stabilization and upregulation of MtCK1 in patient-derived HER2-positive breast cancer xenografts (PDX). “We employed metabolomics and proteomics approaches to identify HER2-dependent metabolic events, and discovered that HER2 signaling activates mitochondrial creatine kinase 1,” says Dr. Hitosugi.

Initial tests demonstrated that knocking down MtCK1 reduced the proliferation of two different HER2-expressing cell lines, and decreased mitochondrial oxygen consumption rate and mitochondrial respiratory capacity. Tests also confirmed that trastuzumab-resistant cell lines exhibited increased levels of phosphorylated HER2 and total MtCK1, while depleting MtCK1 reduced proliferation of the HER2+ tumor cells.

The team found high expression levels of MtCK1 in five of six HER2+ PDX tumors, but could not detect MtCK1 in any normal breast samples. MtCK1 Y153 was also highly phosphorylated in all evaluated HER2-positive PDX tumors that expressed MtCK1, but not in triple-negative PDX tumors, “suggesting that MtCK1 Y153 phosphorylation is specific to HET2+ PDX tumors,” the authors write. Of particular interest, the team found that HER2+ PDX tumors derived from patients whose cancers were resistant to trastuzumab exhibited particularly high MtCK1 Y153 phosphorylation. Again, knockdown of MtCK1 reduced the proliferation of trastuzumab-resistant PDX cells in culture, and also suppressed the growth of these tumors in mice. In contrast, knocking down MtCK1 in a triple-negative breast cancer PDX that expressed MtCK1 had no effect on cell proliferation. “Taken together, these results raise the possibility that MtCK1 Y153 phosphorylation regulates a metabolic pathway that is important in HER2+ breast cancers,” the researchers suggest.

Given the observation that knocking down MtCK1 could reduce the proliferation of HER2+ cell lines and PDX models, the authors wanted to evaluate whether targeting the PCr energy shuttle directly could also reduce tumor growth.

Subsequent tests demonstrated that proliferation of trastuzumab-resistant and trastuzumab-sensitive HER2+ breast cancer cells in vitro was reduced following exposure to the creatine analog cyclocreatine (Cyclo-Cr), but could be restored by adding PCr to the medium, “indicating that the inhibitory effect of Cyclo-Cr was due to the inhibition of the PCr energy shuttle,” the team comments.  Cyclo-Cr also inhibited the growth of trastuzumab-resistant PDX-derived organoids in culture, but had no adverse effects on normal breast epithelial cells, “suggesting that blocking the PCr energy shuttle may have selective anti-tumor activity against HER2+ tumors.”

Importantly, adding Cyclo-Cr to the drinking water of experimental mice led to significantly reduced growth of trastuzumab-resistant PDX tumors, without showing any evidence of toxicity. Combined treatment using Cyclo-Cr and oral lapatinib had even greater anti-proliferative effects in tumor-bearing mice than either therapy alone. Again, the antiproliferative effects of therapy could be reversed by treatment using PCr.

“In summary, our study revealed that MtCK1 stabilization by Y153 phosphorylation is a key molecular mechanism underlying the intersection between HER2 signalling and mitochondrial energy metabolism, and also showed that the resulting enhanced PCr energy transfer creates a druggable metabolic liability in HER2+ breast cancer,” the authors conclude.

They acknowledge that while Cyclo-Cr is used as a dietary supplement and healthy people ingest grams of it in sports drinks, the maximum tolerated dose hasn't yet been determined in humans. Given that the concentrations used in the mouse studies were relatively high, clinical studies will be needed to evaluate the safety of high concentrations in humans, they team points out. Another possibility, they suggest, would be to combine lower doses of Cyclo-Cr with other drugs that act synergistically to reduce tumor growth, similar to the additive antiproliferative effect of combining lapatinib with Cyclo-Cr in mice.

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