Skp2 activates cancer-promoting, glucose-processing Akt

This chain of events, the scientists found, promotes Herceptin resistance in breast cancer and activation of glucose metabolism (glycolysis), which cancer cells primarily rely on to fuel their growth and survive.

Their research focused on Skp2 E3 ligase, a protein that binds to and tags other proteins with molecules called ubiquitins, in this case to activate the Akt kinase. "We discovered a novel function of Skp2 E3 ligase that makes it an important player in cancer development and also identified a crucial role for it as a regulator of the glycolysis pathway," said senior author Hui-Kuan Lin, Ph.D., associate professor in MD Anderson's Department of Molecular and Cellular Oncology.

"This is potentially important for understanding and addressing Herceptin resistance in breast cancer," Lin said. "The effect on glucose metabolism also could have implications for other types of solid tumor cancers, including prostate, because they rely so heavily on glycolysis."

The team also found that Skp2 overexpression is associated with poor prognosis for breast cancer patients and its spread to other organs.

Lin and colleagues are studying potential inhibitors of Skp2 that might be developed for treatment.

The EGFR family of proteins includes HER2, which abundantly coats cancer cells in about a third of breast cancers, making these tumors prime targets for the targeted drug Herceptin. Fundamental discovery about Akt activation

The Akt kinase relays signals by growth factors from outside of the cell into the cell. It regulates cell profliferation and survival, metabolism and tumor development, the authors noted.

To do its work, whether normal or oncogenic signaling, it must move from the cytosol to the plasma membrane. To do that, Lin and colleagues had previously shown that Akt must be ubiquitinated -- and those ubiquitins must be attached in a specific chain formation, the K63-linked polyubiquitin chains.

That earlier finding involved the insulin-like growth factor receptor (IGF-1) and a different E3 ligase. "Finding that the epidermal growth factor receptors also ubiquitinate Akt, and that they do so through the Skp2 E3 ligase, was quite unexpected," Lin said.

Finding two paths to ubiquitination implies that there might be more, Lin said.

Regulating cancer's energy engine

Tumor cells evolve to rely mainly on glucose processing to generate energy, grow and thrive. They increase glucose uptake and glycolysis, a process that doesn't involve oxygen and occurs in the cell's cytosol. Non-cancerous cells rely more heavily on oxygen-based metabolism of fatty acids and other nutrients in the cell's mitochondria. This difference is called the Warburg Effect.

In a series of experiments, the researchers blocked Skp2 expression and found:

• Suppression of glucose uptake and glycolysis in breast cancer cell lines.

• Reduction of tumor size and glucose uptake in a mouse model of HER2-expressing breast cancer.

• Steep declines in the expression of glucose-transporter Glut1. Cells with Skp2 produced more Glut1 when induced by epidermal growth factor signaling. Akt activates Glut1.

• Reduced Akt activation, Glut1 expression and tumor growth plus extended survival in mice genetically engineered to develop breast cancer and metastasis.

Poor prognosis, Herceptin resistance

The team analyzed 213 cases of breast cancer treated with modified radical mastectomy but no chemotherapy or radiation. Of these, 80 were HER2-positive. In these tumors Skp2 expression was correlated with:

• Adverse prognostic factors such as cancer stage, primary tumor status and lymph node metastasis.

• Increased activation of Akt.

A multivariable analyses of prognostic factors showed Skp2 overexpression to be a significant factor affecting metastasis-free survival for HER2-positive breast cancer patients. No prognostic role was found for Skp2 expression among patients with HER2-negative disease. HER2-positive breast cancer patients can develop resistance to Herceptin treatment, the researchers noted, and some encounter this problem right at the start. Knocking down Skp2 made cancer cell lines more susceptible to Herceptin, stifling cancer proliferation. In another mouse model, Herceptin by itself inhibited tumor growth but did not shrink tumors. With Skp2 inhibited, Herceptin treatment caused the tumors to nearly vanish in 22 days.

This sensitivity was blocked when the scientists overexpressed Akt in Skp2-silenced cancer cells.

Overall, the team's findings indicate that blocking Skp2 might inhibit glycolysis and that targeting glycolysis could be an important approach to cancer treatment, Lin said. Co-authors with Lin are first author Chia-Hsin Chan, Ph.D., Wei-Lei Yang, Yuan Gao, Szu-Wei Lee, Zizhen Feng, Dihua Yu, M.D., Ph.D., Dos Sarbassov, Ph.D., and Mien-Chie Hung, Ph.D., all of MD Anderson's Department of Molecular and Cellular Oncology; Leo Flores of MD Anderson's Department of Experimental Diagnostic Imaging; Yiping Shao, Ph.D., and John Hazle, Ph.D., of MD Anderson's Department of Imaging Physics; Chien-Feng Li, M.D., of Chi-Mei Foundational Medical Center in Tainan, Taiwan, and with Kelvin Tsai, M.D., also of the National Institute of Cancer Research in Tainan; Hsuan-Ying Huang, M.D., Chang Gung Memorial Hospital-Kaohsiung Medical Center, Chang Gung University College of Medicine, Taiwan; Wenyi Wei, Ph.D., Beth Israel Deaconess Medical Center and Harvard Medical School; and Keiichi Nakayama, M.D., Ph.D. Medical Institute of Bioregulations, Kyushu University, Fukuoka, Japan.

Yang, Gao and Lee are all students in The University of Texas Graduate School of Biomedical Sciences at Houston, a joint program of MD Anderson and The University of Texas Health Science Center at Houston. Hung, Yu and Sarbassov are on the GSBS faculty.