Scientists have identified a molecule that connects a biochemical pathway used by cancer cells to generate energy and regulates an oncogene that drives the progression of breast cancer and other tumor types. The studies, by a team at Baylor College of Medicine and Roswell Park Comprehensive Cancer Center, showed that PFKFB4, an enzyme in the Warburg pathway, activates an oncogene called SRC-3. The findings throw new light on the long-recognized phenomenon of the Warburg effect—by which cancer cells forego normal mitochondrial metabolism of glucose and instead convert glucose to lactate via fermentation—and suggest potential new drug targets. The team’s in vivo tests showed that inhibiting either PFKFB4 or SRC-3 in cancer-bearing mice led to reduced tumor growth and size and prevented the tumors from metastasizing to the lungs.
The researchers report their findings in Nature, in a paper entitled “Metabolic Enzyme PFKFB4 Activates Transcriptional Coactivator SRC-3 to Drive Breast Cancer.”
“In the 1920s, Otto Warburg and his colleagues discovered that cancer cells consume larger amounts of glucose than normal cells,” explains lead researcher Bert O'Malley, Ph.D., chair and professor of molecular and cellular biology, Thomas C. Thompson Chair in Cell Biology and associate director of basic research in the Dan L Duncan Comprehensive Cancer Center. “This has been a mystery for quite some time. Why would cancer cells, which need large amounts of energy to sustain their growth, prefer to use a pathway that produces less adenosine triphosphate (ATP) than another available pathway? What would be the advantage for cancer cells to use the Warburg pathway?”
Dr. O’Malley’s lab had previously identified SRC-3 (also known as NCOA3), a nuclear receptor coactivator that plays a key role in regulating gene expression and is commonly amplified or overexpressed in breast cancer. SRC-3 phosphorylation triggers the protein to become overactive, which is a common sign of many tumor types. “Phosphorylation of SRC-3 can alter its transcriptional activity, protein stability and subcellular localization, and deregulated kinase signalling hyperactivating SRC-3 is a hallmark of many tumours,” the authors write.
As part of their latest research, the team carried out screening assays to search for kinase enzymes that impacted on SRC-3 activity. Their screens highlighted metabolic kinase PFKFB4 as “the most robust positive regulator of SRC-3,” and indicated that PFKFB4 was “the most dominant kinase that regulates cellular proliferation.” Subsequent in vitro tests showed that silencing PFKFB4 led to decreased SRC-3 activity in a number of cancer cell lines, while overexpression of PFKFB4 boosted SRC-3 activity.
The team then carried out enzymatic reaction experiments and assays to demonstrate that PFKFB4 functions as a protein kinase that transfers a phosphate group from ATP to SRC-3 at position Ser857. “We were surprised to identify an enzyme named PFKFB4 as one of the most dominant regulators of protein SRC-3,” notes first author Subhamoy Dasgupta, Ph.D., who worked on the project at the O’Malley lab, and is now an assistant professor of cell stress biology at Roswell Park Comprehensive Cancer Center. “This was unexpected because PFKFB4 was well known for its ability to only add phosphate groups to sugars in the Warburg pathway. Nobody had described before that this enzyme could also add phosphate groups to proteins.” As Dr. O’Malley adds, “When PFKFB4 adds a phosphate group to SRC-3, it transforms it into a potent driver of breast cancer and other cancers as well.”
Increased glucose metabolism has been shown to stimulate the kinase activity of PFKFB4 that is needed to maintain steady glycolysis. The team next measured levels of phosphorylated SRC-3 in cells cultured in different concentrations of glucose. The results showed that increased glucose concentration was associated with increased levels of SRC-3 phosphorylation. Breast cancer cells grown in low glucose conditions demonstrated lower SRC-3 phosphorylation than tumor cells cultured in normal glucose conditions, while knocking down PFKFB4 in breast cancer cells grown in normal glucose levels abolished pSRC-3-Ser857, “ indicating that PFKFB4-dependent SRC-3 phosphorylation on Ser857 is a highly selective modification under conditions conducive to active glycolysis,” the team writes.
In a separate set of experiments they showed that overexpressing PFKFB4 in cancer cells cultured in media containing a normal glucose concentration resulted in significantly increased SRC-3 transcriptional activity when compared with the same PFKFB4-overexpressing cells cultured in low glucose conditions. This suggests that “PFKFB4-dependent SRC-3 phosphorylation is important for the coactivator-driven transcriptional response,” the researchers state.
Encouragingly, silencing either SRC-3 or PFKFB4 in a mouse model of breast cancer led to “substantially reduced tumor growth and volume.” Immunostaining studies showed that tumors lacking SRC-3 or PFKFB4 had far fewer proliferative cells. And while animals with primary tumors and normal SRC-3 went on to develop lung metastases, suppressing either SRC-3 or PFKFB4 or expressing a phosphorylation-deficient SRC-3 mutant SRC-3(Ser857Ala) inhibited the development of lung metastases. “These findings demonstrate that SRC-3 and PFKFB4 are drivers of basal-subtype breast tumour growth and that phosphorylation of SRC-3 at the Ser857 site is crucial for metastatic progression of the disease,” the team notes.
To identify the clinical relevance of their findings, the team analyzed data from The Cancer Genome Atlas (TCGA) dataset, which confirmed elevated PFKFB4 expression levels were evident across all subtypes of breast cancer. SRC-3 is also a known estrogen receptor (ER) coactivator, so the researchers analyzed expression of pSRC-3-Ser857 and PFKFB4 in ER-positive human primary breast tumors and adjacent normal tissues. These results also showed increased levels of pSRC-3-Ser857, PFKFB4, and SRC-3 in most of the tumor tissue evaluated, when compared with normal tissues. A common PFKFB4-SRC-3 proteomic signature was similarly associated with decreased likelihood of survival in a basal-like subtype triple-negative patient cohort. “These clinical associations are compatible with our in vivo experimental observations substantiating that the PFKFB4–SRC-3 axis is a molecular powerhouse that propels breast tumorigenesis leading it to an aggressive metastatic disease,” the team states. “PFKFB4 and phosphorylated SRC-3 levels are increased and correlate in estrogen receptor-positive tumours, whereas, in patients with the basal subtype, PFKFB4 and SRC-3 drive a common protein signature that correlates with the poor survival of patients with breast cancer.”
“Here we have uncovered an interaction between the glycolytic pathway and the oncogenic activation of the transcriptional coactivator SRC-3,” they conclude. “The Warburg effect is known to be one of the most dominant sugar metabolic pathways across cancers generating energy and macromolecules to sustain rapid proliferation and tumour growth. We now find that a glycolytic stimulator, the bifunctional enzyme PFKFB4, also can operate as a protein kinase, at least in actively glycolytic tumours.…These findings suggest that the Warburg pathway enzyme PFKFB4 acts as a molecular fulcrum that couples sugar metabolism to transcriptional activation by stimulating SRC-3 to promote aggressive metastatic tumours.…Our work suggests that targeting the PFKFB4–SRC-3 axis may be therapeutically valuable in breast tumours that are notably dependent on glucose metabolism.”
“One of the most interesting things to me is that we have solved some of this nearly 100-year-old mystery,” O'Malley states. “Also, our findings give us more potential intervention points for future therapies.”