Blocking PHGDH in tumors that overexpress it halts cell proliferation and cancer growth in vitro and in vivo.
The enzyme phosphoglycerate dehydrogenase (PHGDH) may represent a therapeutic target for breast cancers that overexpress the protein or have PHGDH gene amplifications, researchers claim. PHGDH was found to be elevated at the protein level in 70% of estrogen receptor (ER)-negative breast cancers.
Further investigation showed that suppressing PHGDH in relevant cancer cell lines led to reduced cell proliferation, while blocking PHGDH in mice with existing tumors significantly slowed tumor growth. Richard Possemato, Ph.D., David M. Sabatini, Ph.D., and colleagues from Whitehead Institute for Biomedical Research and MIT report on their findings in Nature. The paper is titled “Functional genomics reveal that serine synthesis pathway is essential in breast cancer.”
The researchers developed lentiviral shRNA libraries targeting 133 metabolic enzyme and transporter genes identified through public databases for their association with aggressive breast cancer and stemness. To find which of these might be essential for tumorigenesis in vivo, the shRNA libraries were injected into human MCF10DCIS.COM breast cancer cells so that each cell carried one viral integrant. The transduced cells were then injected into mouse mammary fat pads and allowed to form into tumors. After 28 days tumor tissue was evaluated using massively parallel DNA sequencing.
Sixteen genes were designated hits in the screen, including a number that have previously been shown to play important roles in cancer as well as genes involved in oxidative stress, the pentose phosphate pathway, glycolysis, and in the proline and serine biosynthetic pathways.
For five of these genes, shRNAs were then tested for their effects on tumor formation in MCF10DCIS.com cells. In each case the shRNA suppressed expression of its target and reduced tumor-forming capacity. Of these five genes, PHDGH was chosen for further study based on published copy number data demonstrating that the gene exists in a chromosome region commonly amplified in breast cancer, melanoma, and other cancer types.
In fact, the authors write, 18% of patient-derived breast cancer cell lines and 6% of primary tumous have amplifications in PHGDH. In contrast, according to the data sets examined, none of the other hit genes identified by the shRNA screen are sited in genomic regions of focal and recurrent copy number gain.
One previous study had also found elevated PHGDH mRNA in ER-negative, basal-type breast cancers that were associated with poor survival. To corroborate this data, the Whitehead/MIT team analyzed 82 human breast tumor samples using an immunohistochemical assay for PHGDH.
They found that PHGDH protein levels correlated significantly with ER-negative status; compared with ER-positive breast tumors, about 70% of ER-negative breast tumors had elevations of PHGDH at the mRNA and protein levels.
PHGDH catalyzes the first step in the serine biosynthesis pathway, and serine is essential for the production of proteins and biomolecules needed for cell proliferation, the authors note. Studies have shown that rat tumor lysates demonstrate elevated serine biosynthetic activity.
To understand the metabolic consequences of increased PHGDH expression the team used metabolite profiling and serine synthesis pathway flux analysis to examine breast cancer cells with and without PHGDH amplifications. They found that cells with PHGDH amplifications had increased flux through the serine synthesis pathway compared to those without PHGDH amplifications.
The researchers next investigated whether cells with elevated PHGDH expression actually required the enzyme for proliferation and survival. They found that in these cell lines, but not in those without increased PHGDH expression, RNAi-mediated suppression of the enzyme caused a significant decrease in cell number and cell death in the absence of apoptotic markers. The sensitivity to PHGDH suppression was evident both in cells with PHGDH amplifications and those those with high PHGDH expression but without PHGDH amplification.
The ultimate test was to see whether suppressing PHGDH would affect the growth of established tumors in vivo. The team generated an inducible shRNA that only reduced PHGDH protein levels on doxycycline treatment. The transduced cancer cells were injected into mice and allowed to form mammary fat pad tumors for 25 days, before some of the mice were given doxycycline.
Compared with control mice, doxycycline-treated animals exhibited substantially reduced tumor growth. Tumors made from cells transduced with a control inducible shRNA also grew equally well in the presence or absence of doxycycline.
Interestingly, the researchers found that PHGDH suppression inhibited proliferation of cancer cells in vitro even when extra serine was provided, suggesting that serine production may not be the only key requirement of cell lines with high PHGDH expression. The researchers therefore examined whether reactions mediated by PHGDH and the other enzymes in the serine biosynthetic pathway, PSAT1 and PSPH, also generate different metabolites that are critical for cell proliferation.
What they found in cancer cells in vitro was that suppressing PHGDH caused a large reduction in levels of alpha-ketoglutarate (aKG), which the serine pathway normally produces in equimolar amounts alongside serine itself.
Proliferating cells use intermediates of the tricarboxylic acid (TCA) cycle, such as aKG, as biosynthetic precursors and upregulate anaplerotic reactions (i.e., reactions that form intermediates of a metabolic pathway) that drive glutamine-derived carbon into the TCA cycle to counterbalance molecules that are used up, the authors explain. In fact, they note, “of the major metabolites measured, aKG was the one with the most significant and largest change upon PHGDH suppression, whereas serine levels were not significantly changed.”
As with suppression of PHGDH, suppression of PSAT1 also caused a significant reduction in serine pathway flux and aKG levels, and further labeling studies showed that the absolute flux from glutamine to aKG and other TCA intermediates was significantly reduced on RNAi-mediated suppression of either PHGDH or PSAT1.
“These data indicate that in cell lines with high PHGDH expression, the serine synthesis pathway is responsible for approximately 50% of the net conversion of glutamate to aKG and that suppression of PHGDH results in a significant loss of TCA intermediate flux and steady-state levels of TCA intermediates,” the team states.
“As about 70% of ER-negative breast cancers exhibit elevated PHGDH , our work suggests that targeting the serine synthesis pathway may be therapeutically valuable in breast cancers with elevated PHGDH expression or PHGDH amplifications.”