Researchers at Johns Hopkins Medicine have discovered how a set of proteins work in tandem to build the vascular supply lines that deliver oxygen and nutrients to tumors, enabling them to survive and grow. The studies, in human cancer cells and mouse models, showed that the protein pair, peptidylarginine deiminase 4 (PADI4) and hypoxia-inducible factor 1 (HIF-1), ramp up their activity under low-oxygen conditions that are typically found in a fast-growing tumor, to allow the formation of new blood vessels that feed the cancer’s growth. The researchers say their findings provide new possible avenues for developing anticancer therapies that interfere with blood vessel development.

“The discovery gives us an opportunity to find combinations of existing or new drugs that target these pathways to treat cancer and prevent drug resistance,” stated research lead Gregg Semenza, MD, PhD, the C. Michael Armstrong professor of genetic medicine, pediatrics, oncology, medicine, radiation oncology, and biological chemistry at the Johns Hopkins University School of Medicine. Semenza and colleagues reported on their study in Science Advances, in a paper titled, “Histone citrullination by PADI4 is required for HIF-dependent transcriptional responses to hypoxia and tumor vascularization.”

Reduced oxygen availability (hypoxia) is a characteristic feature of the tumor microenvironment, the authors explained. “Rapid responses to hypoxia are critical to maintain cellular viability, and the HIF system is poised to respond. In response to oxygen deficit, HIFs activate the transcription of hundreds of genes that play key roles in angiogenesis, metabolic reprogramming, extracellular matrix remodeling, invasion and metastasis, cancer stem cell specification, and immune evasion. “The molecular mechanism by which HIFs activate transcription is an area of active investigation.”

Semenza serves as director of the vascular program at the Johns Hopkins Institute for Cell Engineering, and shared the 2019 Nobel Prize in Physiology or Medicine for the discovery of how HIF-1 controls the ability of cells to adapt to low oxygen levels. His lab and others have found that HIF-1 activates more than 5,000 genes under low-oxygen conditions. However, it was unclear precisely how HIF-1 turned on those genes to spur blood vessel growth.

Within the cell, DNA is negatively charged, which allows it to interact with positively charged proteins called histones. The DNA is wound like a spool of thread around the histones when it is not being used. Specifically, Semenza said, PADI4 incites a reaction that causes the histones to lose their positive charge, allowing the DNA to unwind. “A less well-known posttranslational modification of histones that decreases their net positive charge is the hydrolysis of arginine (Arg) residues to citrulline (Cit), which is catalyzed by the peptidylarginine deiminases PADI2 and PADI4,” the team further noted.

To explore the partnership between HIF-1 and PADI4, the researchers studied human breast cancer—including triple negative breast cancer (TNBC)—and liver cancer (hepatocellular carcinoma; HCC cells) grown in the laboratory. The team first interfered with the cells’ ability to produce PADI4, and then exposed the cells to low oxygen conditions for 24 hours. By analyzing gene activity within these cells, the researchers found that 87% of the 1,300 genes turned on by HIF-1 in response to hypoxia were not turned on in cells lacking PADI4 protein. The results, they said, “demonstrate that PAD14 expression is induced by hypoxia in a HIF-dependent manner in breast and liver cancer cell lines and that, in turn, PAD14 is required for activation of HIF target gene transcription … RNA sequencing revealed that almost all HIF target genes in breast cancer cells are PADI4 dependent.”

The researchers then injected the cancer cells into the breast tissue of mice and tracked tumor growth. Tumors without PADI4 were five times smaller and developed five times fewer blood vessels compared with tumors formed from cells with normal levels of PADI4. This showed that in a living organism, elimination of PADI4 impaired the tumor’s ability to grow. The findings in mice, the researchers said, allow them to tie together studies from human cancers where higher HIF-1 activity in patients’ primary tumor biopsies correlates with higher rates of mortality.

“In this study, we have identified a previously unidentified regulatory feature in which HIF-dependent PADI4 gene transcription and HIF-dependent recruitment of PADI4 to target genes leads to increased HIF occupancy of HREs [hypoxia responsive elements], histone citrullination, additional histone modifications, and transcriptional activation,” the team concluded. They pointed out that the high level of PAD14 expression in breast and liver cancer contrasts with its expression in normal human tissues, which is restricted to bone marrow and spleen. “Further studies are required to identify the oncogenic switch that is responsible for PADI4 gene expression in breast and liver cancers,” they wrote. “ … both HIF-1α and PADI4 expression are correlated with breast and liver cancer vascularization and patient mortality.” And with the relative lack of effective therapies for patients with TNBC or advanced HCC, “The current study suggests that pharmacologic inhibition of HIF and/or PADI4 activity may represent novel therapeutic strategies for these patients.”

The investigators further noted that because both HIF-1α and PAD14 overexpression are associated with patient mortality in other cancers, further studies are warranted to determine whether this pathway plays an important role different tumor types. Semenza concluded, “The more we know about the cellular ecosystem of cancer, the better shot we have at controlling it.”