Do Cell Cycle Inhibitors Promote Angiogenesis by Altering the Bone Marrow Microenvironment?

Abstract

A biochemical partnership between two novel compounds called cell-cycle inhibitors is crucial to the development of blood vessels that help tumors survive and thrive, according to a collaborative Weill Medical College of Cornell University and Memorial Sloan-Kettering Cancer Center study published in the Proceedings of the National Academy of Sciences.

When researchers transplanted tumors into mice genetically engineered to lack two of these inhibitors, those tumors failed to develop much-needed vasculature -- a process called angiogenesis.

"Mice lacking these two inhibitors of cell proliferation had pronounced defects in their ability to mobilize bone marrow stem cells and adjacent blood vessels to create new blood vessels which support both spontaneous tumors and those implanted into the mice," explained co-senior researcher Dr. Andrew Koff, Head of the Cell Cycle Regulation Laboratory at Memorial Sloan-Kettering Cancer Center in New York City.

"This is the first time that anyone has looked at whether disturbing the appropriate cell-cycle controls in tissues supporting tumor growth would affect tumor biology," he added.

What's more, the study suggests that this angiogenic process may be much more complex than previously thought, with one mechanism governing the proliferation of distinct progenitor cell compartments in bone marrow, and another directing the exit of these vessel-growing cells from the marrow into the bloodstream as they head toward the tumor site.

"That means we may have any number of new steps in the angiogenic process, with an interruption in each step representing a potential new anti-cancer target for drug development," explained co-senior researcher Dr. David Lyden, Associate Professor of Pediatrics and Cell and Developmental Biology at Weill Cornell Medical College in New York City.

For over a decade, scientists have sought a safe, effective means of starving malignancies by denying them access to a nutrient-rich blood supply, a theory pioneered by Dr. Judah Folkman, an editor of this manuscript. Much of this research has focused on halting tumor angiogenesis.

Like all new tissues in the body, tumors recruit new blood vessels, helped along by cytokines such as vascular endothelial growth factor (VEGF). Stem cells and their successors, called progenitor cells, proliferate and mobilize under VEGF signaling.

In this collaborative study -- led by Drs. Lyden and Shahin Rafii of Weill Cornell and Drs. Koff and Anxo Vidal of Memorial Sloan-Kettering -- researchers took aim at two different types of VEGF-expressing cells important to angiogenesis: precursor myeloid cells and precursor endothelial cells.

When needed, both of these stem-cell types can spring to life in both bone marrow and in vessels lying adjacent to the new tumor.

The researchers wondered if compounds called cell-cycle inhibitors -- which help regulate progenitor cell activity -- might influence these cells during angiogenesis.

"Looking specifically at two novel cell-cycle inhibitors, p130 and p27, we genetically engineered what we call 'double knock-out' (DKO) mice incapable of expressing either of these enzymes," Dr. Koff explained.

When the researchers transplanted malignant tumors into the p130- and p27-deficient mice, those malignancies failed to create the network of blood vessels they needed to survive.

"First of all, the DKO mice displayed poor proliferation of one cell type, the VEGF-sensitive myeloid progenitor cell, while the endothelial progenitor cells proliferated normally within the bone marrow. However, both cells failed to exit the marrow into the bloodstream," Dr. Lyden said.

"Next, we transferred DKO bone marrow into the normal mouse, which seems to have re-started proliferation of both types of progenitor cells -- especially the myeloid progenitor cells. This suggests that it's the microenvironment in the p130/p27-deficient marrow surrounding these cells that's holding back the proliferation of certain progenitor cells," Dr. Lyden said. "It implies that there are discrete compartments for stem cells within the bone marrow."

Finally, a separate experiment using cells in the lab suggests that a deficiency of p130 and p27, in combination, also inhibits proper endothelial differentiation, with cells from DKO mice failing to form structures that ultimately become blood vessels.

The researchers point out that deficiency in just one of the two cell-cycle inhibitors had little effect on angiogenesis, but when both were removed, the process soon ground to a halt. "They seem to have overlapping functions, cooperating somehow to move the process along," Dr. Koff said.

All of these findings also suggest one thing: the proliferation of cells that go on to form new blood vessels, their migration into the bloodstream and their differentiation in the tumor bed may be three distinct steps, working somewhat independently of each other.

"That's a totally new discovery, and it means we potentially have many more steps in the cell cycle to interrupt the angiogenesis process, giving us multiple 'Achilles' heels' at which to target cancer," Dr. Lyden said.

He noted that the findings may also have implications for leukemias, as well, since blood cancers are usually characterized by the proliferation and migration of blood cells in the marrow.

The study was funded by grants from the National Institutes of Health, the Pew Foundation, The Irma T. Hirschl Trust, the Bane Foundation, the Children's Blood Foundation, the National Cancer Institute, the Emerald Foundation, the Theodore A. Rapp Foundation, Nicolaos Tzimas, the American Hellenic Educational Progressive Association V District, and the Golfers Against Cancer Foundation.

Other co-authors include Dr. Stergios Zacharoulis, Anna H. Bramley, Dr. Kristian K. Jensen, Daniel Kato, Daniel D. McDonald, and Joseph Knowles -- all of Weill Cornell Medical College; Dr. Wenjun Guo, Dr. David Shaffer, Dr. Fillippo Giancotti, and Dr. Nancy Yeh -- all of Memorial Sloan-Kettering Cancer Center; Dr. Carmen de la Hoz of the University of the Basque Country, in Vizaya, Spain; and Dr. Lawrence A. Frohman of the University of Illinois, Chicago. Dr. Vidal, who helped conduct this research while at Memorial Sloan-Kettering, is now at the University of Santiago de Compostela, Spain.