This image of an extracellular matrix – in this case collagen (blue) plus tumor cells (red cells attached to blue fibers, arrow) from a human sarcoma -- shows the mini highway along which tumor cells migrate toward blood vessels in order to metastasize (left panel). The way in which tumor cells use collagen to hitch a ride is still a mystery. Penn Medicine researchers, using a sophisticated laser-based technology called multi-photon second harmonic generation imaging to take pictures of tumor tissue expressing green florescent protein, were able to see tumor cells and collagen in action. The right panel shows a cross section of more normal collagen without tumor cells and with red blood cells populating the opening of the vessel.
With this zoomed-in view, the Abramson Family Cancer Research Institute (AFCRI) lab of Celeste Simon, PhD, and postdoctoral fellow, T.S. Karin Eisinger-Mathason PhD, report in Cancer Discovery about the potential of the collagen-modifying enzyme PLOD2 as a new therapeutic target for the treatment of metastatic undifferentiated pleomorphic sarcoma (UPS). Simon is the AFCRI Scientific Director, an Investigator with the Howard Hughes Medical Institute, and a professor of Cell and Developmental Biology.Sarcomas are relatively rare cancers of bone, fat, muscle, and other tissues. UPS is a commonly diagnosed and particularly aggressive type of adult sarcoma, which often goes to the lung. Metastasis is the most common cause of sarcoma-associated death. What regulates this process is not well understood, but primary sarcomas generate extensive extracellular collagen networks.
The identification of the PLOD2 pathway as a determinant of sarcoma metastatic potential suggests that targeting PLOD2 may be a potential strategy to prevent sarcoma metastasis. “We concluded that PLOD2 is a novel therapeutic target in sarcomas and successful inhibition of this enzyme may reduce tumor cell movement out of the primary tumor,” says Eisinger-Mathason.
Increasing evidence from preclinical studies suggests that remodeling of the collagen-based extracellular matrix can be induced by pockets of low oxygen, called hypoxia, in tumors, that trigger the expression of hypoxia-inducible factor 1a (HIF-1a). Low oxygen in the tumors and the presence of HIF-1a correlate with metastasis and poor survival in patients with sarcoma.
High levels of HIF-1a in the primary tumor induces the expression of PLOD2, which is required for production of disorganized collagen in sarcomas. Loss of HIF-1α or PLOD2 disrupts the formation of this collagen matrix, and therefore tumor cell migration and eventual metastasis to the lung. However, primary tumor growth in mouse models of sarcoma is not affected by loss of HIF-1α or PLOD2.
The researchers inhibited PLOD2 in cells in two ways: Genetically removing HIF-1a and using minoxidil (the main ingredient of the hair loss medication Rogaine). “We found that tumor cells could not migrate and metastasize without PLOD2, but in cells with PLOD2 the collagen becomes easier for tumor cells to latch onto and use as a ride get to the closest blood vessel.” From there, tumor cells can access the main blood stream and the lungs, in the case of many sarcomas.
What’s more, expression of PLOD2 restores migration and metastatic potential in cells within HIF-1α–deficient tumors. The team also saw elevated HIF-1α and PLOD2 expression in metastatic primary tissue from human sarcomas.
Collaborator Sam Yoon from Memorial Sloan-Kettering Cancer Center made a microarray of tumor tissue and found HIF-1a and other downstream proteins overexpressed. The team then screened the overexpressed proteins to see which ones were induced by hypoxia in cell lines and in mice.
“PLOD 2 was the only that really popped out,” says Eisinger-Mathason. The team used a human cell line of fibosarcoma and two mouse cell lines that develop sarcoma and exposed them to low levels of oxygen in a special incubator, at 0.5 percent spell oxygen levels versus 21 percent normal, ambient oxygen levels. PLOD2 was the most upregulated among the proteins in the hypoxia group. mRNA expression of PLOD2 in hypoxic versus normal oxygen-level cells was six times higher.
“We now need to look at minoxidil and other related drugs and enzymes as lead compounds and work with medicinal chemists to optimize the molecule for it to become a viable cancer drug,” says Eisinger-Mathason.Eisinger-Mathason TS, Zhang M, Qiu Q, Skuli N, Nakazawa MS, Karakasheva T, Mucaj V, Shay JE, Stangenberg L, Sadri N, Puré E, Yoon SS, Kirsch DG, & Simon MC (2013). Hypoxia-Dependent Modification of Collagen Networks Promotes Sarcoma Metastasis. Cancer discovery PMID: 23906982