Proline Metabolism in Metastatic Breast Cancer

Institution: The Burnham Institute for Medical Research
Investigator(s): Adam Richardson, Ph.D. -
Award Cycle: 2009 (Cycle 15) Grant #: 15IB-0095 Award: $284,895
Award Type: IDEA
Research Priorities
Biology of the Breast Cell>Pathogenesis: understanding the disease



Initial Award Abstract (2009)

Increased glucose consumption is perhaps the most common phenotype of tumorigenic cells. Although we know that the metabolic program of human tumor cells is critically important to malignant tumors, the molecular details of human tumor metabolic program remain largely unknown. Previously, we studied the changes that occur to glucose metabolic pathways as breast cells become increasingly invasive and them metastatic. Based upon these and other results, we suspect that the proline (one of 20 DNA-encoded amino acids) biosynthetic pathway is a key mediator of breast tumor cell metastasis.

Using IDEA grant funding from CBCRP we will test our hypothesis that altered breast tumor cell proline metabolism is associated mechanistically with metastasis. We will specifically test:

1) To what extent does proline biosynthesis increase metastasis?
2) Does increased proline biosynthesis or an increased cellular proline pool suppress apoptosis and allow breast tumor cells to migrate?

Our experimental approach is based on and utilizes stable isotope labeling, an uncommon but extremely informative branch of metabolomics (i.e., the collection of all metabolites in a cell or organism). By growing human breast tumor cells in the presence of 13C-glucose (which is indistinguishable from 12C-glucose on a cellular level, but more readily observed) we are able to determine not only the relative amounts of metabolites but the rates at which they were produced. We will use this technique to measure proline biosynthetic rates in human cells following gene silencing or over-expression, and in mouse models of metastasis. The importance of proline biosynthesis in metastasis will be tested in a series of human breast cells, derived from the same patient sample, that range from completely non-tumorigenic (normal control) to invasive to metastatic.

While it is well documented that breast cancer cells shift their global metabolic programs, the exact role of these metabolic shifts in tumor invasion and metastasis remains undefined. Determination of the activity of the metabolic program in addition to the expression level of the enzymatic components will describe an important aspect of the breast tumor phenotype and potentially allow us to design therapies that inhibit tumor cell movement. This study will significantly contribute both to our knowledge of metabolic changes associated with the metastatic phenotype in breast cancer, and to the development of anti-metastatic therapies for breast cancer.




Final Report (2011)

Increased glucose consumption (Warburg effect) is perhaps the most common phenotype of cancer cells. This is exemplified by clinical success and importance of FDG-PET imaging, which directly measures cellular glucose uptake. Although we know that the metabolic program of human tumor cells is critically important to malignant tumors, the molecular details of human tumor metabolic program remain largely unknown. Using an advanced metabolomic technique, we have mapped the changes that occur to glucose and glutamine metabolic pathways as breast cells become increasingly invasive and then metastatic. Based upon these and other results, we suspect that the proline biosynthetic pathway is a key mediator of breast tumor cell metastasis.

Our specific aims were composed of two main goals: to determine the extent to which proline biosynthesis increases metastasis in breast cancer cell lines and to test if proline biosynthesis (or an increased pool of proline) suppressed apoptosis. In order to accomplish these aims, it was first necessary to successfully manipulate proline biosynthesis in our MCF10 cell line model. This proved to be a good deal more difficult and complex than anticipated. While learning to control proline biosynthesis through molecular biology, we discovered a previously uncharacterized protein in the proline pathway, pyrroline-5-carboxylate reductase-like (PYCRL). We have since expressed, purified and fully characterized this protein, along with the other two PYCR enzymes (PYCRI and PYCR2). Due to significant homology and functional overlap between these three enzymes, understanding and controlling this system proved too difficult to accomplish in the timeframe of the CBCRP funding.

However, we did succeed in developing new information on this topic, and we are currently completing a manuscript, "Functional Reundancy in the Last Step of Human Proline Biosynthesis." With this new information in hand, we are currently testing the role of each of these three proline biosynthetic enzymes in breast cancer metastasis.




Symposium Abstract (2010)

The goal of this work is to determine the role of proline biosynthesis in breast cancer metastasis and to test if inhibiting proline biosynthesis reduces the ability of breast tumor cells to metastasize. Recently we mapped the central carbon metabolism of several breast carcinoma cell lines using 13C stable isotope labeling. We discovered that de novo proline synthesis is greatly increased in breast cancer lines that are able to metastasize. We propose a model in which proline biosynthesis acts as an anti-apoptotic signal and increases metastatic potential. In this model, epithelial cells that have gained the ability to invade the surrounding stroma also experience an increase in proline pool size through collagen degradation. In non-transformed cells, the resulting enlarged proline pool causes a p53-regulated apoptotic response via reactive oxygen species produced by proline dehydrogenase. Tumor cells are able to avoid death due to down-regulation of proline oxidation. As the tumor progresses to the stage of metastasis and cells detach from the extracellular matrix, they must produce their own proline through de novo synthesis. Therefore, it is plausible that enzymes of the proline biosynthetic pathway are potential targets for the suppression of metastasis in some human cancers.

Pyrroline-5-carboxylase 1 (PYCR1) is considered the rate-limiting step of proline biosynthesis. Although this gene has historically been identified as the major producer of proline in human systems, we now know that two additional PYCR-encoding genes, PYCR2 and PYCRL, also play a significant role. Here we report the characterization of the three human PYCR genes in both a biochemical and cellular context. We have expressed and purified functional PYCR1, PYCR2 and PYCRL using an E. coli system, allowing us to characterize the enzymatic functions of each PYCR. We additionally report on the cellular function of each of the PYCR enzymes, particularly their relative contribution to proline biosynthesis. We have utilized two approaches during this in situ characterization. First, we suppressed the expression of the PYCR genes in the metastatic MCF10-CA1a breast tumor cell line (both individually and in combination) using siRNA and measured proline biosynthesis via 13C labeling and mass spectrometry. Second, we introduced each of the PYCR genes into the MCF10-AT1 breast carcinoma cell line (which does not natively express any of the PYCR enzymes) and again performed flux analysis. The results of these characterizing experiments will be presented, as well as the impact of each PYCR on the ability of breast carcinoma cells to survive, proliferate and metastasize. Overall, this study contributes to our knowledge of metabolic changes associated with the metastatic phenotype in breast cancer and to the development of anti-metastatic therapies.