RESUMO
Bone metastasis of breast cancer results in severe bone loss, fractures, and death. Crosstalk between breast cancer cells and bone resident cells promotes osteoclast activity and the release of growth factors from the bone matrix resulting in aggressive tumor growth and bone loss. We and others have shown that Runt-related transcription factor-2 (Runx2) promotes metastatic tumor growth-associated bone loss. Breast cancer cells also induce autophagy to survive metabolic stress at the metastatic site. Recently, we reported a Runx2-dependent increase in autophagy. In this study, to examine the underlying mechanisms of metastasis and tumor resistance to stress, we used a bone metastatic isogenic variant of breast cancer MDA-MB-231 cells isolated from a xenograft tumor mouse model of metastasis. Our results with immunofluorescence and biochemical approaches revealed that Runx2 promotes microtubule (MT) stability to facilitate autophagy. Stable MTs are critical for autophagosome trafficking and display increased acetylation at Lysine 40 of α-tubulin. Runx2 silencing decreases acetylated α-tubulin levels. The expression levels of HDAC6 and αTAT1, which serve to regulate the acetylation of α-tubulin, were not altered with Runx2 silencing. We found that HDAC6 interaction with α-tubulin is inhibited by Runt-related factor-2 (Runx2). We show that the expression of wild-type Runx2 can restore the acetylated polymer of MTs in Runx2 knockdown cells, while the C-terminal deletion mutant fails to rescue the polymer of MTs. Importantly, cellular stress, such as glucose starvation also increases the acetylation of α-tubulin. We found that the loss of Runx2 increases the sensitivity of breast cancer cells to MT-targeting agents. Overall, our results indicate a novel regulatory mechanism of microtubule acetylation and suggest that Runx2 and acetylated microtubules may serve as therapeutic targets for bone metastatic tumors.
RESUMO
Bone metastasis is a frequent complication of breast cancer with nearly 70% of metastatic breast cancer patients developing bone metastasis during the course of their disease. The bone represents a dynamic microenvironment which provides a fertile soil for disseminated tumor cells, however, the mechanisms which regulate the interactions between a metastatic tumor and the bone microenvironment remain poorly understood. Recent studies indicate that during the metastatic process a bidirectional relationship between metastatic tumor cells and the bone microenvironment begins to develop. Metastatic cells display aberrant expression of genes typically reserved for skeletal development and alter the activity of resident cells within the bone microenvironment to promote tumor development, resulting in the severe bone loss. While transcriptional regulation of the metastatic process has been well established, recent findings from our and other research groups highlight the role of the autophagy and secretory pathways in interactions between resident and tumor cells during bone metastatic tumor growth. These reports show high levels of autophagy-related markers, regulatory factors of the autophagy pathway, and autophagy-mediated secretion of matrix metalloproteinases (MMP's), receptor activator of nuclear factor kappa B ligand (RANKL), parathyroid hormone related protein (PTHrP), as well as WNT5A in bone metastatic breast cancer cells. In this review, we discuss the recently elucidated mechanisms and their crosstalk with signaling pathways, and potential therapeutic targets for bone metastatic disease.
RESUMO
BACKGROUND: Breast cancer is the most commonly diagnosed cancer among women and its metastases results in poor survival rates in patients. The ability to alter metabolism is a key attribute cancer cells use to survive within different metastatic microenvironments and cause organ failure. We hypothesized that evaluation of metabolic alterations within tumor cells could provide a better understanding of cancer metastasis. Therefore, to investigate underlying metabolic alterations during metastases, we utilized human MDA-MB-231 and mouse 4T1 models that closely mimic human breast cancer metastasis. METHODS: The glycolysis and glutamine pathway-related changes were examined in bone metastatic cells by XF-24 extracellular flux analyzer and western blotting. The expression levels of genes related to metabolism were examined by PCR arrays. RESULTS: The MDA-MB-231 cells isolated after bone metastases showed reduced glucose uptake and glycolysis compared to parental cells, suggesting that these cells could alter metabolic requirements for survival. To understand these metabolic changes, we investigated glutamine, a common and naturally occurring non-essential amino acid. Interestingly, in reduced glucose conditions both cell lines showed dependence on glutamine for cell survival, and with glutamine withdrawal significantly increasing apoptotic cell death. Glutamine was also critical for normal cell proliferation even in the presence of high glucose concentrations. To further understand this metabolic switch in metastatic cells, we examined the genes related to metabolism and identified a more than seven-fold downregulation of protein kinase C zeta (PKC-ζ) expression levels in bone-derived MDA-MB-231 cells compared to the parental population. The PKC-ζ levels were also significantly reduced in metastatic 4T1 cells compared to non-metastatic MT1A2 cells. Since PKC-ζ deficiency promotes glutamine utilization via the serine biosynthesis pathway, we examined glutamine metabolism. The ectopic expression of PKC-ζ inhibited glutamine conversion to glutamate, while mutant PKC-ζ reversed this effect. Furthermore, the gene expression levels of enzymes involved in serine biosynthesis, phosphoserine phosphatase (PSPH), phosphoserine aminotransferase (PSAT1), and phosphoglycerate dehydrogenase (PHGDH) showed upregulation following glucose deprivation with PKC-ζ deficiency. The PHGDH upregulation was inhibited by ectopically expressing wild type but not mutated PKC-ζ in glucose-deprived conditions. CONCLUSIONS: Our results support the upregulation of serine biosynthesis pathway genes and downregulation of PKC-ζ as potential metabolic alterations for bone metastatic breast cancer cells.
