These data support the hypothesis that prostate cancer cells containing a mutation in the mitochondrial DNA may be relevant to bone metastasis. The data demonstrate that the growth advantage conferred by mtDNA mutations is most fully realized in the bone stromal microenvironment. This effect has been demonstrated in two model systems: co-inoculation of stromal and epithelial cells subcutaneously in nude mice and the direct intratibial injection of mutant or wild type cancer cells. In addition, we present the findings of differential regulation of genes important in the progression of prostate cancer by the presence of bone stromal cells. Gene expression analysis reveals a 37 gene signature of this highly growth-inductive interaction between prostate cancer cells with mtDNA mutation and bone stromal cells. Amongst these 37 genes, two stand out as significantly overexpressed only when both mtDNA mutations and bone stroma are present and as highly relevant to prostate cancer: FGF-1 and FAK, and others may ultimately be shown to be important.
Both FGF-1 (acidic or aFGF) and FGF-2 (basic or bFGF) are expressed in clinical prostate cancer, along with other FGF family members [16
]. In a series of 72 patients with prostate cancer, aFGF was overexpressed in 62/72 (86.1%) cases while bFGF was only overexpressed in 65.5% of cases. In this detailed analysis of cellular site of aFGF immunoreactivity in clinical prostate specimens, only 6.9% of cases showed aFGF being produced in the stroma, and 7.4% of cases showed aFGF production in the surrounding benign tissue from malignant prostate specimens. In benign prostatic hyperplasia (BPH) specimens, none showed significant aFGF protein. This increase in aFGF in clinical prostate cancer, compared to either benign hypertrophy or surrounding stroma, is consistent with findings in other malignancies such as colorectal and pancreatic carcinomas [17
]. Thus, aFGF appears to be the major FGF isoform in clinical prostate cancer and is secreted predominantly by the malignant epithelium, suggesting autocrine stimulation.
In prostate cancer, FGF-1 interacts (primarily) with FGF-receptor 1 (FGFR-1) to activate it by phosphorylation. Activated FGFR-1 also phosphorylates other intracellular proteins including phospholipase C and extracellular signal-regulated kinases (ERKs), members of the MAP kinase family. FGF-1 stimulation of this pathway results in phosphorylation (and activation) of STAT3 and expression of matrix metalloproteinases MMP 14 and MMP 7 [19
]. MMP 14 is also known as membrane-type 1 matrix metalloproteinase (MT1-MMP) and is a key enzyme in the initiation of extracellular matrix (ECM) protein breakdown. MMP7 (matrilysin) is overexpressed in prostate cancer and increases prostate cancer cell invasion. Thus, in the bone, prostate cancer cells with mtDNA mutations may increase metastatic potential and the ability to breakdown the extracellular matrix via the induction of the FGF-1 signaling pathway.
Focal adhesion kinase (FAK) also known as protein tyrosine kinase 2 (PTK2) is important in cell spreading, cell migration and motility, cell proliferation and suppression of apoptosis. FAK is a non-receptor tyrosine kinase that is ubiquitously expressed and becomes activated upon phosphorylation of tyrosine 397.
FAK has been examined by immunohistochemistry in clinical prostate cancer specimens. In the normal (benign) prostate glands FAK is absent from the luminal epithelium but present at a high level in the basal cells. It is also present in PIN, PIA and prostate cancer. Strong immunostaining was seen in 70% of primary prostate cancers, but the pattern was heterogeneous while metastatic foci had uniform and strong staining, suggesting a selective advantage for the development of metastatic disease [20
]. These findings corroborate an earlier study using RT-PCR that demonstrated increased FAK message levels in metastatic patients compared to patients with localized disease [21
]. FAK can also be upregulated by interaction with the extracellular matrix [22
One result of 8993 mtDNA mutations is an increase in ROS in subcutaneous tumors developing from the mutant cybrids [3
]. This mutation has been studied in cybrid cell lines and shown to cause the hyperpolarization of the mitochondrial inner membrane and increase mitochondrial ROS production [26
]. Similarly, mutations in respiratory complex I have been shown to increase metastatic potential of cancers cells in vivo, an effect that is mediated by ROS [27
]. We have also demonstrated that respiratory complex IV mutations increase ROS (data not shown). Thus, one common result of mtDNA mutations in cancer is to increase cellular ROS. There is an intimate relationship between ROS and FAK. Overexpression of FAK protects cells against oxidative-stress induced apoptosis and inhibits the activation of caspase-3 protease by hydrogen peroxide. In order for FAK to be in the active form, it must remain phosphorylated, a process that is enhanced in the presence of ROS and the oxidative inhibition of FAK tyrosine phosphatase is required for cell adhesion. Multiple studies document that ROS trigger FAK phosphorylation, including in pulmonary artery endothelium [23
] and human umbilical vein endothelial cells [24
]. ROS from mitochondria trigger FAK phosphorylation
in endothelial cells, a response that arises in response to mechanical stretch and involves protein kinase C (PKC) [25
While there is currently ample documentation of mtDNA mutations in prostate cancer there remains a great need to determine their functionality. Because of our previous work showing that mtDNA mutations confer only minimal growth advantage in vitro
but are responsible for a large, significant and reproducible growth advantage in vivo
], we sought to further explore the impact of mtDNA on stromal-epithelial interactions in prostate cancer.
Clinically, prostate cancer metastases show a consistent tropism for bone. The specific causes of this site-specific metastatic pattern remain an area of active investigation. Our data show that mtDNA mutations in the potentially bone-metastatic cell may preferentially enhance growth in the bone and bone stromal microenvironment, an effect likely mediated by upregulation of FGF-1 and FAK. We therefore propose that mtDNA mutations in prostate cancer are functional, enhancing bone metastasis. These findings are corroborated by the recent discovery of the importance of mtDNA mutations in metastatic capacity of murine cancer cell lines [27