RUNX2 and the PI3K/AKT axis reciprocal activation as a driving force for tumor progression

The products of RUNX (Runt-related transcription factor) genes comprise a family of three closely related transcription factors, RUNX1, RUNX2
and RUNX3. These genes are defined by a highly conserved 128 amino acid DNA binding/protein-protein
interaction domain, called the Runt-homology domain 1],2]. RUNX2 is a major determinant of osteoblast differentiation and regulates chondrocyte
proliferation, differentiation and hypertrophy during endochondral bone formation
3]–6].

The involvement of RUNX2 in tumor development, progression and metastasis is largely
documented. Some evidence indicates a role for RUNX2 in T-cell lymphoma, acute myeloid
leukemia and multiple myeloma 1],7]–9] and bone metastasis in advanced mammary and prostate cancer 1],10]–12]. However, the role of RUNX2 in promoting tumor development in mammary and prostate
cancer extends beyond its pro-bone metastatic effects 10],11] and the osteolytic disease associated with these cancers 13],14]. RUNX2 regulates the expression of genes intimately associated with tumor progression,
invasion and metastasis and its role in migration and invasion has been documented
in different tumor cell types. RUNX2 siRNA treatment of the prostate cancer cell line
PC3 decreases migration and invasion through Matrigel in vitro13]. Overexpression of RUNX2 in the prostate cancer cell line C4-2B enhances its invasiveness
15]. In addition, RUNX2 overexpression upregulates transcription factors (SOX9, SNAI2 and SMAD3) implicated in the process of epithelial to mesenchymal transition (EMT), whose features
include increased motility and invasion potential. Furthermore, RUNX2 overexpression
upregulates genes involved in cellular movement and cytoskeleton remodeling 15]. High RUNX2 levels in PC3 cells are associated with development of large tumors,
and increased expression of the two metalloproteinases MMP9 and MMP13, OPN and VEGF, and secreted bone-resorbing factors (PTHrP, IL-8) promoting osteolytic disease 13]. Similarly, in human breast cancer, RUNX2 directly regulates the expression of MMP9 and MMP1316]–18], bone sialoprotein and OPN16], IL-819] and the TGF?-induced PTHrP levels 14] and mediates invasion of the human breast cancer cell lines MDA-MB-231 and MCF7 17]. Interestingly, aberrant RUNX2 expression induces EMT-like changes in normal mammary
epithelial cells 20] and disrupts normal acini structure in three-dimensional cultures 19], suggesting a role for RUNX2 in promoting the early events of breast cancer progression.
RUNX2 also plays a central role in mediating the pro-migratory and pro-invasive function
of thyroid tumor cells, by activating the expression of MMP2, MMP13, MMP14 and OPN21]. SiRNA-mediated knockdown of RUNX2 in human colon carcinoma cells leads to decreased
migration and invasion 22]. The U2OS osteosarcoma cells also demonstrate reduced motility following siRNA-mediated
depletion of RUNX2. In addition, genomic promoter occupancy of RUNX2 in osteosarcoma
cells identifies genes involved in motility, such as FAK/PTK2 or talin (TNL1)23]. We also demonstrated that RUNX2 knock down in melanoma cell lines significantly
inhibits their migration and invasion potential 24].

In addition, the pro-angiogenic effects of RUNX2 are highly suggestive of RUNX2 as
a major player in tumor promotion. These effects include endothelial cell proliferation,
migration and invasion 25],26], induction of VEGF expression or RUNX2 physical and functional interactions with another major pro-angiogenic
factor, hypoxia-inducible factor 1-a (HIF1-a) 27],28]. These studies altogether define the transcription factor RUNX2 as pro-migratory,
pro-invasive and pro-angiogenic, in addition to its role in promoting the early steps
of tumorigenesis in breast cancer and driving the metastatic bone disease in prostate
and breast cancer. In this review, we are focusing on the functional interrelations
between RUNX2 and the PI3K/AKT pathway, which contribute to cancer progression.

The oncogenic role of the PI3K/AKT axis on tumor growth extends beyond its pro-proliferative
and survival effects and includes migration and invasion. One important contribution
is the demonstration that PI3K function is required for TGF?-mediated epithelial to
mesenchymal transition (EMT) of the NMuMG mammary epithelial cell line. In addition,
PI3K inhibition blocks both basal and TGF?-induced cell migration of mouse breast
cancer cell lines 29]. The collaboration of TGF? autocrine signaling and the activated PI3K/AKT pathway
plays a key role in cancer progression, causing the shift in TGF?/SMAD signaling from
its tumor suppressive to its tumor promoting mode 30]. As another illustration of this concept, AKT-mediated phosphorylation of the EMT
transcription factor TWIST1 leads to transcriptional activation of the TGF?2 promoter and activated TGF? signaling promoting EMT and breast cancer metastasis
31].

Numerous studies describe the requirement of AKT signaling for the migration and invasion
of tumor cells. Overexpression of AKT or myristylated AKT (MyrAKT), which is anchored
to the plasma membrane and has a constitutively active kinase activity, increases
the migration and invasion of a human fibrosarcoma cell line. This study demonstrates
that AKT promotes migration and invasion in a manner depending on both its membrane-translocating
ability and its kinase activity. In addition, cell migration and invasion require
PI3K-dependent translocation of AKT at the cell membrane, as evidenced by the inhibition
of AKT translocation, cell migration and invasion by the PI3K inhibitor LY294002 32]. The expression of subunit p110? (PI3K) siRNA or AKT1 siRNA in a human ovarian cancer
cell line significantly decreases its migration and invasion 33]. CXCL12-mediated MMP-9 expression and chemoinvasion is sensitive to PI3K inhibitors in various prostate
cancer cell lines 34]. Also in prostate cancer cells, oncogenic ETS transcription factors require AKT signaling
to activate a cell migration gene expression program through ETS/AP-1 binding sequences
35]. Adenoviral transfer of PTEN into melanoma cells leads to inhibition of AKT phosphorylation
and suppression of melanoma cell invasion 36]. Similarly, PTEN loss increases invasion of human melanoma cells and non-transformed
melanocytes, with a concomitant shift to phosphorylation of AKT2 37]. The adaptor GAB2 involved in the activation of both RAS-ERK and PI3K/AKT signaling
pathways, is overexpressed in metastatic melanoma, promoting migration and invasion
of melanoma cells 38]. In addition, AKT is involved in TGF?-mediated migration of human osteosarcoma cells
39]. Therefore, the activation of AKT promotes the EMT, migration and invasion programs
in a PI3K-dependent manner.

The implication of RUNX2 in signaling pathways involved in tumorigenesis, such as
the transforming growth factor beta (TGF?) signaling pathway 40],41], the WNT pathway 40] and the p53 pathway 42] has been reviewed thoroughly. Mechanistic studies mainly performed in osteoblasts
and chondrocytes and in different cancer cellular systems shed light into a functional
interaction and cooperation between RUNX2 and the PI3K/AKT pathway. In the context
of cancer cells, this interaction might feed a positive feedback loop for the benefit
of tumor progression.