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Abstract
Inflammatory breast cancer (IBC) is the most aggressive and lethal manifestation of locally advanced breast cancer with the ability to rapidly metastasize throughout the body. The phosphoinositide 3-kinase/Akt (Protein Kinase B) signaling pathway has been shown to be involved in cellular motility, invasion, and metastasis. We have found that Akt is highly active in SUM149 IBC cells and increased Akt activity results in high levels of serine phosphorylated RhoC GTPase. In order for Rho proteins such as RhoC GTPase to become active in the cell, they must be translocated from the cytosol and localized to the inner plasma membrane by an isoprene moiety for entrance into the GTPase cycle. This is done via prenylation, either with the addition of a farnesyl (F) or geranylgeranyl (GG) group. Farnesyltransferase inhibitors (FTIs) interfere with GTPase farnesylation and activity and we have shown that FTI treatment of SUM149 IBC cells leads to inhibition of the RhoC GTPase-induced invasive phenotype of IBC.
On the basis of preliminary observations and with consideration of the highly metastatic behavior of inflammatory breast cancer cells, we hypothesize in this study that FTI treatment increases levels of geranylgeranylated RhoB GTPase in inflammatory breast cancer cells, aberrantly localizing Akt1, and leads to repression of the RhoC GTPase-induced metastatic phenotype of inflammatory breast cancer.
Inflammatory breast cancer is a phenotypically distinct form of breast cancer that is characterized by primary skin changes, including redness and thickening of the skin, nipple retraction, and peau d'aurange. IBC is clinically distinguished by rapid onset of these skin changes, which typically occur within six months. IBC is relatively rare, accounting for approximately 1–3% of newly diagnosed breast cancers in the United States annually, and carries an extremely poor prognosis. The low survival rate of IBC is due to the highly invasive nature of the disease, and its proclivity to invade and block the dermal lymphatics of the skin overlying the breast and metastasize rapidly throughout the body makes it the most lethal form of breast cancer.
The exact roles each of the three Akt kinase isoforms (Akt1, Akt2, and Akt3) in the PI3K/Akt pathway play in mediating cellular processes of motility and invasion are fairly unclear. Although each Akt kinase is activated through similar mechanisms and responds similarly to stimuli, their different tissue-specific expression patterns suggest distinct roles. Akt1 has traditionally been implicated to be a key signaling protein involved in the inhibition of apoptotic processes to promote cellular survival and tumor progression through the survival pathway. Akt2 has more recently been implicated in having a pro-invasive role in non-inflammatory breast cancer. However, our data show that the opposite appears to be true in IBC, where Akt1 is the protein kinase involved in cellular motility and invasion. Using siRNA to specifically knock down Akt1 levels, we found that the in vitro invasive capabilities of IBC cells were significantly inhibited, while the invasiveness of the non-inflammatory breast cancer cells was not affected.
RhoC GTPase has been suggested to be an essential molecular marker for IBC. RhoC contains a putative Akt phosphorylation site at its serine 73 residue within the switch II region. We have demonstrated a role for RhoC in conferring a highly motile and invasive phenotype to IBC cells and our studies suggest that Akt phosphorylation of RhoC leads to cellular motility and invasion. Furthermore, we have shown that mutation of the Akt phosphorylation site of RhoC with a RhoCS73A phosphorylation mutant significantly decreases the invasive capabilities of IBC cells.
RhoC (geranylgeranylated) is not a direct target of FTI action, however, its relative, RhoB (geranylgeranylated or farnesylated), is a putative FTI target. It has been shown that FTI inhibition of farnesylated RhoB (RhoB-F) leads to an accumulation of geranylgeranylated RhoB (RhoB-GG) in the cell, which can interfere with GTPase signals and mimics the effects of FTI in SUM149 IBC cells. RhoB is an intracellular trafficking GTPase and accumulation of RhoB-GG leads to redistribution of other proteins within the cell. Of particular interest is that RhoB GTPase regulates cellular localization of Akt1, thus affecting its activation. We demonstrate that treatment of SUM149 IBC cells with FTI leads to an increase in RhoB GTPase expression. In addition, FTI treatment results in a significant decrease in Akt expression and localization of Akt to the nucleus with a resultant decrease in serine phosphorylation of RhoC GTPase.
Aggressive, deadly, and typically misdiagnosed, inflammatory breast cancer is a biological entity that is phenotypically distinct from other forms of breast cancer and must be treated as such. As with nearly all cancers, metastasis is the primary cause of death of IBC patients. The difference lies in the rapidity at which IBC progresses, appearing metastatic upon inception. The present study examines potential essential mechanisms for the invasive and metastatic phenotype of IBC. Understanding the underpinning mechanisms of the metastatic phenotype will provide potential drug targets. We are able to demonstrate that AKT1/PKB&agr; phosphorylation of RhoC GTPase drives IBC invasion. We also report that the PI3K/AKT1 signaling pathway involved in cytoskeletal reorganization is upregulated in IBC patients. Furthermore, we show that there is a possible use for farnesyltransferase inhibitors in IBC treatment, as we have begun to delineate the molecular mechanisms of the FTIs through RhoB GTPase and Akt1 in this study. Our data demonstrate an integral role for Akt1 and RhoC GTPase in IBC and suggest a key molecular mechanism for therapeutic target.