Previous reports suggest that AAMP can function in part by promoting migratory activity in vascular endothelial, smooth muscle, breast, and non-small cell lung cancer cells (Holvoet and Sinnaeve, 2008;Vogt et al., 2008;Yin et al., 2013;Hu et al., 2016;Yao et al., 2021). The overexpression of AAMP results in enhanced migratory activity in vascular smooth muscle cells, while antibody or siRNA transfection mediated inhibition of AAMP can reverse this effect (Holvoet and Sinnaeve, 2008). AAMP downregulation results in decreases in RhoA activity levels within vascular smooth muscle cell membrane components (Holvoet and Sinnaeve, 2008;Vogt et al., 2008), consistent with the ability of this protein to regulate angiogenesis and migration activity in vascular endothelial cells by controlling RhoA activation (Hu et al., 2016). We have previously reported that reductions in RhoA activity can suppress YAP activity via the RhoA/ ROCK2/LIMK2/CFL1 pathway, thereby compromising the ability of OS cells to tolerate photodynamic therapy (Zhan et al., 2021). AAMP promotes proliferative activity and chemoresistance within NSCLC cells through interactions with epidermal growth factor (Yao et al., 2019), and it has also been suggested to interact with CDC42 within NSCLC cells to drive their invasive and migratory activity (Yao et al., 2021). Herein, we found that OS patients exhibiting high levels of AAMP expression exhibited a worse prognosis than patients expressing lower levels of this protein, with such upregulation being more pronounced in patients with recurrent or lung metastatic OS relative to patients with primary OS. The knockdown of AAMP significantly suppressed OS cell migratory and invasive activity in addition to blunting EMT induction. Given the central role of the EMT in tumor metastatic progression (Lin and Wu, 2020), we posit that AAMP may function as a key regulator of OS cell metastasis.
The terminal Rho GTPase signaling cascade effector protein CFL1 is an important regulator of actin dynamics within cells, functioning by cutting F-actin and promoting filament depolymerization (Sousa-Squiavinato et al., 2019). The overexpression of CFL1 has been reported in many tumors wherein it regulates cytoskeletal recombination, EMT induction, and lamellipodium formation (Xu et al., 2021). The inactivation of CFL1 is mediated by its Ser3 phosphorylation by LIMK1/LIMK2 and by upstream Rho GTPase signaling intermediates such as Rac1, RhoA, and Cdc42 (Xu et al., 2021). The phosphorylation of CFL1 causes it to lose its affinity for actin, leading to the inhibition of depolymerization and cleavage, while its phosphatase-mediated dephosphorylation results in CFL1 activation. AAMP is an important upstream regulator of RhoA activation (Holvoet and Sinnaeve, 2008;Vogt et al., 2008;Hu et al., 2016). Herein, we found AAMP and CFL1 expression levels to be positively correlated in OS patients. Following AAMP knockdown, p-CFL1 and F-actin levels declined, and lamellipodia formation was reduced. These data align well with NSCLC-related findings published by Yao et al. (Yao et al., 2021). As such, we hypothesized that AAMP may control OS cell migration and invasion by regulating RhoA activity and influencing cytoskeletal dynamics via the RhoA /ROCK/LIMK/CFL1 axis.
The mitochondrial are key coordinators of cellular metabolic activity, generating ATP through the TCA cycle and oxidative phosphorylation in addition to regulating diverse cellular processes(Foo et al., 2021;Ji et al., 2021). Tumor cell invasion/migration and associated F-actin polymerization consume large quantities of ATP (Garde and Sherwood, 2021). CFL1 has previously been shown to maintain mitochondrial morphology and to regulate mitochondrial dynamics (Rehklau et al., 2017;Hoffmann et al., 2019). When activated, CFL1 is transported to the mitochondrial outer membrane where it is able to interact with dynamin-related protein 1 (Drp1), inducing mitochondrial fission, cytochrome C release, and consequent tumor cell apoptosis (Hoffmann et al., 2019;Hu et al., 2020). CFL1 can further regulate EMT induction within colorectal cancer (CRC) cells by promoting cytoskeletal rearrangement and cell-cell interactions (Sousa-Squiavinato et al., 2019). Herein, we found that the knockdown of AAMP was associated with mitochondrial swelling, a loss of mitochondrial cristae, impaired mitochondrial fusion, and reduced ATP production, thereby inhibiting OS cell migration and invasion. CFL1 has recently been suggested to function as a regulator of chemoresistance and metastasis in prostate cancer (Chen et al., 2020). In metastatic HCC, Zhang et al. have suggested that excessive mitochondrial fission is predominant (Zhang et al., 2020), which may have important research implications. Moreover, Chuang et al. (Chuang et al., 2020) have reported that ROS generation can disrupt normal mitochondrial dynamics, resulting in increased fission, autophagic activity, and mitochondrial dysfunction within skin cancer cells. These results suggest that AAMP regulates CFL1 phosphorylation, resulting in changes in mitochondrial morphology, function, and ATP production that ultimately shape the migration and invasion of OS cells (Fig. 6e).
YAP is a transcriptional coactivator that functions as a downstream component of the Hippo signaling pathway (Zanconato et al., 2019), and it is an important mediator of organ development and tissue regeneration(Moya and Halder, 2019). Prolonged, excessive YAP activation is a common hallmark of oncogenesis (Moya and Halder, 2019). Mechanistically, Shi et al. (Shi et al., 2018) found that YAP was able to regulate the CFL/F-actin/lamellipodium axis to drive metastatic activity in hepatocellular carcinoma (HCC) cells. Qiao et al. (Qiao et al., 2017) further reported YAP to regulate ARHGAP29 within gastric cancer cells and to thereby control F-actin dynamics to regulate metastatic progression. AAMP has previously been shown to regulate RhoA activity within vascular endothelial and smooth muscle cells (Holvoet and Sinnaeve, 2008;Vogt et al., 2008;Hu et al., 2016). Our prior work suggests that reductions in RhoA activity within OS cells can inhibit the activity of YAP via a RhoA/ROCK2/LIMK2/CFL1 signaling axis (Zhan et al., 2021). We thus hypothesized that AAMP may be able to regulate YAP to control the migratory and invasive activity of OS cells. To test this hypothesis, we conducted bioinformatics analyses which revealed YAP and AAMP expression levels to be strongly positively correlated in OS. Patients with high levels of YAP expression exhibited significantly lower rates of metastasis-free survival relative to patients with lower levels of YAP expression. Knocking down YAP was sufficient to reduce the migratory and invasive activity of OS cells in vitro, whereas its overexpression had the opposite effect. YAP protein levels and those of downstream target genes also fell significantly following the knockdown of AAMP, together with reductions in EMT marker protein expression. These data are consistent with recent evidence published by Morice et al. (Morice et al., 2021), who demonstrated that YAP can bind to Smad3 in response to TGF-B signaling, thereby promoting OS pulmonary metastasis. Overall, these data suggest that AAMP can promote OS invasion and migration at least in part by enhancing YAP activation.
To further confirm the functional importance of AAMP as a mediator of OS pulmonary metastasis, we established a murine model of lung metastasis. In this experimental context, AAMP knockdown significantly reduced the number of metastatic lung nodules observed in mice without any concomitant reduction in murine body weight, potentially as a consequence of the selected experimental time point. These results thus suggest that AAMP is an important mediator of OS cell metastasis, and that the therapeutic targeting of AAMP may be a viable approach to suppressing the metastatic progression of this form of cancer.
There are certain limitations to the present study. For one, no large-scale analyses of clinical samples were performed, although corresponding analyses of a limited subset of primary tumors and lung metastases were conducted along with supporting bioinformatics research. Secondly, no rescue experiments associated with YAP or AAMP were conducted, although the overexpression and knockdown of these genes were evaluated in two cell lines, and the impact of AAMP on OS cell migratory and invasive activity was assessed in the context of AAMP knockdown-associated mitochondrial dysfunction, with animal studies further being used to validate these results.