Depletion of mitochondrial protease OMA1 alters proliferative properties and promotes metastatic growth of breast cancer cells

Metastatic competence of cancer cells is influenced by many factors including metabolic alterations and changes in mitochondrial biogenesis and protein homeostasis. While it is generally accepted that mitochondria play important roles in tumorigenesis, the respective molecular events that regulate aberrant cancer cell proliferation remain to be clarified. Therefore, understanding the mechanisms underlying the role of mitochondria in cancer progression has potential implications in the development of new therapeutic strategies. We show that low expression of mitochondrial quality control protease OMA1 correlates with poor overall survival in breast cancer patients. Silencing OMA1 in vitro in patientderived metastatic breast cancer cells isolated from the metastatic pleural effusion and atypical ductal hyperplasia mammary tumor specimens (21MT-1 and 21PT) enhances the formation of filopodia, increases cell proliferation (Ki67 expression), and induces epithelial-mesenchymal transition (EMT). Mechanistically, loss of OMA1 results in alterations in the mitochondrial protein homeostasis, as reflected by enhanced expression of canonic mitochondrial unfolded protein response genes. These changes significantly increase migratory properties in metastatic breast cancer cells, indicating that OMA1 plays a critical role in suppressing metastatic competence of breast tumors. Interestingly, these results were not observed in OMA1-depleted non-tumorigenic MCF10A mammary epithelial cells. This newly identified reduced activity/levels of OMA1 provides insights into the mechanisms leading to breast cancer development, promoting malignant progression of cancer cells and unfavorable clinical outcomes, which may represent possible prognostic markers and therapeutic targets for breast cancer treatment

Depletion of mitochondrial protease OMA1 alters proliferative properties and promotes metastatic growth of breast cancer cells

Metastatic competence of cancer cells is influenced by many factors including metabolic alterations and changes in mitochondrial biogenesis and protein homeostasis. While it is generally accepted that mitochondria play important roles in tumorigenesis, the respective molecular events that regulate aberrant cancer cell proliferation remain to be clarified. Therefore, understanding the mechanisms underlying the role of mitochondria in cancer progression has potential implications in the development of new therapeutic strategies. We show that low expression of mitochondrial quality control protease OMA1 correlates with poor overall survival in breast cancer patients. Silencing OMA1 in vitro in patientderived metastatic breast cancer cells isolated from the metastatic pleural effusion and atypical ductal hyperplasia mammary tumor specimens (21MT-1 and 21PT) enhances the formation of filopodia, increases cell proliferation (Ki67 expression), and induces epithelial-mesenchymal transition (EMT). Mechanistically, loss of OMA1 results in alterations in the mitochondrial protein homeostasis, as reflected by enhanced expression of canonic mitochondrial unfolded protein response genes. These changes significantly increase migratory properties in metastatic breast cancer cells, indicating that OMA1 plays a critical role in suppressing metastatic competence of breast tumors. Interestingly, these results were not observed in OMA1-depleted non-tumorigenic MCF10A mammary epithelial cells. This newly identified reduced activity/levels of OMA1 provides insights into the mechanisms leading to breast cancer development, promoting malignant progression of cancer cells and unfavorable clinical outcomes, which may represent possible prognostic markers and therapeutic targets for breast cancer treatment

Protease OMA1 modulates mitochondrial bioenergetics and ultrastructure through dynamic association with MICOS complex

Remodeling of mitochondrial ultrastructure is a process that is critical for organelle physiology and apoptosis. Although the key players in this process—mitochondrial contact site and cristae junction organizing system (MICOS) and Optic Atrophy 1 (OPA1)—have been characterized, the mechanisms behind its regulation remain incompletely defined. Here, we found that in addition to its role in mitochondrial division, metallopeptidase OMA1 is required for the maintenance of intermembrane connectivity through dynamic association with MICOS. This association is independent of OPA1, mediated via the MICOS subunit MIC60, and is important for stability of MICOS and the intermembrane contacts. The OMA1-MICOS relay is required for optimal bioenergetic output and apoptosis. Loss of OMA1 affects these activities; remarkably it can be alleviated by MICOSemulating intermembrane bridge. Thus, OMA1-dependent ultrastructure support is required for mitochondrial architecture and bioenergetics under basal and stress conditions, suggesting a previously unrecognized role for OMA1 in mitochondrial physiology

Metalloproteases of the inner mitochondrial membrane

The inner mitochondrial membrane (IM) is among most protein-rich cellular compartments. The metastable IM sub-proteome where the concentration of proteins is approaching oversaturation creates a challenging protein folding environment with high probability for protein malfunction or aggregation. Failure to maintain protein homeostasis in such a setting can impair functional integrity of the mitochondria and drive clinical manifestations. The IM is equipped with a series of highly conserved, proteolytic complexes dedicated to the maintenance of normal protein homeostasis within this mitochondrial sub-compartment. Particularly important is a group of membrane-anchored metallopeptidases commonly known as m-AAA and i-AAA proteases, and the ATP-independent Oma1 protease. Herein, we will summarize current biochemical knowledge about these proteolytic machines and discuss recent advances toward understanding mechanistic aspects of their functioning

A conserved motif in the ITK PH-domain is required for phosphoinositide binding and TCR signaling but dispensable for adaptor protein interactions.

Binding of the membrane phospholipid phosphatidylinositol 3,4,5-trisphosphate (PIP(3)) to the Pleckstrin Homology (PH) domain of the Tec family protein tyrosine kinase, Inducible T cell Kinase (ITK), is critical for the recruitment of the kinase to the plasma membrane and its co-localization with the TCR-CD3 molecular complex. Three aromatic residues, termed the FYF motif, located in the inner walls of the phospholipid-binding pocket of the ITK PH domain, are conserved in the PH domains of all Tec kinases, but not in other PH-domain containing proteins, suggesting an important function of the FYF motif in the Tec kinase family. However, the biological significance of the FYF amino acid motif in the ITK-PH domain is unknown. To elucidate it, we have tested the effects of a FYF triple mutant (F26S, Y90F, F92S), henceforth termed FYF-ITK mutant, on ITK function. We found that FYF triple mutation inhibits the TCR-induced production of IL-4 by impairing ITK binding to PIP(3), reducing ITK membrane recruitment, inducing conformational changes at the T cell-APC contact site, and compromising phosphorylation of ITK and subsequent phosphorylation of PLCγ(1). Interestingly, however, the FYF motif is dispensable for the interaction of ITK with two of its signaling partners, SLP-76 and LAT. Thus, the FYF mutation uncouples PIP(3)-mediated ITK membrane recruitment from the interactions of the kinase with key components of the TCR signalosome and abrogates ITK function in T cells

FYF-ITK mutant is deficient in its ability to become phosphorylated upon TCR stimulation.

<p><i>(A)</i>, Jurkat cells transfected with cDNA constructs encoding CFP/YFP chimeric WT-, F26S- and FYF-ITK mutants were stimulated with anti-CD3ε (+) or isotype control (-) antibodies, lysed, and ITK (both transfected and endogenous) immuno-precipitated (IP) with anti-ITK antibodies. The immune complexes were resolved by SDS-PAGE, proteins transferred onto PVDF membranes, and immuno-blotted (IB) sequentially with anti-phosphotyrosine and anti-ITK antibodies as indicated. The upper sets of panels represent transfected and the lower sets endogenous ITK. Signals were developed by chemiluminescence. <i>(B)</i>, Bands from three replicate experiments (including the one displayed in panel A) performed as in (A) were quantified using ImageJ software and displayed as the percentage of transfected WT-ITK phosphorylation calculated as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045158#s4" target="_blank">materials and methods</a> section. The * denotes that the difference between FYF-mutant and WT or F26S is statistically significant at p<0.05 determined by the student’s t test.</p

TCR-induced association of FYF-ITK mutant with SLP-76 and LAT is intact.

<p><i>(A),</i> Jurkat cells transfected with the indicated CFP/YFP chimeric ITK mutant constructs were stimulated with anti-CD3ε or isotype control antibodies, lysed, and immunoprecipitated (IP) with anti-SLP-76 antibodies. Immune complexes were resolved by PAGE, proteins transferred onto PVDF membranes, and immunoblotted (IB) sequentially with anti-ITK and anti-SLP-76 antibodies (loading control) as indicated. <i>(B)</i>, lysates of cells similarly transfected, stimulated, and lysed were immunoprecipitated with anti-LAT antibodies and immune complexes resolved and sequentially immunoblotted with anti-ITK and anti-LAT antibodies. Bands were visualized by chemiluminescence as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045158#s4" target="_blank">Materials and Methods</a> section. Results are representative of three replicate experiments with the exception of F26S that represents a single experiment.</p

TCR-induced changes in E_app at the T cell-APC contact site.

<p><i>(A)</i>, Jurkat T cells transfected with WT-ITK were incubated with SEE-pretreated Raji cells and E_app at the center and periphery (average of right and left sides) of the contact site was assessed as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045158#s4" target="_blank">Materials and Methods</a> section. Red and black dots represent the E_app at the periphery and center of the same conjugate, respectively. The values displayed are those of 55 representative conjugates out of 114 total. Differences in E_app at the periphery and center of the contact of each individual conjugate are significant (p<0.0001, paired student’s t test). <i>(B),</i> Jurkat cells transfected with FYF-ITK and treated as in (A). The values of 55 conjugates are displayed. Differences in E_app at the periphery and center of the contact of each individual conjugate are not significant (p = 0.5973, paired student’s t test). <i>(C),</i> Jurkat cells transfected with pYC control (construct containing only the two fluorescent proteins separated by a short linker) and treated as in (A). The values of 21 conjugates are displayed. Differences in E_app at the periphery and center of the contact of each individual conjugate are not significant (p = 0.6366, paired student’s t test).</p