Metastasis Suppressor Protein Research Articles

Abstract B023: Ectopic Expression of the Obscurin PH-domain in Aggressive Breast Cancer Cells Modulates PI3K/Akt Activity and Inhibits Cellular Migration and Invasion

Abstract New therapies targeting metastatic spread are greatly needed to improve breast cancer patient survival. Recently, obscurin, an ~800kDa protein localized to the cell membrane, has risen to the spotlight as an overlooked metastasis suppressor commonly lost in breast cancer. Specifically, breast epithelial cells lacking obscurin undergo epithelial-mesenchymal transition (EMT) and demonstrate increased migration and invasion. Biochemical studies in our lab have linked breast epithelial obscurin loss to increased activation of the pro-metastatic PI3K/Akt signaling pathway. Mechanistically, the obscurin pleckstrin homology (PH)-domain directly binds the SH3-domain of the p85 regulatory subunit of PI3K. Thus far, due to obscurin’s massive size, gain of function/rescue experiments of obscurin have not been feasible. To combat this challenge, we have generated a mini-obscurin protein construct composed of an obscurin core functional unit: a myristilated obscurin PH-domain (Myr-PH). We hypothesized that expression of Myr-PH in obscurin-low breast cancer cells will reduce PI3K/Akt pathway activity and suppress metastasis. Remarkably, we report that ectopic expression of Myr-PH in the obscurin-low MDA-MB-231 (triple negative) and SKBr3 (HER2 positive) breast cancer cell lines diminishes PI3K/Akt pathway activity. Furthermore, Myr-PH sequesters p85 to the cell membrane, anchoring actin at the cell periphery. Functionally, we demonstrate that expression of Myr-PH in both cell line receptor subtypes: 1) reduces cellular migration collectively through decreased scratch assay wound closure and decreased transwell assay 8um pore migration or at the single cell level, through reduced 3um diameter microchannel migration, 2) diminishes cell seeding on multiple extracellular matrix (ECM) substrates known to populate metastatic microenvironments, and 3) decreases cell dissemination and tumorsphere invasion into a 3D collagen-1 matrix. Underlying these functional changes, we show that Myr-PH inhibits actin-dependent filopodia and invadopodia formation and decreases the expression of downstream PI3K/Akt target matrix metalloproteinases, all of which are potent drivers of metastatic dissemination. To this end, we are currently delivering the obscurin-PH domain in animals via lipid nanoparticles, aiming to examine its anti-metastatic efficacy in vivo. If successful, this work would pioneer the first gene therapy-based, targeted use of a single metastasis suppressor protein domain to block PI3K signaling and inhibit breast cancer metastasis. Citation Format: Matthew K Eason, Se-Jong Lee, Poornima Dubey, Anthony Kim, Konstantinos Konstantopoulos, Aikaterini Kontrogianni-Konstantopoulos. Ectopic Expression of the Obscurin PH-domain in Aggressive Breast Cancer Cells Modulates PI3K/Akt Activity and Inhibits Cellular Migration and Invasion [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr B023.

A potential new biomarker in HNSCC: metastasis suppressor protein 1 (MTSS1)

ObjectiveMetastasis suppressor protein 1 (MTSS1) is a prognostic tumour marker in different malignant epithelial tumour entities and previously mainly the MTSS1 expression was analyzed. This study evaluated the best analysis method as a prognosis and aggressiveness tumour marker in head and neck squamous cell carcinoma (HNSCC). Study DesignMTSS1 expression, MTSS1 intensity, interpretation MTSS1 score and MTSS1 edging score were analysed in formalin-fixed paraffin-embedded tissue slices of 60 patients with proven HNSCC and correlated with clinical and pathological outcome parameters. ResultsA lack of MTSS1 expression showed tumour aggressiveness, but surprisingly, mainly MTSS1 intensity was correlated with a worse patient outcome. There was a significant correlation between higher MTSS1 intensity and an increased risk for lymph node metastasis (p = 0.027) and a significant increased risk for extracapsular growth (p = 0.016). Furthermore, disease-specific survival was worse in cases with higher MTSS1 intensity (p = 0.001). ConclusionMTSS1 intensity has a high scientific potential for further studies and could potentially be used as a prognostic marker in diagnostic and therapeutic decision making.

Characterization of Vemurafenib-Resistant Melanoma Cell Lines Reveals Novel Hallmarks of Targeted Therapy Resistance.

Regardless of the significant improvements in treatment of melanoma, the majority of patients develop resistance whose mechanisms are still not completely understood. Hence, we generated and characterized two melanoma-derived cell lines, primary WM793B and metastatic A375M, with acquired resistance to the RAF inhibitor vemurafenib. The morphology of the resistant primary WM793B melanoma cells showed EMT-like features and exhibited a hybrid phenotype with both epithelial and mesenchymal characteristics. Surprisingly, the vemurafenib-resistant melanoma cells showed a decreased migration ability but also displayed a tendency to collective migration. Signaling pathway analysis revealed the reactivation of MAPK and the activation of the PI3K/AKT pathway depending on the vemurafenib-resistant cell line. The acquired resistance to vemurafenib caused resistance to chemotherapy in primary WM793B melanoma cells. Furthermore, the cell-cycle analysis and altered levels of cell-cycle regulators revealed that resistant cells likely transiently enter into cell cycle arrest at the G0/G1 phase and gain slow-cycling cell features. A decreased level of NME1 and NME2 metastasis suppressor proteins were found in WM793B-resistant primary melanoma, which is possibly the result of vemurafenib-acquired resistance and is one of the causes of increased PI3K/AKT signaling. Further studies are needed to reveal the vemurafenib-dependent negative regulators of NME proteins, their role in PI3K/AKT signaling, and their influence on vemurafenib-resistant melanoma cell characteristics.

Progesterone receptor isoform ratio dictates antiprogestin/progestin effects on breast cancer growth and metastases: A role for NDRG1.

Progesterone receptors (PRs) ligands are being tested in luminal breast cancer. There are mainly two PR isoforms, PRA and PRB, and their ratio (PRA/PRB) may be predictive of antiprogestin response. Our aim was to investigate: the impact of the PR isoform ratio on metastatic behaviour, the PR isoform ratio in paired primary tumours and lymph node metastases (LNM) and, the effect of antiprogestin/progestins on metastatic growth. Using murine and human metastatic models, we demonstrated that tumours with PRB > PRA (PRB-H) have a higher proliferation index but less metastatic ability than those with PRA > PRB (PRA-H). Antiprogestins and progestins inhibited metastatic burden in PRA-H and PRB-H models, respectively. In breast cancer samples, LNM retained the same PRA/PRB ratio as their matched primary tumours. Moreover, PRA-H LNM expressed higher total PR levels than the primary tumours. The expression of NDRG1, a metastasis suppressor protein, was higher in PRB-H compared to PRA-H tumours and was inversely regulated by antiprogestins/progestins. The binding of the corepressor SMRT at the progesterone responsive elements of the NDRG1 regulatory sequences, together with PRA, impeded its expression in PRA-H cells. Antiprogestins modulate the interplay between SMRT and AIB1 recruitment in PRA-H or PRB-H contexts regulating NDRG1 expression and thus, metastasis. In conclusion, we provide a mechanistic interpretation to explain the differential role of PR isoforms in metastatic growth and highlight the therapeutic benefit of using antiprogestins in PRA-H tumours. The therapeutic effect of progestins in PRB-H tumours is suggested.

Regulation of metastasis suppressor NME1 by a key metabolic cofactor coenzyme A

The metastasis suppressor protein NME1 is an evolutionarily conserved and multifunctional enzyme that plays an important role in suppressing the invasion and metastasis of tumour cells. The nucleoside diphosphate kinase (NDPK) activity of NME1 is well recognized in balancing the intracellular pools of nucleotide diphosphates and triphosphates to regulate cytoskeletal rearrangement and cell motility, endocytosis, intracellular trafficking, and metastasis. In addition, NME1 was found to function as a protein-histidine kinase, 3′-5′ exonuclease and geranyl/farnesyl pyrophosphate kinase. These diverse cellular functions are regulated at the level of expression, post-translational modifications, and regulatory interactions. The NDPK activity of NME1 has been shown to be inhibited in vitro and in vivo under oxidative stress, and the inhibitory effect mediated via redox-sensitive cysteine residues. In this study, affinity purification followed by mass spectrometric analysis revealed NME1 to be a major coenzyme A (CoA) binding protein in cultured cells and rat tissues. NME1 is also found covalently modified by CoA (CoAlation) at Cys109 in the CoAlome analysis of HEK293/Pank1β cells treated with the disulfide-stress inducer, diamide. Further analysis showed that recombinant NME1 is efficiently CoAlated in vitro and in cellular response to oxidising agents and metabolic stress. In vitro CoAlation of recombinant wild type NME1, but not the C109A mutant, results in the inhibition of its NDPK activity. Moreover, CoA also functions as a competitive inhibitor of the NME1 NDPK activity by binding non-covalently to the nucleotide binding site. Taken together, our data reveal metastasis suppressor protein NME1 as a novel binding partner of the key metabolic regulator CoA, which inhibits its nucleoside diphosphate kinase activity via non-covalent and covalent interactions.

The metastasis suppressor protein NM23-H1 modulates the PI3K-AKT axis through interaction with the p110\u03b1 catalytic subunit

The PI3K pathway is one of the most deregulated pathways in cancer, which is predominantly due to gain of function mutations or altered expression of the PI3KCA gene. This is codified by what is seen for the class I PI3K catalytic subunit p110α, a common feature of many cancers. The metastasis suppressor protein NM23-H1 (NME1), whose ability to suppress the metastasis activities of different tumors has been widely described and was previously reported to alter phosphatidylinositol signaling. Here, we show interaction of NM23-H1 with the p110α subunit and the functional consequence of this interaction. This interaction is predominantly localized at the plasma membrane with some signals seen in the cytoplasmic compartment. Analysis of NM23-H1 levels showed a negative correlation between NM23-H1 expression and Akt phosphorylation, the key marker of PI3K pathway activation. Investigating the functional consequence of this interaction using cell motility and clonogenicity assays showed that expression of NM23-H1 reversed the enhanced migration, invasion, adhesion, and filopodia structure formation in cells expressing the p110α catalytic subunit. A similar trend was seen in anchorage-independent assays. Notably, differential analyses using NM23-H1 mutants which lacked the enzymatic and metastasis suppressor activity, showed no detectable interaction between p110α and the NM23-H1 mutant proteins P96S, H118F, and S120G, as well as no dysregulation of the PI3K-AKT axis.

CTCF and EGR1 suppress breast cancer cell migration through transcriptional control of Nm23-H1

Tumor metastasis remains an obstacle in cancer treatment and is responsible for most cancer-related deaths. Nm23-H1 is one of the first metastasis suppressor proteins discovered with the ability to inhibit metastasis of many cancers including breast, colon, and liver cancer. Although loss of Nm23-H1 is observed in aggressive cancers and correlated with metastatic potential, little is known regarding the mechanisms that regulate its cellular level. Here, we examined the mechanisms that control Nm23-H1 expression in breast cancer cells. Initial studies in aggressive MDA-MB-231 cells (expressing low Nm23-H1) and less invasive MCF-7 cells (expressing high Nm23-H1) revealed that mRNA levels correlated with protein expression, suggesting that transcriptional mechanisms may control Nm23-H1 expression. Truncational analysis of the Nm23-H1 promoter revealed a proximal and minimal promoter that harbor putative binding sites for transcription factors including CTCF and EGR1. CTCF and EGR1 induced Nm23-H1 expression and reduced cell migration of MDA-MB-231 cells. Moreover, CTCF and EGR1 were recruited to the Nm23-H1 promoter in MCF-7 cells and their expression correlated with Nm23-H1 levels. This study indicates that loss of Nm23-H1 in aggressive breast cancer is apparently caused by downregulation of CTCF and EGR1, which potentially drive Nm23-H1 expression to promote a less invasive phenotype.

Bacillus anthracis' PA63 Delivers the Tumor Metastasis Suppressor Protein NDPK-A/NME1 into Breast Cancer Cells.

Some highly metastatic types of breast cancer show decreased intracellular levels of the tumor suppressor protein NME1, also known as nm23-H1 or nucleoside diphosphate kinase A (NDPK-A), which decreases cancer cell motility and metastasis. Since its activity is directly correlated with the overall outcome in patients, increasing the cytosolic levels of NDPK-A/NME1 in such cancer cells should represent an attractive starting point for novel therapeutic approaches to reduce tumor cell motility and decrease metastasis. Here, we established the Bacillus anthracis protein toxins’ transport component PA63 as transporter for the delivery of His-tagged human NDPK-A into the cytosol of cultured cells including human MDA-MB-231 breast cancer cells. The specifically delivered His6-tagged NDPK-A was detected in MDA-MB-231 cells via Western blotting and immunofluorescence microscopy. The PA63-mediated delivery of His6-NDPK-A resulted in reduced migration of MDA-MB-231 cells, as determined by a wound-healing assay. In conclusion, PA63 serves for the transport of the tumor metastasis suppressor NDPK-A/NME1 into the cytosol of human breast cancer cells In Vitro, which reduced the migratory activity of these cells. This approach might lead to development of novel therapeutic options.

MTSS1 suppresses mammary tumor-initiating cells by enhancing RBCK1-mediated p65 ubiquitination.

Tumor-initiating cells (TICs) are considered the culprits of cancer development and progression. Dysregulation of metastasis suppressor protein 1 (MTSS1) has been widely observed in tumor metastasis, but its functional contribution and mechanism in cancer is poorly understood. Here we report a role of MTSS1 in suppressing TICs in breast cancer. Mtss1 knockout (KO) enhances the mammary epithelial TIC subpopulation in both luminal and basal-like breast cancer mouse models. MTSS1 also suppresses tumorsphere formation in breast cancer cells. Mechanistically, MTSS1 interacts with the E3 ligase RanBP2-type and C3HC4-type zinc finger containing 1 (RBCK1) to facilitate RBCK1-mediated p65 ubiquitination and degradation, thus suppressing the NF-κB signaling pathway and tumorigenesis. In addition, actin beta-like 2 (ACTBL2) competes with RBCK1 for MTSS1 binding, leading to p65 stabilization. Importantly, MTSS1 silencing promotes patient-derived organoid formation and xenograft growth. MTSS1 downregulation in clinical tumors is also linked to worse prognosis. Overall our data reveal a new paradigm of NF-κB regulation and may have important implications in therapeutics targeting TICs.

MiRNA-182 regulated MTSS1 inhibits proliferation and invasion in Glioma Cells.

Human glioma is the most common malignant and fatal primary tumor in the central nervous system. Currently, the high incidence and low cure rate of glioma make it a considerable threat to human health. Thus, elucidating the molecular mechanisms of glioma development and progression has become a major focus to identify new and effective biomarkers and improve the comprehensive neurosurgical treatment of glioma from the basic research and clinical perspectives. In our present study, we aimed to investigate the expression pattern and biological function of Metastasis suppressor protein 1(MTSS1) in glioma and to further explore whether miRNAs were involved in the deregulation of MTSS1. By overexpressing MTSS1 in highly malignant human glioma cells, we discovered a role for MTSS1 in suppressing the proliferation and invasion of glioma cells, and we showed that MTSS1 participated in transforming growth factor-beta 1 (TGF‐β1) -induced epithelial‐mesenchymal transition (EMT) in glioma cells. Biochemical analyses suggested that miR-182 may target MTSS1 and that miR-182 expression is negatively correlated with MTSS1 expression in glioma tissues. This finding was further confirmed by luciferase reporter experiments. Furthermore, a miR-182 inhibitor induced glioma cell proliferation and invasion by increasing MTSS1 expression. In conclusion, we believed that miR-182 modulates glioma cell migration and invasion by targeting the MTSS1 and suggested that miR-182 was a potential therapeutic target for gliomas.

KISS1 in breast cancer progression and autophagy.

Tumor suppressors are cellular proteins typically expressed in normal (non-cancer) cells that not only regulate such cellular functions as proliferation, migration and adhesion, but can also be secreted into extracellular space and serve as biomarkers for pathological conditions or tumor progression. KISS1, a precursor for several shorter peptides, known as metastin (Kisspeptin-54), Kisspeptin-14, Kisspeptin-13 and Kisspeptin-10, is one of those metastasis suppressor proteins, whose expression is commonly downregulated in the metastatic tumors of various origins. The commonly accepted role of KISS1 in metastatic tumor progression mechanism is the ability of this protein to suppress colonization of disseminated cancer cells in distant organs critical for the formation of the secondary tumor foci. Besides, recent evidence suggests involvement of KISS1 in the mechanisms of tumor angiogenesis, autophagy and apoptosis regulation, suggesting a possible role in both restricting and promoting cancer cell invasion. Here, we discuss the role of KISS1 in regulating metastases, the link between KISS1 expression and the autophagy-related biology of cancer cells and the perspectives of using KISS1 as a potential diagnostic marker for cancer progression as well as a new anti-cancer therapeutics.

Abstract 1127: A rare subpopulation of melanoma cells with low expression of the metastasis suppressor gene NME1 is highly invasive

Abstract Malignant melanoma is an extremely aggressive form of cancer, resulting in the majority of all skin-cancer related deaths. Despite developments in melanoma treatment, tumor recurrence and metastasis, and resistance to therapy remain serious challenges to successful management of the disease. Melanoma tumors contain subpopulations of cells that display genomic and phenotypic variation. NME1 is an established metastasis suppressor protein, which inhibits the metastatic spread of melanoma cells. In the current study, we sought to determine whether melanoma cell lines contain a subpopulation of cells that express reduced expression of NME1 and, thus, potentially exhibit aggressive tumor and metastatic activities. Through immunostaining and flow cytometry, we discovered a rare subpopulation of cells with markedly reduced NME1 expression (NME1Low) in two melanoma cell lines (WM9 and WM278). To enable the isolation and further characterization of NME1-based subpopulations, we employed CRISPR-Cas9 technology to introduce the EGFP coding sequence at the C-terminus of the endogenous NME1 gene in both WM9 and WM278 cell lines. The resulting NME1-EGFP fusion protein is thus expressed under the regulatory control of the endogenous NME1 promoter. Fluorescence-activated Cell Sorting (FACS) was used to isolate cells that expressed the NME1-EGFP fusion protein at different levels of expression, from which multiple clones were isolated and expanded for analysis of their tumor growth and metastatic properties. Clones were sequenced and genotyped to ensure accurate EGFP incorporation at the NME1 locus. Clones derived from cells expressing low levels of NME1-EGFP (NME1Low) retained their low expression phenotype over 10 passages in monolayer culture conditions. NME1Low clones derived from both melanoma cell lines were much more invasive than clones expressing normal levels of NME1-EGFP (NME1Normal) when embedded in Matrigel. In addition, RNA-seq analysis demonstrated that NME1Low and NME1Normal clones differed greatly in their RNA expression profiles (>200 differentially expressed genes), with NME1Low clones expressing elevated levels of genes associated with morphogenesis (GO: 0009653). In particular, genes were identified relating to development of neural, bone and heart tissues, suggesting relevance to neural crest cell differentiation. Studies are ongoing to assess the growth and metastatic properties of these distinct cell subpopulations in vivo using a xenograft approach in immunocompromised mice. Overall, these studies strongly suggest that a subpopulation of cells that expresses low levels of NME1 represents a primitive and malignant component of melanoma lesions. These cells have potential for providing new therapeutic targets and molecular markers in advanced melanoma. Citation Format: Devin E. Snyder, Ying Wang, David M. Kaetzel. A rare subpopulation of melanoma cells with low expression of the metastasis suppressor gene NME1 is highly invasive [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1127.

Hepatitis C virus core protein interacts with cellular metastasis suppressor Nm23-H1 and promotes cell migration and invasion.

Hepatitis C virus (HCV) is the major etiological agent of hepatocellular carcinoma (HCC), which is the fourth most common cause of cancer-related deaths worldwide and second in terms of deaths of males (Bray et al. in CA Cancer J Clin 68(6):394-424, 2018). HCV-induced HCC is a multi-step process that involves alteration of several host regulatory pathways. One of the key features of HCV-associated hepatocellular carcinoma is the metastasis of cancer cells to different organs. Human Nm23-H1 is one of the best-studied metastasis suppressor proteins, and it has been shown to be modulated in many human cancers. Our study shows that the core protein of HCV genotype 2a can co-localize and interact directly with Nm23-H1 within cancer cells, resulting in modulation of the anti-metastasis properties of Nm23-H1. The HCV core protein promotes SUMOylation and degradation of the Nm23-H1 protein, as well as transcriptional downregulation. This study provides evidence that the HCV core protein is a pro-metastatic protein that can interact directly with and modulate the functions of cellular metastasis suppressor Nm23-H1.

Two mechanisms involving the autophagic and proteasomal pathways process the metastasis suppressor protein, N-myc downstream regulated gene 1

N-myc downstream regulated gene 1 (NDRG1) is an intriguing metastasis suppressor protein, which plays an important role in suppressing multiple oncogenic signaling pathways. Interestingly, multiple isoforms of NDRG1 have been identified, although the molecular mechanisms involved in their generation remains elusive. Herein, we demonstrate the role of two mechanisms involving autophagic and proteasomal machinery as part of an intricate system to generate different NDRG1 isoforms. Examining multiple pancreatic cancer cell-types using immunoblotting demonstrated three major isoforms of NDRG1 at approximately 41-, 46- and 47-kDa. The top NDRG1 band at 47-kDa was shown to be processed by the proteasome, followed by autophagic metabolism of the middle NDRG1 band at 46-kDa. The role of the proteasomal and autophagic pathways in NDRG1 processing was further confirmed by co-localization analysis of confocal images using PSMD9 and LC3 as classical markers of these respective pathways. All NDRG1 isoforms were demonstrated to be, at least in part, phosphorylated forms of the protein. Inhibition of two well-characterized upstream kinases of NDRG1, namely GSK3β and SGK1, resulted in decreased levels of the top NDRG1 band. Studies demonstrated that inhibition of GSK3β decreased levels of the top 47-kDa NDRG1 band, independent of its kinase activity, and this effect was not mediated via the proteasomal pathway. In contrast, the decrease in the top NDRG1 band at 47-kDa after SGK1 inhibition, was due to suppression of its kinase activity. Overall, these studies elucidated the complex and intricate regulatory pathways involving both proteasomal and autophagic processing of the metastasis suppressor protein, NDRG1.

Metastasis suppressor protein 1 regulated by PTEN suppresses invasion, migration, and EMT of gastric carcinoma by inactivating PI3K/AKT signaling.

Epithelial-mesenchymal transition (EMT) is a crucial event for cancer progression and metastasis. Metastasis suppressor protein 1 (MTSS1) is a metastasis suppressor in several cancers. In this study, we elucidated the potential physiological function of MTSS1 in the invasion and migration of gastric cancer (GC), and its distinct role in EMT and subsequently determined the potential molecular mechanism. We observed that MTSS1 expression was downregulated in GC tissues and several GC cell lines (SGC-7901, MGC-803, MKN-28, MKN-45, and BGC-823). Importantly, forced expression of MTSS1 drastically diminished the cell viability in both SGC-7901 and MKN-45 cells. Moreover, overexpression of MTSS1 attenuated the invasion ability of these two cell lines. In addition to the invasive capability, introduction of MTSS1 led to a loss of migratory potential. Furthermore, augmentation of MTSS1 exhibited the typical EMT phenotype switch, accompanied by enhanced the expression of vimentin and N-cadherin and reduced E-cadherin expression. Interestingly, MTSS1 also repressed transforming growth factor beta 1 (TGF-β1)-induced EMT. Our mechanistic investigations revealed that MTSS1 was positively regulated by the phosphatase and tensin homolog (PTEN), and it functioned as a tumor suppressor, possibly by inactivating the phosphoinositide 3-kinase (PI3K)/v-akt murine thymoma viral oncogene (AKT) pathway in GC cells. Collectively, our data provide insight into an important role for MTSS1 in suppressing tumor cell invasion, migration and EMT, which indicates that MTSS1 may act as a prospective prognostic biological marker and a promising therapeutic target for GC.

Abstract 2696: Deciphering the interactome of the metastasis-suppressor protein RAI2

Abstract We have identified low RAI2 expression as a novel metastasis-associated genetic alteration especially related to the presence of disseminated tumor cells in the bone marrow of breast cancer patients and poor outcome (Werner et al, Cancer Discovery, 2015). In hormone-dependent breast and prostate cancer cell lines RAI2 depletion induces down-regulation of hormone receptor expression. Furthermore, RAI2 depletion is associated with chromosomal instability. Nevertheless, to date the exact molecular mechanisms behind these observations remain largely elusive. To better define the RAI2 interactome we performed SILAC-immunoprecipitation quantitative proteomics. HEK293T cells were stably isotope labeled with amino acids in cell culture and transfected with a plasmid for expression of a HA-tagged RAI2 protein. Whole protein extracts were used for co-immunoprecipitation and possible interacting proteins of RAI2 were determined by mass spectrometry based on their SILAC ratios in enriched cell lysates from RAI2 over-expressing and control cells. Subsequently, the DAVID functional annotation tool was used for annotation of co-precipitated proteins. For validation of newly identified potential interactions, we applied combined co-immunoprecipitation, immunoblot and microscopic imaging analysis. The quantitative proteomics approach revealed high confidence interactions of the RAI2 protein with nine gene products (CTBP1, CLTC, HSPA8, NONO, HSPA9, HSPA1A, TNRC6B, UPF1, AP2B1). Additionally, eleven low confidence interactions (HSPA5, PARP1, SFPQ, NUDT21, KRT8, DDX24, FXR1, HSPB1, COL17A1, SLC25A11, FARSA) were identified. The highest SILAC ratio was obtained for CTBP1. Functional annotation showed significant enrichment for the GO terms mRNA processing (GO:0006397, p=0.018), paraspeckles (GO:0042382, p<0.001) and polyA RNA binding (GO:0044822, p<0.001). We validated molecular interactions of RAI2 with CTBP1 and the paraspeckles proteins NONO and SFPQ by combined co-immunoprecipitation and immunoblot analysis. Immunofluorescence staining showed that the RAI2 protein is predominantly co-localized with CTBP1 in nuclear speckles, which is dependent on an intact non-consensus bipartite CTBP-binding domain of the RAI2 protein. In conclusion, the RAI2 protein interacts with different scaffolding proteins and various RNA binding proteins that are involved in mRNA processing, i.e. post-transcriptional regulation. Based on these findings we conclude that the tumor suppressive function of the RAI2 protein is mainly associated with post-transcriptional regulation probably by orchestrating protein scaffolds and subnuclear compartmentalization. Citation Format: Stefan Werner, Kai Bartkowiak, Pascal Steffen, Katharina Besler, Lena Böttcher, Hartmut Schlüter, Klaus Pantel, Harriet Wikman. Deciphering the interactome of the metastasis-suppressor protein RAI2 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2696.

Deficiency of tumor suppressor Merlin facilitates metabolic adaptation by co-operative engagement of SMAD-Hippo signaling in breast cancer.

The NF2 gene encodes the tumor and metastasis suppressor protein Merlin. Merlin exerts its tumor suppressive role by inhibiting proliferation and inducing contact-growth inhibition and apoptosis. In the current investigation, we determined that loss of Merlin in breast cancer tissues is concordant with the loss of the inhibitory SMAD, SMAD7, of the TGF-β pathway. This was reflected as dysregulated activation of TGF-β signaling that co-operatively engaged with effectors of the Hippo pathway (YAP/TAZ/TEAD). As a consequence, the loss of Merlin in breast cancer resulted in a significant metabolic and bioenergetic adaptation of cells characterized by increased aerobic glycolysis and decreased oxygen consumption. Mechanistically, we determined that the co-operative activity of the Hippo and TGF-β transcription effectors caused upregulation of the long non-coding RNA Urothelial Cancer-Associated 1 (UCA1) that disengaged Merlin's check on STAT3 activity. The consequent upregulation of Hexokinase 2 (HK2) enabled a metabolic shift towards aerobic glycolysis. In fact, Merlin deficiency engendered cellular dependence on this metabolic adaptation, endorsing a critical role for Merlin in regulating cellular metabolism. This is the first report of Merlin functioning as a molecular restraint on cellular metabolism. Thus, breast cancer patients whose tumors demonstrate concordant loss of Merlin and SMAD7 may benefit from an approach of incorporating STAT3 inhibitors.

MTSS1 and SCAMP1 cooperate to prevent invasion in breast cancer

Cell–cell adhesions constitute the structural “glue” that retains cells together and contributes to tissue organisation and physiological function. The integrity of these structures is regulated by extracellular and intracellular signals and pathways that act on the functional units of cell adhesion such as the cell adhesion molecules/adhesion receptors, the extracellular matrix (ECM) proteins and the cytoplasmic plaque/peripheral membrane proteins. In advanced cancer, these regulatory pathways are dysregulated and lead to cell–cell adhesion disassembly, increased invasion and metastasis. The Metastasis suppressor protein 1 (MTSS1) plays a key role in the maintenance of cell–cell adhesions and its loss correlates with tumour progression in a variety of cancers. However, the mechanisms that regulate its function are not well-known. Using a system biology approach, we unravelled potential interacting partners of MTSS1. We found that the secretory carrier-associated membrane protein 1 (SCAMP1), a molecule involved in post-Golgi recycling pathways and in endosome cell membrane recycling, enhances Mtss1 anti-invasive function in HER2+/ER−/PR− breast cancer, by promoting its protein trafficking leading to elevated levels of RAC1-GTP and increased cell–cell adhesions. This was clinically tested in HER2 breast cancer tissue and shown that loss of MTSS1 and SCAMP1 correlates with reduced disease-specific survival. In summary, we provide evidence of the cooperative roles of MTSS1 and SCAMP1 in preventing HER2+/ER−/PR− breast cancer invasion and we show that the loss of Mtss1 and Scamp1 results in a more aggressive cancer cell phenotype.

MicroRNA-411 promoted the osteosarcoma progression by suppressing MTSS1 expression.

MicroRNAs (miRNAs) play crucial roles in the progression of different tumors. In our study, we investigated the expression and roles of miR-411 in human osteosarcoma. In this study, we first confirmed that the miR-411 expression was higher in the serum of patients with osteosarcoma than in the serum of healthy volunteers. In addition, we found that the miR-411 expression was upregulated in the osteosarcoma tissues compared to that in the matched normal bone tissues. We also demonstrated that the miR-411 expression was upregulated in the four osteosarcoma cell lines. Elevated expression of miR-411 promoted osteosarcoma cell proliferation and migration. Moreover, we identified that metastasis suppressor protein 1 (MTSS1) was a direct target gene of miR-411 in the osteosarcoma cell. We also demonstrated that the MTSS1 expression was downregulated in the osteosarcoma tissues compared to that in the matched normal bone tissues. In addition, MTSS1 expression level was inversely correlated with miR-411 expression in the osteosarcoma tissues. Furthermore, elevated expression of miR-411 enhanced the osteosarcoma cell proliferation and migration through inhibiting the MTSS1 expression. These data suggested that miR-411 played as oncogene in the osteosarcoma partly by inhibiting the MTSS1 expression.

PTEN-Dependent Stabilization of MTSS1 Inhibits Metastatic Phenotype in Pancreatic Ductal Adenocarcinoma

Pancreatic ductal adenocarcinoma (PDAC) presents at metastatic stage in over 50% of patients. With a survival rate of just 2.7% for patients presenting with distant disease, it is imperative to uncover novel mechanisms capable of suppressing metastasis in PDAC. Previously, we reported that the loss of metastasis suppressor protein 1 (MTSS1) in PDAC cells results in significant increase in cellular migration and invasion. Conversely, we also found that overexpressing MTSS1 in metastatic PDAC cell lines corresponds with not only decreased metastatic phenotype, but also greater overall survival. While it is known that MTSS1 is downregulated in late-stage PDAC, the mechanism behind that loss has not yet been elucidated. Here, we build off our previous findings to present a novel regulatory mechanism for the stabilization of MTSS1 via the tumor suppressor protein phosphatase and tensin homolog (PTEN). We show that PTEN loss in PDAC cells results in a decrease in MTSS1 expression and increased metastatic potential. Additionally, we demonstrate that PTEN forms a complex with MTSS1 in order to stabilize and protect it from proteasomal degradation. Finally, we show that the inflammatory tumor microenvironment, which makes up over 90% of PDAC tumor bulk, is capable of downregulating PTEN expression through secretion of miRNA-23b, potentially uncovering a novel extrinsic mechanism of MTSS1 regulation. Collectively, these data offer new insight into the role and regulation of MTSS1in suppressing tumor cell invasion and migration and help shed light as to what molecular mechanisms could be leading to early cell dissemination in PDAC.