Posttranslational modifications of serine protease TMPRSS13 regulate zymogen activation, proteolytic activity, and cell surface localization.

TMPRSS13, a member of the type II transmembrane serine protease (TTSP) family, harbors four N-linked glycosylation sites in its extracellular domain. Two of the glycosylated residues are located in the scavenger receptor cysteine-rich (SRCR) protein domain, while the remaining two sites are in the catalytic serine protease (SP) domain. In this study, we examined the role of N-linked glycosylation in the proteolytic activity, autoactivation, and cellular localization of TMPRSS13. Individual and combinatory site-directed mutagenesis of the glycosylated asparagine residues indicated that glycosylation of the SP domain is critical for TMPRSS13 autoactivation and catalytic activity toward one of its protein substrates, the prostasin zymogen. Additionally, SP domain glycosylation-deficient TMPRSS13 displayed impaired trafficking of TMPRSS13 to the cell surface, which correlated with increased retention in the endoplasmic reticulum. Importantly, we showed that N-linked glycosylation was a critical determinant for subsequent phosphorylation of endogenous TMPRSS13. Taken together, we conclude that glycosylation plays an important role in regulating TMPRSS13 activation and activity, phosphorylation, and cell surface localization.

TMPRSS13 zymogen activation, surface localization, and shedding is regulated by proteolytic cleavage within the non-catalytic stem region.

TMPRSS13 is a member of the type II transmembrane serine protease (TTSP) family. Here we characterize a novel post-translational mechanism important for TMPRSS13 function: proteolytic cleavage within the extracellular TMPRSS13 stem region located between the transmembrane domain and the first site of N-linked glycosylation at asparagine (N)-250 in the scavenger receptor cysteine rich (SRCR) domain. Importantly, the catalytic competence of TMPRSS13 is essential for stem region cleavage, suggesting an autonomous mechanism of action. Site-directed mutagenesis of the 10 basic amino acids (four arginine and six lysine residues) in this region abrogated zymogen activation and catalytic activity of TMPRSS13, as well as phosphorylation, cell surface expression, and shedding. Mutation analysis of individual arginine residues identified R223, a residue located between the low-density lipoprotein receptor class A domain and the SRCR domain, as important for stem region cleavage. Mutation of R223 causes a reduction in the aforementioned functional processing steps of TMPRSS13. These data provide further insight into the roles of different post-translational modifications as regulators of the function and localization of TMPRSS13. Additionally, the data suggest the presence of complex interconnected regulatory mechanisms that may serve to ensure the proper levels of cell-surface and pericellular TMPRSS13-mediated proteolysis under homeostatic conditions.

Cell surface-anchored serine proteases in cancer progression and metastasis.

Over the last two decades, a novel subgroup of serine proteases, the cell surface-anchored serine proteases, has emerged as an important component of the human degradome, and several members have garnered significant attention for their roles in cancer progression and metastasis. A large body of literature describes that cell surface-anchored serine proteases are deregulated in cancer and that they contribute to both tumor formation and metastasis through diverse molecular mechanisms. The loss of precise regulation of cell surface-anchored serine protease expression and/or catalytic activity may be contributing to the etiology of several cancer types. There is therefore a strong impetus to understand the events that lead to deregulation at the gene and protein levels, how these precipitate in various stages of tumorigenesis, and whether targeting of selected proteases can lead to novel cancer intervention strategies. This review summarizes current knowledge about cell surface-anchored serine proteases and their role in cancer based on biochemical characterization, cell culture-based studies, expression studies, and in vivo experiments. Efforts to develop inhibitors to target cell surface-anchored serine proteases in cancer therapy will also be summarized.

Phosphorylation of the type II transmembrane serine protease, TMPRSS13, in hepatocyte growth factor activator inhibitor-1 and -2-mediated cell-surface localization.

TMPRSS13 is a member of the type II transmembrane serine protease (TTSP) family. Although various TTSPs have been characterized in detail biochemically and functionally, the basic properties of TMPRSS13 remain unclear. Here, we investigate the activation, inhibition, post-translational modification, and localization of TMPRSS13. We show that TMPRSS13 is a glycosylated, active protease and that its own proteolytic activity mediates zymogen cleavage. Full-length, active TMPRSS13 exhibits impaired cell-surface expression in the absence of the cognate Kunitz-type serine protease inhibitors, hepatocyte growth factor activator inhibitor (HAI)-1 or HAI-2. Concomitant presence of TMPRSS13 with either HAI-1 or -2 mediates phosphorylation of residues in the intracellular domain of the protease, and it coincides with efficient transport of the protease to the cell surface and its subsequent shedding. Cell-surface labeling experiments indicate that the dominant form of TMPRSS13 on the cell surface is phosphorylated, whereas intracellular TMPRSS13 is predominantly non-phosphorylated. These data provide novel insight into the cellular properties of TMPRSS13 and highlight phosphorylation of TMPRSS13 as a novel post-translational modification of this TTSP family member and potentially other members of this family of proteases.

Cell Surface Human Airway Trypsin-Like Protease Is Lost During Squamous Cell Carcinogenesis.

Cancer progression is accompanied by increased levels of extracellular proteases that are capable of remodeling the extracellular matrix, as well as cleaving and activating growth factors and receptors that are involved in pro-cancerous signaling pathways. Several members of the type II transmembrane serine protease (TTSP) family have been shown to play critical roles in cancer progression, however, the expression or function of the TTSP Human Airway Trypsin-like protease (HAT) in carcinogenesis has not been examined. In the present study we aimed to determine the expression of HAT during squamous cell carcinogenesis. HAT transcript is present in several tissues containing stratified squamous epithelium and decreased expression is observed in carcinomas. We determined that HAT protein is consistently expressed on the cell surface in suprabasal/apical layers of squamous cells in healthy cervical and esophageal epithelia. To assess whether HAT protein is differentially expressed in normal tissue versus tissue in different stages of carcinogenesis, we performed a comprehensive immunohistochemical analysis of HAT protein expression levels and localization in arrays of paraffin embedded human cervical and esophageal carcinomas compared to the corresponding normal tissue. We found that HAT protein is expressed in the non-proliferating, differentiated cellular strata and is lost during the dedifferentiation of epithelial cells, a hallmark of squamous cell carcinogenesis. Thus, HAT expression may potentially be useful as a marker for clinical grading and assessment of patient prognosis in squamous cell carcinomas.

Type II transmembrane serine proteases as potential targets for cancer therapy.

Carcinogenesis is accompanied by increased protein and activity levels of extracellular cell-surface proteases that are capable of modifying the tumor microenvironment by directly cleaving the extracellular matrix, as well as activating growth factors and proinflammatory mediators involved in proliferation and invasion of cancer cells, and recruitment of inflammatory cells. These complex processes ultimately potentiate neoplastic progression leading to local tumor cell invasion, entry into the vasculature, and metastasis to distal sites. Several members of the type II transmembrane serine protease (TTSP) family have been shown to play critical roles in cancer progression. In this review the knowledge collected over the past two decades about the molecular mechanisms underlying the pro-cancerous properties of selected TTSPs will be summarized. Furthermore, we will discuss how these insights may facilitate the translation into clinical settings in the future by specifically targeting TTSPs as part of novel cancer treatment regimens.

The matriptase-prostasin proteolytic cascade in epithelial development and pathology.

The type II transmembrane serine protease matriptase has an essential role in the integrity and function of multiple epithelial tissues. In the epidermis, matriptase activates the glycosylphosphatidylinositol (GPI) anchored membrane serine protease prostasin to initiate a proteolytic cascade that is required for the development of the stratum corneum barrier function. Accordingly, mice deficient for matriptase phenocopy mice deficient for epidermal prostasin and present with impaired corneocyte differentiation, imparied lipid matrix formation, loss of profilaggrin processing and loss of tight junction formation and function. Together, these defects lead to a compromised epidermal barrier and result in fatal dehydration during the neonatal period. Proteolytic activity of the matriptase-prostasin cascade is regulated in the epidermis via inhibition by the Kunitz-type serine protease inhibitor hepatocyte growth factor activator inhibitor-1 (HAI-1). Importantly, targeted post-natal ablation of matriptase in mice perturbs the function of multiple adult tissues, indicating an ongoing requirement for matriptase proteolysis in the maintenance of diverse types of epithelia. Impaired matriptase proteolytic activity has been linked to human Autosomal Recessive Icthyosis with Hypotrichosis (ARIH), whereas aberrant matriptase activity has been implicated in Netherton's Syndrome. This review will summarize information pertaining to the role of matriptase in epithelial biology and will discuss recent advancements in our understanding of how matriptase activity is regulated and the down-stream effectors of matriptase proteolysis.

Membrane-anchored serine protease matriptase regulates epithelial barrier formation and permeability in the intestine.

The intestinal epithelium serves as a major protective barrier between the mammalian host and the external environment. Here we show that the transmembrane serine protease matriptase plays a pivotol role in the formation and integrity of the intestinal epithelial barrier. St14 hypomorphic mice, which have a 100-fold reduction in intestinal matriptase mRNA levels, display a 35% reduction in intestinal transepithelial electrical resistance (TEER). Matriptase is expressed during intestinal epithelial differentiation and colocalizes with E-cadherin to apical junctional complexes (AJC) in differentiated polarized Caco-2 monolayers. Inhibition of matriptase activity using a specific peptide inhibitor or by knockdown of matriptase by siRNA disrupts the development of TEER in barrier-forming Caco-2 monolayers and increases paracellular permeability to macromolecular FITC-dextran. Loss of matriptase was associated with enhanced expression and incorporation of the permeability-associated, "leaky" tight junction protein claudin-2 at intercellular junctions. Knockdown of claudin-2 enhanced the development of TEER in matriptase-silenced Caco-2 monolayers, suggesting that the reduced barrier integrity was caused, at least in part, by an inability to regulate claudin-2 expression and incorporation into junctions. We find that matriptase enhances the rate of claudin-2 protein turnover, and that this is mediated indirectly through an atypical PKCzeta-dependent signaling pathway. These results support a key role for matriptase in regulating intestinal epithelial barrier competence, and suggest an intriguing link between pericellular serine protease activity and tight junction assembly in polarized epithelia.

Strong expression association between matriptase and its substrate prostasin in breast cancer.

Breast cancer tumorigenesis is accompanied by increased levels of extracellular proteases that are capable of remodeling the extracellular matrix as well as cleaving and activating growth factors and signaling receptors that are critically involved in neoplastic progression. Multiple studies implicate the membrane anchored serine protease matriptase (also known as MT-SP1 and epithin) in breast cancer. The pro-form of the GPI-anchored serine protease prostasin has recently been identified as a physiological substrate of matriptase and the two proteases are co-expressed in multiple healthy tissues. In this study, the inter-relationship between the two membrane-anchored serine proteases in breast cancer was investigated using breast cancer cell lines and breast cancer patient samples to delineate the association between matriptase and prostasin. We used Western blotting to determine the expression of matriptase and prostasin proteins in a panel of breast cancer cell lines and immunohistochemistry to assess the expression in serial sections from breast cancer tissue arrays. We demonstrate that the expression of matriptase and prostasin is closely correlated in breast cancer cell lines as well as in breast cancer tissue samples. Furthermore, matriptase and prostasin display a near identical spatial expression pattern in the epithelial compartment of breast cancer tissue. These data suggest that the matriptase-prostasin cascade might play a critical role in breast cancer.

Fibroblast-secreted hepatocyte growth factor mediates epidermal growth factor receptor tyrosine kinase inhibitor resistance in triple-negative breast cancers through paracrine activation of Met.

INTRODUCTION: Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) have shown clinical efficacy in lung, colon, and pancreatic cancers. In lung cancer, resistance to EGFR TKIs correlates with amplification of the hepatocyte growth factor (HGF) receptor tyrosine kinase Met. Breast cancers do not respond to EGFR TKIs, even though EGFR is overexpressed. This intrinsic resistance to EGFR TKIs in breast cancer does not correlate with Met amplification. In several tissue monoculture models of human breast cancer, Met, although expressed, is not phosphorylated, suggesting a requirement for a paracrine-produced ligand. In fact, HGF, the ligand for Met, is not expressed in epithelial cells but is secreted by fibroblasts in the tumor stroma. We have identified a number of breast cancer cell lines that are sensitive to EGFR TKIs. This sensitivity is in conflict with the observed clinical resistance to EGFR TKIs in breast cancers. Here we demonstrate that fibroblast secretion of HGF activates Met and leads to EGFR/Met crosstalk and resistance to EGFR TKIs in triple-negative breast cancer (TNBC). METHODS: The SUM102 and SUM149 TNBC cell lines were used in this study. Recombinant HGF as well as conditioned media from fibroblasts expressing HGF were used as sources for Met activation. Furthermore, we co-cultured HGF-secreting fibroblasts with Met-expressing cancer cells to mimic the paracrine HGF/Met pathway, which is active in the tumor microenvironment. Cell growth, survival, and transformation were measured by cell counting, clonogenic and MTS assays, and soft agar colony formation, respectively. Student's t test was used for all statistical analysis. RESULTS: Here we demonstrate that treatment of breast cancer cells sensitive to EGFR TKIs with recombinant HGF confers a resistance to EGFR TKIs. Interestingly, knocking down EGFR abrogated HGF-mediated cell survival, suggesting a crosstalk between EGFR and Met. HGF is secreted as a single-chain pro-form, which has to be proteolytically cleaved in order to activate Met. To determine whether the proteases required to activate pro-HGF were present in the breast cancer cells, we utilized a fibroblast cell line expressing pro-HGF (RMF-HGF). Addition of pro-HGF-secreting conditioned fibroblast media to TNBC cells as well as co-culturing of TNBC cells with RMF-HGF fibroblasts resulted in robust phosphorylation of Met and stimulated proliferation in the presence of an EGFR TKI. CONCLUSIONS: Taken together, these data suggest a role for Met in clinical resistance to EGFR TKIs in breast cancer through EGFR/Met crosstalk mediated by tumor-stromal interactions.

Loss of the matriptase inhibitor HAI-2 during prostate cancer progression.

BACKGROUND: Prostate cancer progression is accompanied by increased levels of extracellular proteases that are capable of remodeling the extracellular matrix as well as cleaving and activating growth factors and their receptors that are critically involved in pro-cancerous signaling pathways. The membrane anchored serine protease matriptase (also known as MT-SP1, epithin, and TADG15) has been implicated in prostate cancer. Several studies have demonstrated that the expression of this protease, both on the RNA and protein level is significantly increased during prostate cancer progression. Hepatocyte activator growth factor inhibitor-2 (HAI-2) has recently been identified as a physiological inhibitor of matriptase. It has been proposed that the increase of matriptase with a concomitant loss of its inhibitors may play a critical role in cancer progression. METHODS: In this study, we used immunohistochemistry to determine the expression of HAI-2 protein in 136 prostate cancer samples, 20 prostate benign prostatic hyperplasia (BPH) samples, and 31 normal or tumor-adjacent prostate samples. Specificity of detection was ensured by using two unrelated HAI-2 antibodies and corresponding non-immune IgG antibodies. RESULTS: We demonstrate that HAI-2 protein is significantly decreased in malignant lesions as compared to normal and BPH lesions, and that the most poorly differentiated tumors (Gleason score 8-10) have the lowest level of HAI-2 expression. CONCLUSION: These data suggest that the loss of HAI-2 may be actively involved in prostate cancer progression by causing a reduced inhibitory capacity of proteolysis possibly of the physiological target for HAI-2 matriptase.

Loss of matriptase suppression underlies spint1 mutation-associated ichthyosis and postnatal lethality.

Hepatocyte growth factor activator inhibitor-1 (HAI)-1 is an epithelial Kunitz-type transmembrane serine protease inhibitor that is encoded by the SPINT1 gene. HAI-1 displays potent inhibitory activity toward a large number of trypsin-like serine proteases. HAI-1 was recently shown to play an essential role in postnatal epithelial homeostasis. Thus, Spint1-deficient mice were found to display severe growth retardation and are unable to survive beyond postnatal day 16. The mice present histologically with overt hyperkeratosis of the forestomach, hyperkeratosis and acanthosis of the epidermis, and hypotrichosis associated with abnormal cuticle development. In this study, we show that loss of inhibition of a proteolytic pathway that is dependent on the type II transmembrane serine protease, matriptase, underlies the detrimental effects of postnatal Spint1 deficiency. Matriptase and HAI-1 precisely co-localize in all tissues that are affected by the Spint1 disruption. Spint1-deficient mice that have low matriptase levels, caused by a hypomorphic mutation in the St14 gene that encodes matriptase, not only survived the neonatal period but were healthy and displayed normal long-term survival. Furthermore, a detailed histological analysis of neonatal, young adult, as well as aged mice did not reveal any abnormalities in Spint1-deficent mice that have low matriptase levels. This study identifies matriptase suppression as an essential function of HAI-1 in postnatal tissue homeostasis.

Epithelial integrity is maintained by a matriptase-dependent proteolytic pathway.

A pericellular proteolytic pathway initiated by the transmembrane serine protease matriptase plays a critical role in the terminal differentiation of epidermal tissues. Matriptase is constitutively expressed in multiple other epithelia, suggesting a putative role of this membrane serine protease in general epithelial homeostasis. Here we generated mice with conditional deletion of the St14 gene, encoding matriptase, and show that matriptase indeed is essential for the maintenance of multiple types of epithelia in the mouse. Thus, embryonic or postnatal ablation of St14 in epithelial tissues of diverse origin and function caused severe organ dysfunction, which was often associated with increased permeability, loss of tight junction function, mislocation of tight junction-associated proteins, and generalized epithelial demise. The study reveals that the homeostasis of multiple simple and stratified epithelia is matriptase-dependent, and provides an important animal model for the exploration of this membrane serine protease in a range of physiological and pathological processes.

The cell-surface anchored serine protease TMPRSS13 promotes breast cancer progression and resistance to chemotherapy.

Breast cancer progression is accompanied by increased expression of extracellular and cell-surface proteases capable of degrading the extracellular matrix as well as cleaving and activating downstream targets. The type II transmembrane serine proteases (TTSPs) are a family of cell-surface proteases that play critical roles in numerous types of cancers. Therefore, the aim of this study was to identify novel and uncharacterized TTSPs with differential expression in breast cancer and to determine their potential roles in progression. Systematic in silico data analysis followed by immunohistochemical validation identified increased expression of the TTSP family member, TMPRSS13 (transmembrane protease, serine 13), in invasive ductal carcinoma patient tissue samples compared to normal breast tissue. To test whether loss of TMPRSS13 impacts tumor progression, TMPRSS13 was genetically ablated in the oncogene-induced transgenic MMTV-PymT tumor model. TMPRSS13 deficiency resulted in a significant decrease in overall tumor burden and growth rate, as well as a delayed formation of detectable mammary tumors, thus suggesting a causal relationship between TMPRSS13 expression and the progression of breast cancer. Complementary studies using human breast cancer cell culture models revealed that siRNA-mediated silencing of TMPRSS13 expression decreases proliferation, induces apoptosis, and attenuates invasion. Importantly, targeting TMPRSS13 expression renders aggressive triple-negative breast cancer cell lines highly responsive to chemotherapy. At the molecular level, knockdown of TMPRSS13 in breast cancer cells led to increased protein levels of the tumor-suppressive protease prostasin. TMPRSS13/prostasin co-immunoprecipitation and prostasin zymogen activation experiments identified prostasin as a potential novel target for TMPRSS13. Regulation of prostasin levels may be a mechanism that contributes to the pro-oncogenic properties of TMPRSS13 in breast cancer. TMPRSS13 represents a novel candidate for targeted therapy in combination with standard of care chemotherapy agents in patients with hormone receptor-negative breast cancer or in patients with tumors refractory to endocrine therapy.

TMPRSS13 promotes cell survival, invasion, and resistance to drug-induced apoptosis in colorectal cancer.

Cancer progression is often accompanied by increased levels of extracellular proteases capable of remodeling the extracellular matrix and promoting pro-cancerous signaling pathways by activating growth factors and receptors. The type II transmembrane serine protease (TTSP) family encompasses several proteases that play critical roles in cancer progression; however, the expression or function of the TTSP TMPRSS13 in carcinogenesis has not been examined. In the present study, we found TMPRSS13 to be differentially expressed at both the transcript and protein levels in human colorectal cancer (CRC). Immunohistochemical analyses revealed consistent high expression of TMPRSS13 protein on the cancer cell surface in CRC patient samples; in contrast, the majority of normal colon samples displayed no detectable expression. On a functional level, TMPRSS13 silencing in CRC cell lines increased apoptosis and impaired invasive potential. Importantly, transgenic overexpression of TMPRSS13 in CRC cell lines increased tolerance to apoptosis-inducing agents, including paclitaxel and HA14-1. Conversely, TMPRSS13 silencing rendered CRC cells more sensitive to these agents. Together, our findings suggest that TMPRSS13 plays an important role in CRC cell survival and in promoting resistance to drug-induced apoptosis; we also identify TMPRSS13 as a potential new target for monotherapy or combination therapy with established chemotherapeutics to improve treatment outcomes in CRC patients.

Loss of proteolytically processed filaggrin caused by epidermal deletion of Matriptase/MT-SP1.

Profilaggrin is a large epidermal polyprotein that is proteolytically processed during keratinocyte differentiation to release multiple filaggrin monomer units as well as a calcium-binding regulatory NH2-terminal filaggrin S-100 protein. We show that epidermal deficiency of the transmembrane serine protease Matriptase/MT-SP1 perturbs lipid matrix formation, cornified envelope morphogenesis, and stratum corneum desquamation. Surprisingly, proteomic analysis of Matriptase/MT-SP1-deficient epidermis revealed the selective loss of both proteolytically processed filaggrin monomer units and the NH2-terminal filaggrin S-100 regulatory protein. This was associated with a profound accumulation of profilaggrin and aberrant profilaggrin-processing products in the stratum corneum. The data identify keratinocyte Matriptase/MT-SP1 as an essential component of the profilaggrin-processing pathway and a key regulator of terminal epidermal differentiation.

uPARAP/Endo180 is essential for cellular uptake of collagen and promotes fibroblast collagen adhesion.

The uptake and lysosomal degradation of collagen by fibroblasts constitute a major pathway in the turnover of connective tissue. However, the molecular mechanisms governing this pathway are poorly understood. Here, we show that the urokinase plasminogen activator receptor-associated protein (uPARAP)/Endo180, a novel mesenchymally expressed member of the macrophage mannose receptor family of endocytic receptors, is a key player in this process. Fibroblasts from mice with a targeted deletion in the uPARAP/Endo180 gene displayed a near to complete abrogation of collagen endocytosis. Furthermore, these cells had diminished initial adhesion to a range of different collagens, as well as impaired migration on fibrillar collagen. These studies identify a central function of uPARAP/Endo180 in cellular collagen interactions.

Matriptase: a culprit in cancer?

Pericellular proteases can degrade extracellular matrix proteins and reshape their microenvironment, as well as cleave and activate signaling molecules such as growth factors and their receptors. In this capacity, pericellular proteolysis is essential for multiple biological processes, including development, tissue homeostasis and tissue repair. On the flip side, dysregulated pericellular proteolysis is a hallmark in many pathological conditions including cancer, and is believed to be critically involved in tumor growth, invasion and dissemination of cancer cells to other organs. Matriptase is a member of the family of Type II transmembrane serine proteases, and has been implicated in a variety of epithelial cancers. This review summarizes current knowledge about matriptase and its role in cancer based on expression studies, biochemical characterization, cell-culture based studies and in vivo experiments.

Matriptase-dependent cell surface proteolysis in epithelial development and pathogenesis.

Matriptase is an epithelial type II transmembrane serine protease with a complex modular structure and sophisticated activation mechanism. Reduced matriptase activity in mice or humans is associated with incomplete terminal differentiation of epidermis, epidermal appendages, oral epithelium, and, likely, other epithelial structures. Preliminary evidence indicates that matriptase is part of a serine protease zymogen activation cascade that regulates epithelial cell proliferation and fate. Matriptase activity must be tightly controlled in epithelial tissues by transcriptional and posttranslational mechanisms, as matriptase dysregulation can cause embryonic demise as well as malignant transformation.

The role of type II transmembrane serine protease-mediated signaling in cancer.

Pericellular proteases have long been implicated in carcinogenesis. Previous research focused on these proteins, primarily as extracellular matrix (ECM) protein-degrading enzymes which allowed cancer cells to breach the basement membrane and invade surrounding tissue. However, recently, there has been a shift in the view of cell surface proteases, including serine proteases, as proteolytic modifiers of particular targets, including growth factors and protease-activated receptors, which are critical for the activation of oncogenic signaling pathways. Of the 176 human serine proteases currently identified, a subset of 17, known as type II transmembrane serine proteases (TTSPs). Many have been shown to be relevant to cancer progression since they were first identified as a family around the turn of the century. To this end, altered expression of TTSPs appeared as a trademark of several tumor types. However, the substrates and underlying signaling pathways remained unclear. Localization of these proteins to the cell surface places them in the unique position to mediate signal transduction between the cell and its surrounding environment. Many of the TTSPs have already been shown to play key roles in processes such as postnatal development, tissue homeostasis, and tumor progression, which share overlapping molecular mechanisms. In this review, we summarize the current knowledge regarding the role of the TTSP family in pro-oncogenic signaling.