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Supplemental Material and Methods. Cell lines, Transfections, Precipitation of Proteins by TCA-DOC , Co-immunoprecipitation (Co-IP) from Cell Lysates, Preparation of Cell Lysates

Supplemental Figure Legends S1-S7. Supplemental Figure Legends

Supplemental Figures S1-S7: Supplemental Figure S1: The effect of γ-secretase inhibition on ectodomain cleavage Supplemental Figure S2. Regulated cleavage targets substrates after surface expression Supplemental Figure S3: Interaction between CD44 and Merlin. Supplemental Figure S4: The tumor suppressor merlin (Nf2) inhibits CD44 cleavage. Supplemental Figure S5. Exemplified analysis of the constructs used in the migration assay of Figure 7A and Supplemental Figure 6 Supplemental Figure S6: Examplified photographs of one of the scratch assays with MEFs as shown in Figure 7A. Supplemental Figure S7: Loading diagrams of Figures 3B and 4C

Supplemental Materials & Methods and the references cited within.

Legends of supplemental figures S1-S5

Overview of human testis biopsy samples, which were analyzed for RHAMM parameters (mRNA expression, protein localization, locus variants, allele frequencies) as well as for expression of CFIm25 and of 12 TGCT susceptibility genes.

Its centrosome-targeting domain is indispensable for RHAMM spindle localization and it contributes to maintenance of spindle integrity in vitro.

Hypofertility is a risk factor for the development of testicular germ cell tumors (TGCT), but the initiating event linking these pathologies is unknown. We hypothesized that excessive planar division of undifferentiated germ cells promotes their self-renewal and TGCT development. However, our results obtained from mouse models and seminoma patients demonstrated the opposite. Defective planar divisions of undifferentiated germ cells caused their premature exit from the seminiferous tubule niche, resulting in germ cell depletion, hypofertility, intratubular germ cell neoplasias, and seminoma development. Oriented divisions of germ cells, which determine their fate, were regulated by spindle-associated RHAMM—a function we found to be abolished in 96% of human seminomas. Mechanistically, RHAMM expression is regulated by the testis-specific polyadenylation protein CFIm25, which is downregulated in the human seminomas. These results suggested that spindle misorientation is oncogenic, not by promoting self‐renewing germ cell divisions within the niche, but by prematurely displacing proliferating cells from their normal epithelial milieu. Furthermore, they suggested RHAMM loss-of-function and spindle misorientation as an initiating event underlying both hypofertility and TGCT initiation. These findings identify spindle-associated RHAMM as an intrinsic regulator of male germ cell fate and as a gatekeeper preventing initiation of TGCTs. Cancer Res; 76(21); 6382–95. ©2016 AACR.

The small G-protein Ras is a tightly controlled regulator of cell fate. Prolonged or persistent arrest in the activated GTP-loaded state by mutation of Ras as in lung cancer or in a Ras–GTPase-activating protein as in neurofibromatosis type 1 promotes tumorigenesis. We now show that the tumor-suppressor protein merlin (mutated in neurofibromatosis type 2) also controls Ras activity. Systematic analysis of growth factor signaling located the step of merlin interference to the activation of Ras and Rac. Merlin independently uncouples both Ras and Rac from growth factor signals. In the case of Ras, merlin acts downstream of the receptor tyrosine kinase-growth factor receptor binding protein 2 (Grb2)-SOS complex. However, merlin does not bind either SOS or Ras, but it counteracts the ERM (ezrin, radixin, moesin)–dependent activation of Ras, which correlates with the formation of a complex comprising ERM proteins, Grb2, SOS, Ras, and filamentous actin. Because efficient signaling from Ras requires Rac-p21-activated kinase–dependent phosphorylations of Raf and mitogen-activated protein/extracellular signal-regulated kinase kinase, merlin can also inhibit signal transfer from dominantly active Ras mutants. We propose that the interference of merlin with Ras- and Rac-dependent signal transfer represents part of the tumor-suppressive action of merlin. [Cancer Res 2007;67(2):520–7]

Supplementary Figure 3 from Merlin/Neurofibromatosis Type 2 Suppresses Growth by Inhibiting the Activation of Ras and Rac

Supplementary Figure 1 from Merlin/Neurofibromatosis Type 2 Suppresses Growth by Inhibiting the Activation of Ras and Rac

Supplementary Figure 2 from Merlin/Neurofibromatosis Type 2 Suppresses Growth by Inhibiting the Activation of Ras and Rac

Supplementary Figure 1 Legend from Merlin/Neurofibromatosis Type 2 Suppresses Growth by Inhibiting the Activation of Ras and Rac

Osteosarcoma is the most common type of pediatric bone tumor. Despite great advances in chemotherapy during the past decades, the survival rates of osteosarcoma patients remain unsatisfactory. Drug resistance is one of the main reasons, leading to treatment failure and poor prognosis. Previous reports correlated expression of cluster of differentiation 44 (CD44) with drug resistance and poor survival of osteosarcoma patients, however the underlying mechanisms are poorly defined. Here, we investigated the role of CD44 in the regulation of drug chemoresistance, using osteosarcoma cells isolated from mice carrying a mutation of the tumor suppressor neurofibromatosis type 2 (Nf2) gene. CD44 expression was knocked-down in the cells using CRISPR/Cas9 approach. Subsequently, CD44 isoforms and mutants were re-introduced to investigate CD44-dependent processes. Sensitivity to doxorubicin was analyzed in the osteosarcoma cells with modified CD44 expression by immunoblot, colony formation- and WST-1 assay. To dissect the molecular alterations induced by deletion of Cd44, RNA sequencing was performed on Cd44-positive and Cd44-negative primary osteosarcoma tissues isolated from Nf2-mutant mice. Subsequently, expression of candidate genes was evaluated by quantitative reverse transcription PCR (qRT-PCR). Our results indicate that CD44 increases the resistance of osteosarcoma cells to doxorubicin by up-regulating the levels of multidrug resistance (MDR) 1 protein expression, and suggest the role of proteolytically released CD44 intracellular domain, and hyaluronan interactions in this process. Moreover, high throughput sequencing analysis identified differential regulation of several apoptosis-related genes in Cd44-positive and -negative primary osteosarcomas, including p53 apoptosis effector related to PMP-22 (Perp). Deletion of Cd44 in osteosarcoma cells led to doxorubicin-dependent p53 activation and a profound increase in Perp mRNA expression. Overall, our results suggest that CD44 might be an important regulator of drug resistance and suggest that targeting CD44 can sensitize osteosarcoma to standard chemotherapy.

Hypofertility is a risk factor for the development of testicular germ cell tumors (TGCT), but the initiating event linking these pathologies is unknown. We hypothesized that excessive planar division of undifferentiated germ cells promotes their self-renewal and TGCT development. However, our results obtained from mouse models and seminoma patients demonstrated the opposite. Defective planar divisions of undifferentiated germ cells caused their premature exit from the seminiferous tubule niche, resulting in germ cell depletion, hypofertility, intratubular germ cell neoplasias, and seminoma development. Oriented divisions of germ cells, which determine their fate, were regulated by spindle-associated RHAMM—a function we found to be abolished in 96% of human seminomas. Mechanistically, RHAMM expression is regulated by the testis-specific polyadenylation protein CFIm25, which is downregulated in the human seminomas. These results suggested that spindle misorientation is oncogenic, not by promoting self-renewing germ cell divisions within the niche, but by prematurely displacing proliferating cells from their normal epithelial milieu. Furthermore, they suggested RHAMM loss-of-function and spindle misorientation as an initiating event underlying both hypofertility and TGCT initiation. These findings identify spindle-associated RHAMM as an intrinsic regulator of male germ cell fate and as a gatekeeper preventing initiation of TGCTs. Cancer Res; 76(21); 6382–95. ©2016 AACR.

Release of cytokines, growth factors and other life-essential molecules from precursors by a-disintegrin-and-metalloproteases (ADAMs) is regulated with high substrate-specificity. We hypothesized that this is achieved by cleavage-regulatory intracellular-domain (ICD)-modifications of the precursors. We show here that cleavage-stimuli-induced specific ICD-modifications cause structural substrate changes that enhance ectodomain sensitivity of neuregulin-1 (NRG1; epidermal-growth-factor) or CD44 (receptor-tyrosine-kinase (RTK) co-receptor) to chymotrypsin/trypsin or soluble ADAM. This inside-out signal transfer required substrate homodimerization and was prevented by cleavage-inhibitory ICD-mutations. In chimeras, regulation could be conferred to a foreign ectodomain, suggesting a common higher-order structure. We predict that substrate-specific protease-accessibility-regulation controls release of numerous ADAM substrates.

The postnatal mammalian ovary contains the primary follicles, each comprising an immature oocyte surrounded by a layer of somatic granulosa cells. Oocytes reach meiotic and developmental competence via folliculogenesis. During this process, the granulosa cells proliferate massively around the oocyte, form an extensive extracellular matrix (ECM) and differentiate into cumulus cells. As the ECM component hyaluronic acid (HA) is thought to form the backbone of the oocyte-granulosa cell complex, we deleted the relevant domain of the Receptor for HA Mediated Motility (RHAMM) gene in the mouse. This resulted in folliculogenesis defects and female hypofertility, although HA-induced signalling was not affected. We report that wild-type RHAMM localises at the mitotic spindle of granulosa cells, surrounding the oocyte. Deletion of the RHAMM C-terminus in vivo abolishes its spindle association, resulting in impaired spindle orientation in the dividing granulosa cells, folliculogenesis defects and subsequent female hypofertility. These data reveal the first identified physiological function for RHAMM, during oogenesis, and the importance of this spindle-associated function for female fertility.

Ectodomain cleavage (shedding) of transmembrane proteins by metalloproteases (MMP) generates numerous essential signaling molecules, but its regulation is not totally understood. CD44, a cleaved transmembrane glycoprotein, exerts both antiproliferative or tumor-promoting functions, but whether proteolysis is required for this is not certain. CD44-mediated contact inhibition and cellular proliferation are regulated by counteracting CD44 C-terminal interacting proteins, the tumor suppressor protein merlin (NF2) and ERM proteins (ezrin, radixin, moesin). We show here that activation or overexpression of constitutively active merlin or downregulation of ERMs inhibited 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced [as well as serum, hepatocyte growth factor (HGF), or platelet-derived growth factor (PDGF)] CD44 cleavage by the metalloprotease ADAM10, whereas overexpressed ERM proteins promoted cleavage. Merlin- and ERM-modulated Ras or Rac activity was not required for this function. However, latrunculin (an actin-disrupting toxin) or an ezrin mutant which is unable to link CD44 to actin, inhibited CD44 cleavage, identifying a cytoskeletal C-terminal link as essential for induced CD44 cleavage. Cellular migration, an important tumor property, depended on CD44 and its cleavage and was inhibited by merlin. These data reveal a novel function of merlin and suggest that CD44 cleavage products play a tumor-promoting role. Neuregulin, an EGF ligand released by ADAM17 from its pro-form NRG1, is predominantly involved in regulating cellular differentiation. In contrast to CD44, release of neuregulin from its pro-form was not regulated by merlin or ERM proteins. Disruption of the actin cytoskeleton however, also inhibited NRG1 cleavage. This current study presents one of the first examples of substrate-selective cleavage regulation. Implications: Investigating transmembrane protein cleavage and their regulatory pathways have provided new molecular insight into their important role in cancer formation and possible treatment. Mol Cancer Res; 13(5); 879–90. ©2015 AACR.