Gene Expression and DNA Methylation in Human Papillomavirus Positive and Negative Head and Neck Squamous Cell Carcinomas.

High-risk human papillomaviruses (HPV) are important agents, responsible for a large percentage of the 745,000 cases of head and neck squamous cell carcinomas (HNSCC), which were identified worldwide in 2020. In addition to being virally induced, tobacco and heavy alcohol consumption are believed to cause DNA damage contributing to the high number of HNSCC cases. Gene expression and DNA methylation differ between HNSCC based on HPV status. We used publicly available gene expression and DNA methylation profiles from the Cancer Genome Atlas and compared HPV positive and HPV negative HNSCC groups. We used differential gene expression analysis, differential methylation analysis, and a combination of these two analyses to identify the differences. Differential expression analysis identified 1854 differentially expressed genes, including PCNA, TNFRSF14, TRAF1, TRAF2, BCL2, and BIRC3. SYCP2 was identified as one of the top deregulated genes in the differential methylation analysis and in the combined differential expression and methylation analyses. Additionally, pathway and ontology analyses identified the extracellular matrix and receptor interaction pathway as the most altered between HPV negative and HPV positive HNSCC groups. Combining gene expression and DNA methylation can help in elucidating the genes involved in HPV positive HNSCC tumorigenesis, such as SYCP2 and TAF7L.

A meta-analysis of BRF2 as a prognostic biomarker in invasive breast carcinoma.

BACKGROUND: Deregulation of the RNA polymerase III specific TFIIIB subunit BRF2 occurs in subtypes of human cancers. However, correlations between BRF2 alterations and clinical outcomes in breast cancer are limited. We conducted this review to analyze BRF2 alterations in genomic data sets housed in Oncomine and cBioPortal to identify potential correlations between BRF2 alterations and clinical outcomes. METHODS: The authors queried both Oncomine and cBioPortal for alterations in BRF2 in human cancers and performed meta-analyses identifying significant correlations between BRF2 and clinical outcomes in invasive breast cancer (IBC). RESULTS: A meta cancer outlier profile analysis (COPA) of 715 data sets (86,733 samples) in Oncomine identified BRF2 as overexpressed in 60% of breast cancer data sets. COPA scores in IBC data sets (3594 patients) are comparable for HER2 (24.211, median gene rank 60) and BRF2 (29.656, median gene rank 36.5). Overall survival in IBC patients with BRF2 alterations (21%) is significantly decreased (p = 9.332e-3). IBC patients with BRF2 alterations aged 46 to 50 have a significantly poor survival outcome (p = 7.093e-3). Strikingly, in metastatic breast cancer, BRF2 is altered in 33% of women aged 45-50. BRF2 deletions are predominant in this age group. CONCLUSION: This study suggests BRF2 may be an prognostic biomarker in invasive breast carcinoma.

A Dual Role for the Nonreceptor Tyrosine Kinase Pyk2 during the Intracellular Trafficking of Human Papillomavirus 16.

UNLABELLED: The infectious process of human papillomaviruses (HPVs) has been studied considerably, and many cellular components required for viral entry and trafficking continue to be revealed. In this study, we investigated the role of the nonreceptor tyrosine kinase Pyk2 during HPV16 pseudovirion infection of human keratinocytes. We found that Pyk2 is necessary for infection and appears to be involved in the intracellular trafficking of the virus. Small interfering RNA-mediated reduction of Pyk2 resulted in a significant decrease in infection but did not prevent viral entry at the plasma membrane. Pyk2 depletion resulted in altered endolysosomal trafficking of HPV16 and accelerated unfolding of the viral capsid. Furthermore, we observed retention of the HPV16 pseudogenome in the trans-Golgi network (TGN) in Pyk2-depleted cells, suggesting that the kinase could be required for the viral DNA to exit the TGN. While Pyk2 has previously been shown to function during the entry of enveloped viruses at the plasma membrane, the kinase has not yet been implicated in the intracellular trafficking of a nonenveloped virus such as HPV. Additionally, these data enrich the current literature on Pyk2's function in human keratinocytes. IMPORTANCE: In this study, we investigated the role of the nonreceptor tyrosine kinase Pyk2 during human papillomavirus (HPV) infection of human skin cells. Infections with high-risk types of HPV such as HPV16 are the leading cause of cervical cancer and a major cause of genital and oropharyngeal cancer. As a nonenveloped virus, HPV enters cells by interacting with cellular receptors and established cellular trafficking routes to ensure that the viral DNA reaches the nucleus for productive infection. This study identified Pyk2 as a cellular component required for the intracellular trafficking of HPV16 during infection. Understanding the infectious pathways of HPVs is critical for developing additional preventive therapies. Furthermore, this study advances our knowledge of intracellular trafficking processes in keratinocytes.

Intermediate heparan sulfate binding during HPV-16 infection in HaCaTs.

Human papillomavirus (HPV) is the most prevalent sexually transmitted disease in the United States and can cause cancer with persistent infection. The most common cancer caused by HPV is cervical carcinoma with an average of 12,000 cases reported every year in the United States. Worldwide, over 500,000 cases of cervical cancer are reported yearly with over 250,000 deaths attributed to the disease. Although much is known about the serious health risks associated with HPV infection, there is still much to be discovered about how HPV binds and enters target cells. Understanding is required on how HPV infections will lead to strategies and therapies for reducing the number of infections and HPV-related diseases, including cancers. The HPV viral particle is composed of 2 viral proteins, L1 and L2. Data suggest that binding of the viral capsid to cells is dependent on the L1 protein. We hypothesize that this initial binding to a heparan sulfate is composed of 2 independent events: the first results in a structural change that exposes a hidden portion of the L1 protein leading to a second binding event on the heparan sulfate. Our experiments tested if this "hidden" portion of L1 is necessary for infection and explored the nature of this binding. We generated a peptide with the sequence of the "hidden" portion of L1. Infection of HaCaT cells in the presence of this peptide is highly reduced. Our results suggest that the binding of the L1 C-terminal domain is dependent on amino acid sequence and is necessary for infection.

The Process of Filopodia Induction during HPV Infection.

Human Papillomavirus 16 (HPV16) infects mucosal and epithelial cells and has been identified as a high-risk HPV type that is an etiologic agent of human cancers. The initial infectious process, i.e., the binding of the virus particle and its entry into the host cell, has been studied extensively, although it is not fully understood. There is still a gap in understanding the steps by which the virus is able to cross the plasma membrane after receptor binding. In this study, we demonstrate that after HPV16 comes into contact with a plasma membrane receptor, there are cytoskeletal changes resulting in an increase of filopodia numbers. This increase in filopodia numbers was transient and was maintained during the first two hours after virus addition. Our data show that there is a statistically significant increase in infection when filopodia numbers are increased by the addition of drug and virus simultaneously, and a decrease in virus infection when filopodia formation is inhibited. We describe that HPV16 binding results in the activation of Cdc42 GTPase that in turn results in an increase in filopodia. siRNA directed at Cdc42 GTPase resulted in a statistically significant reduction of infection and a corresponding lack of filopodia induction.

Human Papillomavirus Minor Capsid Protein L2 Mediates Intracellular Trafficking into and Passage beyond the Endoplasmic Reticulum.

Human papillomaviruses (HPVs) consist of two capsid proteins: major capsid protein L1 and minor capsid protein L2. The L2 protein has been shown to be involved in intracellular trafficking events that lead to the deposition of the viral DNA into the nucleus. In this study, we investigate the role of HPV16 L2 residues 43-DQILQ-47 during intracellular trafficking in human keratinocytes. We demonstrate that the highly conserved amino acids aspartic acid, isoleucine, and leucine are involved with the intracellular trafficking of the virus. Amino acid substitution of the isoleucine and leucine residues with alanine residues results in a significant decrease in infectivity of the pseudovirions without any changes to the binding or internalization of the virus. The pseudovirions containing these substitutions exhibit an altered trafficking pattern and do not deposit the viral pseudogenome into the nucleus. Instead, these mutated pseudovirions display a lack of interaction with syntaxin 18, an ER SNARE protein, are unable to progress past the endoplasmic reticulum (ER) and are redirected to the lysosomes. The results of this study help to elucidate the role and potential involvement of the 43-DQILQ-47 sequence during intracellular trafficking, specifically during trafficking beyond the ER. IMPORTANCE High-risk types of human papillomaviruses (HPVs), such as HPV16, are highly associated with cervical, anogenital, and oropharyngeal cancers. The minor capsid protein L2 is essential for the intracellular trafficking of the viral DNA to the nucleus. This study investigates the role of amino acid residues 43-DQILQ-47 of the HPV16 L2 protein in the intracellular trafficking of the virus. Understanding how the virus traffics through the cell is a key factor in the development of additional preventative antiviral therapies. This study illustrates, through modification of the 43-DQILQ-47 sequence in pseudovirions, the importance of the 43-DQILQ-47 sequence in the trafficking of the virus beyond the endoplasmic reticulum.

Smurf2 alters BPV1 trafficking and decreases infection.

Papillomavirus capsid proteins L1 and L2 mediate virion attachment, internalization and trafficking. In our studies of the capsid proteins, we identified an interaction of L2 with the E3 ligase Smad ubiquitin regulatory factor 2 (Smurf2). Smurf2 expression alters BPV1 virion trafficking and L2 protein levels. Using BPV1 pseudovirions (PSVs) containing a GFP or DSRed transgene encapsidated by L1 and L2 proteins, our data showed that although only BPV1 L2 interacts with Smurf2, both L1 and L2 levels decrease in a Smurf2- and ubiquitin-dependent manner. The decrease in L2 protein levels corresponded to a decrease in infection (i.e., loss of GFP or DSRed expression). We propose that Smurf2 regulates L2 protein cellular localization and therefore alters L2 protein levels. This change in trafficking and protein level decreases nuclear delivery and transcription of encapsidated pseudoviral transgenes and thus decreases BPV1 infection levels.

Human papillomavirus type 16 infection of human keratinocytes requires clathrin and caveolin-1 and is brefeldin a sensitive.

Human papillomavirus type 16 (HPV16) has been identified as being the most common etiological agent leading to cervical cancer. Despite having a clear understanding of the role of HPV16 in oncogenesis, details of how HPV16 traffics during infection are poorly understood. HPV16 has been determined to enter via clathrin-mediated endocytosis, but the subsequent steps of HPV16 infection remain unclear. There is emerging evidence that several viruses take advantage of cross talk between routes of endocytosis. Specifically, JCV and bovine papillomavirus type 1 have been shown to enter cells by clathrin-dependent endocytosis and then require caveolin-1-mediated trafficking for infection. In this paper, we show that HPV16 is dependent on caveolin-1 after clathrin-mediated endocytosis. We provide evidence for the first time that HPV16 infection is dependent on trafficking to the endoplasmic reticulum (ER). This novel trafficking may explain the requirement for the caveolar pathway in HPV16 infection because clathrin-mediated endocytosis typically does not lead to the ER. Our data indicate that the infectious route for HPV16 following clathrin-mediated entry is caveolin-1 and COPI dependent. An understanding of the steps involved in HPV16 sorting and trafficking opens up the possibility of developing novel approaches to interfere with HPV16 infection and reduce the burden of papillomavirus diseases including cervical cancer.

Dynasore inhibits removal of wild-type and DeltaF508 cystic fibrosis transmembrane conductance regulator (CFTR) from the plasma membrane.

Dynasore, a small molecule inhibitor of dynamin, was used to probe the role of dynamin in the endocytosis of wild-type and mutant CFTR (cystic fibrosis transmembrane conductance regulator). Internalization of both wild-type and 'temperature-corrected' DeltaF508 CFTR was markedly inhibited by a short exposure to dynasore, implicating dynamin as a key element in the endocytic internalization of both wild-type and mutant CFTR. The inhibitory effect of dynasore was readily reversible upon washout of dynasore from the growth media. Corr-4 ({2-(5-chloro-2-methoxy-phenylamino)-4'-methyl-[4,5']-bithiazolyl-2'-yl}-phenyl-methanonone), a pharmacological corrector of DeltaF508 CFTR biosynthesis, caused a marked increase in the cell surface expression of mutant CFTR. Co-incubation of DeltaF508 CFTR expressing cells with Corr-4 and dynasore caused a significantly greater level of cell surface CFTR than that observed in the presence of Corr-4 alone. These results argue that inhibiting the endocytic internalization of mutant CFTR provides a novel therapeutic target for augmenting the benefits of small molecule correctors of mutant CFTR biosynthesis.

Bovine papillomavirus type 1: from clathrin to caveolin.

Viruses may infect cells through clathrin-dependent, caveolin-dependent, or clathrin- and caveolin-independent endocytosis. Bovine papillomavirus type 1 (BPV1) entry into cells has been shown to occur by clathrin-dependent endocytosis, a pathway that involves the formation of clathrin-coated pits and fusion to early endosomes. Recently, it has been demonstrated that the closely related JC virus can enter cells in clathrin-coated vesicles and subsequently traffic to caveolae, the organelle where vesicles of the caveolin-dependent pathway deliver their cargo. In this study, we use immunofluorescence staining of BPV1 pseudovirions to show that BPV1 overlaps with the endosome marker EEA1 early during infection and later colocalizes with caveolin-1. We provide evidence through the colocalization of BPV1 with transferrin and cholera toxin B that BPVl trafficking may not be restricted to the clathrin-dependent pathway. Disrupting the entry of caveolar vesicles did not affect BPV1 infection; however, we show that blocking the caveolar pathway postentry results in a loss of BPV1 infection. These data indicate that BPV1 may enter by clathrin-mediated endocytosis and then utilize the caveolar pathway for infection, a pattern of trafficking that may explain the slow kinetics of BPV1 infection.

The role of NH4Cl and cysteine proteases in Human Papillomavirus type 16 infection.

BACKGROUND: The infectious pathway of the non-enveloped Human Papillomavirus Type 16 (HPV16) includes binding to the cell surface, clathrin-mediated endocytosis, and penetration into an endosome. HPV16 infection was shown to decrease in the presence of the lysosomotrophic neutralizing agent ammonium chloride (NH4Cl). NH4Cl neutralizes acidic endo-lysosome compartments, thus suggesting that pH was responsible for PV capsid conformational changes leading endosome escape. RESULTS: However, our data suggested that NH4Cl blocked infection by preventing the movement of PV viral particles from the early endosome to the caveosome as was shown for JC virus 12. We have confirmed that HPV 16 infection requires the trafficking of reporter-virions to the caveosome as is the case for BPV1 34. In this manuscript we propose that the observed decrease in infection of PV in the presence of NH4Cl was due to a loss of movement of reporter-virions to caveosomes. We also demonstrate that cysteine proteases are involved in the infectious process, and that cathepsin B treatment of viral particles was shown to overcome the block of infection observed in the presence of furin inhibition. We confirmed the need for cathepsin B in HPV16 infection using cathepsin B null mouse embryonic fibroblasts. CONCLUSION: We present data that suggest HPV16 infection is in part mediated by cysteine proteases, and that NH4Cl blocks the intracellular trafficking of infectious viral particles. To our knowledge this is the first demonstration that cysteine proteases influence the infection of a non-enveloped virus.

Guanine exchange factor Vav2: a novel potential target for the development of drugs effective in the prevention of papillomavirus infection and disease.

The bovine papillomavirus capsid protein L2 has no homology to known cellular proteins. We identified the interaction of bovine papillomavirus type 1 L2 with the guanine exchange factor Vav2. We determined that the interaction of L2 with Vav2 was mediated by the N-terminus of L2 and independent of the N-terminus of Vav2. L2 expression resulted in a change in the intracellular expression of Vav2 from diffuse to punctate cytoplasmic pattern. Our experiments in human epithelial cells showed that L2 expression results in a loss of phosphorylation of cofilin, an actin depolymerizing factor through the inactivation of Vav2 and RhoA. Cofilin and RhoA have been implicated in mediating endocytosis and in the differentiation mechanism of keratinocytes. We show that bovine papillomavirus type 1pseudovirions interact with Vav2 during infection and that infection efficiency is dependent on the RhoA activation state. Our experiments suggest that L2, through Vav2/RhoA/cofilin, may regulate endocytosis of viral particles and the mechanism of cellular proliferation and differentiation during virus production. Our data propose the Vav2-related pathway as a potential therapeutic target for papillomavirus infection and oncogenic development as has been shown for breast cancer invasiveness.

Nm23-H1 modulates the activity of the guanine exchange factor Dbl-1.

Cytoskeleton rearrangement is necessary for tumor invasion and metastasis. Cellular molecules whose role is to regulate components of the cytoskeletal structure can dictate changes in cellular morphology. One of these molecules is the suppressor of tumor metastasis Nm23-H1. The level of Nm23-H1 expression has been linked to the invasiveness and metastatic potential of human cancers including melanoma and breast cancer. In this report, we demonstrate an interaction between the suppressor of tumor metastasis Nm23-H1, and Dbl-1, an oncoprotein which is associated with guanine exchange and belongs to a family of Guanine Exchange Factors (GEF). Nm23-H1 also was shown to bind pDbl which is the proto-oncoprotein of Dbl. Interestingly, the interaction between Nm23-H1 and Dbl-1 rescues the suppression of the cell motility activity Nm23-H1. Moreover, this interaction results in loss of the ability of the Dbl-1 oncoprotein to function as a GEF for the critical Rho-GTPase family member Cdc42. The loss of GTP loading onto Cdc42 resulted in a dramatic reduction in adhesion stimulated ruffles and suggests that Nm23-H1 can negatively regulate cell migration and tumor metastasis by modulating the activity of Cdc42 through direct interaction with Dbl-1.

HPV16 and BPV1 infection can be blocked by the dynamin inhibitor dynasore.

The initial entry of papillomaviruses into their target cells has been shown to occur by clathrin-mediated endocytosis and caveolae-mediated endocytosis. These mechanisms entail the formation of nascent-coated vesicles at the plasma membrane. Such coated vesicles, clathrin or caveolin, form and pinch-off in a controlled mechanism that involves several proteins including dynamin. Dynamin is a GTPase that forms a dynamin ring at the stem connecting the nascent vesicle to the plasma membrane. In a still not fully characterized mechanism, dynamin's contraction and twisting results in the scission of the vesicle. In an effort to better characterize the role and molecular mechanisms of dynamin's function, researchers have identified dynasore, a dynamin GTPase inhibitor that prevents the scission of dynamin-dependent endocytic vesicles. Here, we have tested if infection by pseudovirus corresponding to the oncogenic human papillomavirus type 16 and bovine papillomavirus type 1 can be blocked by dynasore. We present data demonstrating that dynasore can block infection of human papillomavirus type 16 and bovine papillomavirus type 1 pseudovirions in a dose- and time-dependent manner with equal efficiency. Presently, there is no available therapy that can block infection by a wide range of papillomavirus regardless of species or genotypes. Targeting dynamin may lead to the rational design of drug able to prevent infection by papillomaviruses, and by other infectious agents dependent on this protein for initial internalization into target cells. Whether such an approach will prove successful needs further investigation.

Host cell transcriptome modification upon exogenous HPV16 L2 protein expression.

Human papillomavirus type 16 minor capsid protein L2 has been shown to assist in the initial entry and intracellular trafficking events leading to nuclear translocation of the viral genome. During our investigations of L2 function, we observed that expression of L2 in a keratinocyte cell line (HaCaT) resulted in phenotypic changes. In this manuscript, we present data that expression of the L2 protein in this cellular model system HaCaTs resulted in a shift from G0/G1 phase to mitotic S phase, as well as a reduced amount of retinoblastoma protein (Rb) and an increase in Cdc2 phosphorylation. We performed genome-wide host cell mRNA sequencing and identified 2586 differentially expressed genes upon HPV16 L2 expression. Via machine learning and protein network analysis, genes involved in cellular differentiation and proliferation were highlighted as impacted by L2. Our results have implications for the role of L2 at the viral production stages when the virus needs to prevent cellular differentiation while maintaining the cells ability to replicate DNA. Our study suggests a potential novel function of the L2 protein, as a regulator of cellular gene transcription.

The Role of DCT in HPV16 Infection of HaCaTs.

Persistent infection with high-risk human papillomavirus (HPV) genotype is a major factor leading to many human cancers. Mechanisms of HPV entry into host cells and genome trafficking towards the nucleus are incompletely understood. Dopachrome tautomerase (DCT) was identified as a cellular gene required for HPV infection in HeLa cells on a siRNA screen study. Here, we confirm that DCT knockdown significantly decreases HPV infection in the human keratinocyte HaCaT cells as was observed in HeLas. We investigated the effects of DCT knockdown and found that DCT depletion caused increased reactive oxygen species (ROS) levels, DNA damage and altered cell cycle in HaCaT cells. We observed increased viral DNA localization at the endoplasmic reticulum but an overall decrease in infection in DCT knockdown cells. This observation suggests that viral DNA might be retained in the ER due to altered cell cycle, and viral particles are incapable of further movement towards the nucleus in DCT knockdown cells.

HPV entry into cells.

Human papillomavirus (HPV) is a sexually transmitted virus responsible for the development of cervical cancer, anal cancer, head and throat cancers, as well as genital area warts. A major focus of current HPV research is on preventing the virus from entering a cell and transferring its genetic material to the nucleus, thus potentially preventing the development of cancer. Although the available HPV vaccines are extremely successful, approximately 15 additional cancer-causing HPVs have been identified that the vaccines do not protect against. Therefore, roughly 150,000 cancer cases will not be prevented annually with the current vaccines. Research efforts focused on the basic cell biology of HPV infection have a goal of identifying common infectious events that may lead to inexpensive vaccines or anti-virals to prevent infection by most, if not all, HPVs. In this review we attempt to summarize what is known regarding the process of HPV binding, entry, and intracellular trafficking.

HPV16 infection of HaCaTs is dependent on ß4 integrin, and α6 integrin processing.

Our understanding of human papillomavirus (HPV) is still evolving. To further study the field, our laboratory has focused on determining the role of integrins in the initial steps of viral endocytosis into HaCaT cells. Our and others' previous findings have shown that α6 is necessary for infection. Here we show that α3 and ß1 were dispensable, and we identified integrin α6ß4 complex as necessary for infection in HaCaTs. ß4 knock down resulted in a significant decrease in HPV16 PsV infection and perhaps most importantly resulted in defective post-translational α6 processing. We showed that the unprocessed α6 does not localize to the cell surface. We propose that the α6ß4 complex is necessary for the formation of an endocytic complex that results in the signaling transduction events necessary for initial endocytosis.

Usage of heparan sulfate, integrins, and FAK in HPV16 infection.

Human papillomavirus type 16 (HPV16) is the major causative agent of cervical cancer. Studies regarding the early binding and signaling molecules that play a significant role in infection are still lacking. The current study analyzes the role of heparan sulfate, integrins, and the signaling molecule FAK in HPV16 infection of human adult keratinocytes cell line (HaCaTs). Our data demonstrate that infection requires the binding of viral particles to heparan sulfate followed by activation of focal adhesion kinase through an integrin. Infections were reduced in the presence of the FAK inhibitor, TAE226. TAE226 was observed to inhibit viral entry to the early endosome a known infectious route. These findings suggest that FAK can serve as a novel target for antiviral therapy.

The suppressor of metastasis Nm23-H1 interacts with the Cdc42 Rho family member and the pleckstrin homology domain of oncoprotein Dbl-1 to suppress cell migration.

Tumor invasion and metastasis is regulated by a number of cellular molecules known to be involved in signaling and cytoskeletal rearrangement. One of these molecules is the suppressor of tumor metastasis Nm23-H1 which linked to invasiveness and metastatic potential of human cancers. Nm23-H1 expression is down-regulated in human melanoma and invasive breast carcinoma. Recent studies have shown an association between the Nm23-H1 and oncoprotein Dbl-1 which is associated with guanine exchange and belongs to a family of Guanine Exchange Factors (GEF). In this report we show a direct interaction in vitro and in human B cells and specifically identified the pleckstrin homology domain of Dbl-1 as the domain which binds to Nm23-H1. Furthermore, Nm23-H1 and Dbl-1 colocalized in the cytoplasm of COS-7 cells when expressed exogenously and showed predominant signals at the periphery of the cells particularly at the plasma membrane. Interestingly, Dbl-1 and Cdc42 expression rescued the suppressive activities of Nm23-H1 in cell migration assays. We show that Cdc42 a regulatory protein involved in cytoskeletal reorganization, cell growth and development can bind to Nm23-H1 and the kinase deficient mutant H118F but only weakly to the mutant P96S which lacks the ability to suppress cell migration and metastasis. Cdc42 also colocalized with Nm23-H1 and the Dbl-1 proteins as specific punctate signals in the cytoplasm and at the cell membrane. Nm23-H1 also lead to the reduction in membrane ruffles and protuberances when expressed with Dbl-1 and Cdc42. Surprisingly, Nm23-H1 interacted with Cdc42 as well as Rac1 but somewhat weaker with RhoA. These studies suggests that Nm23-H1 can negatively regulate cell migration and tumor metastasis by modulating the activity of Cdc42 and possibly other Rho family members through interaction with Dbl-1.