Mary E. Anderson

Germline mutations of the gene encoding bone morphogenetic protein receptor 1A in juvenile polyposis.

Juvenile polyposis (JP; OMIM 174900) is an autosomal dominant gastrointestinal hamartomatous polyposis syndrome in which patients are at risk for developing gastrointestinal cancers. Previous studies have demonstrated a locus for JP mapping to 18q21.1 (ref. 3) and germline mutations in the homolog of the gene for mothers against decapentaplegic, Drosophila, (MADH4, also known as SMAD4) in several JP families. However, mutations in MADH4 are only present in a subset of JP cases, and although mutations in the gene for phosphatase and tensin homolog (PTEN) have been described in a few families, undefined genetic heterogeneity remains. Using a genome-wide screen in four JP kindreds without germline mutations in MADH4 or PTEN, we identified linkage with markers from chromosome 10q22–23 (maximum lod score of 4.74, θ=0.00). We found no recombinants using markers developed from the vicinity of the gene for bone morphogenetic protein receptor 1A (BMPR1A), a serine–threonine kinase type I receptor involved in bone morphogenetic protein (BMP) signaling. Genomic sequencing of BMPR1A in each of these JP kindreds disclosed germline nonsense mutations in all affected kindred members but not in normal control individuals. These findings indicate involvement of an additional gene in the transforming growth factor-β (TGF-β) superfamily in the genesis of JP, and document an unanticipated function for BMP in colonic epithelial growth control.

Dystroglycan Function Requires Xylosyl- and Glucuronyltransferase Activities of LARGE

Going LARGE Dystroglycan (DG) is a highly glycosylated extracellular matrix (ECM) receptor involved in a variety of physiological processes, including maintenance of skeletal muscle membrane integrity and the structure and function of the central nervous system. The like-acetylglucosaminyltransferase (LARGE) is responsible for posttranslational modifications of alpha-dystroglycan (α-DG) required for its function. Now, Inamori et al. (p. 93) demonstrate that LARGE is a bifunctional glycosyltransferase able to transfer xylose and glucuronic acid. These modifications allow α-DG to bind the laminin-G domain–containing ECM ligands: laminin, agrin, and neurexin. A bifunctional enzyme adds a heteropolysaccharide to an extracellular matrix receptor, enabling it to bind laminin. Posttranslational modification of alpha-dystroglycan (α-DG) by the like-acetylglucosaminyltransferase (LARGE) is required for it to function as an extracellular matrix (ECM) receptor. Mutations in the LARGE gene have been identified in congenital muscular dystrophy patients with brain abnormalities. However, the precise function of LARGE remains unclear. Here we found that LARGE could act as a bifunctional glycosyltransferase, with both xylosyltransferase and glucuronyltransferase activities, which produced repeating units of [–3-xylose–α1,3-glucuronic acid-β1–]. This modification allowed α-DG to bind laminin-G domain–containing ECM ligands.

Germline SMAD4 or BMPR1A mutations and phenotype of juvenile polyposis

BackgroundJuvenile polyposis (JP) is an inherited condition predisposing to upper gastrointestinal (UGI) polyps and colorectal cancer. Two genes are known to predisose to JP,SMAD4 and bone morphogenetic protein receptor type 1A (BMPR1A). The object of this study was to determine the differences in phenotype of patients withSMAD4 orBMPR1A mutations (MUT+) compared with those without (MUT-).MethodsDNA was extracted from 54 JP probands and used for polymerase chain reaction of all exons ofSMAD4 andBMPRIA. Products were then sequenced and analyzed for mutations. Medical record data were used to create a JP database, and statistical analysis was performed using Fishers exact and unpairedt-tests.ResultsNine of 54 patients had germlineSMAD4 mutations, 13 hadBMPR1A mutations, and 32 had neither. There were no significant differences betweenSMAD4+ andBMPR1A+ cases in terms of clinical factors examined, except for a family history of UGI involvement (P<.01). There was a higher prevalence of familial cases in MUT+ patients (P=.09),>10 lower gastrointestinal polyps (P=.06), and frequency of family history of gastrointestinal cancer compared with MUT-patients (P=.01).ConclusionsPatients with germlineSMAD4 orBMPR1A mutations have a more prominent JP phenotype than those without, andSMAD4 mutations predispose to UGI polyposis.

Immune Response During Therapy With Cisplatin or Radiation for Human Papillomavirus–Related Head and Neck Cancer

BACKGROUND Human papillomavirus (HPV) is the most identifiable cause of head and neck squamous cell cancer (HNSCC). Compared with HPV-negative HNSCC, HPV-positive HNSCC presents at an advanced stage but with significantly better survival. We created a syngeneic mouse model of HPV-positive and HPV-negative HNSCC by transforming mouse primary tonsil epithelial cells with either HPV oncogenes or a nonantigenic RNA interference strategy that affects similar oncogenic pathways. OBJECTIVES To examine the effect of radiation therapy on HPV-positive and HPV-negative tumors in immune-competent and immune-incompetent mice and to examine responses in human cancer cell lines. DESIGN Prospective in vivo murine model. MAIN OUTCOME MEASURES Survival and tumor growth. RESULTS For human and murine transformed cell lines, HPV-positive cells were more resistant to radiation and cisplatin therapy compared with HPV-negative cells. In vivo, HPV-positive tumors were more sensitive to radiation, with complete clearance at 20 Gy, compared with their HPV-negative counterparts, which showed persistent growth. Cisplatin in vivo cleared HPV-positive tumors but not HPV-negative tumors. However, neither radiation or cisplatin therapy cured immune-incompetent mice. Adoptive transfer of wild-type immune cells into immune-incompetent mice restored HPV-positive tumor clearance with cisplatin therapy. CONCLUSIONS The HPV-positive tumors are not more curable based on increased epithelial sensitivity to cisplatin or radiation therapy. Instead, radiation and cisplatin induce an immune response to this antigenic cancer. The implications of these results may lead to novel therapies that enhance tumor eradication for HPV-positive cancers.

SGK196 Is a Glycosylation-Specific O-Mannose Kinase Required for Dystroglycan Function

Dissecting Dystrophies Defects in α-dystroglycan lead to various congenital muscular dystrophies, and its ability to bind to extracellular matrix (ECM) is dependent on formation of a specific O-linked sugar structure. Previous efforts to understand the molecular mechanisms underlying α-dystroglycans ability to bind to the ECM led to the identification of a phosphorylated O-mannosyl trisaccharide on α-dystroglycan and to the demonstration that addition of this residue is a prerequisite for formation of the ligand-binding motif. However, the biosynthetic pathway that leads to production of the phosphorylated O-mannosyl glycan has not been delineated. Yoshida-Moriguchi et al. (p. 896, published online 8 August) elucidate the functions of three genes recently found to cause dystroglycan-related disorders and explain the defects in the production of the phosphorylated O-mannosyl glycan that underlie the pathologies of patients with the relevant mutations. An atypical kinase genetically associated with muscular dystrophies recognizes a unique trisaccharide structure. Phosphorylated O-mannosyl trisaccharide [N-acetylgalactosamine–β3-N-acetylglucosamine–β4-(phosphate-6-)mannose] is required for dystroglycan to bind laminin-G domain–containing extracellular proteins with high affinity in muscle and brain. However, the enzymes that produce this structure have not been fully elucidated. We found that glycosyltransferase-like domain–containing 2 (GTDC2) is a protein O-linked mannose β 1,4-N-acetylglucosaminyltransferase whose product could be extended by β 1,3-N-acetylgalactosaminyltransferase2 (B3GALNT2) to form the O-mannosyl trisaccharide. Furthermore, we identified SGK196 as an atypical kinase that phosphorylated the 6-position of O-mannose, specifically after the mannose had been modified by both GTDC2 and B3GALNT2. These findings suggest how mutations in GTDC2, B3GALNT2, and SGK196 disrupt dystroglycan receptor function and lead to congenital muscular dystrophy.

The PDZ binding motif of human papillomavirus type 16 E6 induces PTPN13 loss, which allows anchorage-independent growth and synergizes with ras for invasive growth.

ABSTRACT The human papillomavirus (HPV) oncogene E6 has been shown to perform multiple functions (p53 degradation, telomerase activation, etc.) that play a role in oncogenic transformation. Beyond known E6 functions, an undefined mechanism that allows cellular invasion requires the E6 PDZ binding motif (PDZBM). Here, we show that HPV type 16 (HPV16) E6 interacts with and induces loss of a protein tyrosine phosphatase (PTPN13) in a PDZBM-dependent manner. PTPN13 loss induced either by the presence of E6 or by a short hairpin RNA strategy allows for anchorage-independent growth (AIG) and synergy with a known oncogene, Rasv12, resulting in invasive growth in vivo. Restoring PTPN13 expression reverses AIG in cells lacking PTPN13. A genomic analysis of colorectal carcinoma has identified an association between PTPN13 loss-of-function mutations and aberrant Ras signaling. Our findings support this correlation and provide methods for further evaluation of the mechanisms by which PTPN13 loss/Ras expression leads to invasive growth, the results of which will be important for treatment of HPV-related and non-HPV cancer.

The L4 22-Kilodalton Protein Plays a Role in Packaging of the Adenovirus Genome

ABSTRACT Packaging of the adenovirus (Ad) genome into a capsid is absolutely dependent upon the presence of a cis-acting region located at the left end of the genome referred to as the packaging domain. The functionally significant sequences within this domain consist of at least seven similar repeats, referred to as the A repeats, which have the consensus sequence 5′ TTTG-N8-CG 3′. In vitro and in vivo binding studies have demonstrated that the adenovirus protein IVa2 binds to the CG motif of the packaging sequences. In conjunction with IVa2, another virus-specific protein binds to the TTTG motifs in vitro. The efficient formation of these protein-DNA complexes in vitro was precisely correlated with efficient packaging activity in vivo. We demonstrate that the binding activity to the TTTG packaging sequence motif is the product of the L4 22-kDa open reading frame. Previously, no function had been ascribed to this protein. Truncation of the L4 22-kDa protein in the context of the viral genome did not reduce viral gene expression or viral DNA replication but eliminated the production of infectious virus. We suggest that the L4 22-kDa protein, in conjunction with IVa2, plays a critical role in the recognition of the packaging domain of the Ad genome that leads to viral DNA encapsidation. The L4 22-kDa protein is also involved in recognition of transcription elements of the Ad major late promoter.

PRECLINICAL MODELS OF HPV+ AND HPV― HNSCC IN MICE: AN IMMUNE CLEARANCE OF HPV+ HNSCC

To investigate whether human papilloma virus (HPV)‐specific immune mechanisms can result in tumor clearance, we have created HPV+ and HPV− tonsil cells that form squamous cancers in immune‐competent mice. Here, we determine that an immune‐specific response can clear HPV+ tumor cells and the cellular requirements to mediate this tumor clearance.

Impaired PTPN13 phosphatase activity in spontaneous or HPV-induced squamous cell carcinomas potentiates oncogene signaling through the MAP kinase pathway

Human papillomaviruses (HPVs) are a causative factor in over 90% of cervical and 25% of head and neck squamous cell carcinomas (HNSCCs). The C terminus of the high-risk HPV 16 E6 oncoprotein physically associates with and degrades a non-receptor protein tyrosine phosphatase (PTPN13), and PTPN13 loss synergizes with H-RasV12 or ErbB2 for invasive growth in vivo. Oral keratinocytes that have lost PTPN13 and express H-RasV12 or ErbB2 show enhanced Ras/RAF/MEK/Erk signaling. In co-transfection studies, wild-type PTPN13 inhibited Ras/RAF/MEK/Erk signaling in HEK 293 cells that overexpress ErbB2, EGFR or H-RasV12, whereas an enzymatically inactive PTPN13 did not. Twenty percent of HPV-negative HNSCCs had PTPN13 phosphatase mutations that did not inhibit Ras/RAF/MEK/Erk signaling. Inhibition of Ras/RAF/MEK/Erk signaling using MEK inhibitor U0126 blocked anchorage-independent growth in cells lacking PTPN13. These findings show that PTPN13 phosphatase activity has a physiologically significant role in regulating MAP kinase signaling.

Deletion of the PDZ motif of HPV16 E6 preventing immortalization and anchorage-independent growth in human tonsil epithelial cells

Human papillomavirus 16 (HPV16) has been associated with head and neck squamous cell carcinoma (HNSCC) in up to 60% of sampled specimens.