ABSTRACT
Urothelial carcinoma of the renal pelvis is a deadly disease with an unclear tumorigenic mechanism. We conducted gene expression profiling on a set of human tumors of this type and identified a phosphatidylinositol 3-kinase (PI3K)/AKT activation expression signature in 76.9% (n = 13) of our samples. Sequence analysis found both activating mutations of PIK3CA (13.6%, n = 22) and loss of heterozygosity at the PTEN locus (25%, n = 8). In contrast, none of the other subtypes of kidney neoplasms (e.g., clear-cell renal cell carcinoma) harbored PIK3CA mutations (n = 87; P < 0.001). Immunohistochemical analysis of urothelial carcinoma samples found loss of PTEN protein expression (36.4%, n = 11) and elevation of phosphorylated mammalian target of rapamycin (mTOR; 63.6%, n = 11). To confirm the role of the PI3K/AKT pathway in urothelial carcinoma, we generated mice containing biallelic inactivation of Pten in the urogenital epithelia. These mice developed typical renal pelvic urothelial carcinomas, with an incidence of 57.1% in mice older than 1 year. Laser capture microdissection followed by PCR confirmed the deletion of Pten exons 4 and 5 in the animal tumor cells. Immunohistochemical analyses showed increased phospho-mTOR and phospho-S6K levels in the animal tumors. Renal lymph node metastases were found in 15.8% of the animals with urothelial carcinoma. In conclusion, we identified and confirmed an important role for the PI3K/AKT pathway in the development of urothelial carcinoma and suggested that inhibitors of this pathway (e.g., mTOR inhibitor) may serve as effective therapeutic agents.
Subject(s)
Kidney Neoplasms/pathology , Kidney Pelvis/pathology , Phosphatidylinositol 3-Kinases/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Urinary Bladder Neoplasms/metabolism , Urinary Bladder Neoplasms/pathology , Animals , Biomarkers, Tumor/genetics , Biomarkers, Tumor/metabolism , Carcinoma, Papillary/genetics , Carcinoma, Papillary/metabolism , Carcinoma, Papillary/pathology , Carcinoma, Renal Cell/genetics , Carcinoma, Renal Cell/metabolism , Carcinoma, Renal Cell/pathology , Female , Gene Expression Profiling , Humans , Immunoenzyme Techniques , Integrases/metabolism , Kidney Neoplasms/genetics , Kidney Neoplasms/metabolism , Kidney Pelvis/metabolism , Lasers , Loss of Heterozygosity , Male , Mice , Mice, Transgenic , Microdissection , Oligonucleotide Array Sequence Analysis , PTEN Phosphohydrolase/physiology , Phosphatidylinositol 3-Kinases/genetics , Protein Kinases/genetics , Protein Kinases/metabolism , Proto-Oncogene Proteins c-akt/genetics , RNA, Messenger/genetics , RNA, Messenger/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Ribosomal Protein S6 Kinases, 70-kDa/genetics , Ribosomal Protein S6 Kinases, 70-kDa/metabolism , TOR Serine-Threonine Kinases , Urinary Bladder Neoplasms/geneticsABSTRACT
Intricate interactions between kinetochores and microtubules are essential for the proper distribution of chromosomes during mitosis. A crucial long-standing question is how vertebrate kinetochores generate chromosome motion while maintaining attachments to the dynamic plus ends of the multiple kinetochore MTs (kMTs) in a kinetochore fibre. Here, we demonstrate that individual kMTs in PtK(1) cells are attached to the kinetochore outer plate by several fibres that either embed the microtubule plus-end tips in a radial mesh, or extend out from the outer plate to bind microtubule walls. The extended fibres also interact with the walls of nearby microtubules that are not part of the kinetochore fibre. These structural data, in combination with other recent reports, support a network model of kMT attachment wherein the fibrous network in the unbound outer plate, including the Hec1-Ndc80 complex, dissociates and rearranges to form kMT attachments.
