Search

Supplementary Information, Methods, Table 1, Figures 1-7 from 3-Phosphoinositide–Dependent Kinase 1 Potentiates Upstream Lesions on the Phosphatidylinositol 3-Kinase Pathway in Breast Carcinoma

Lesions of ERBB2, PTEN, and PIK3CA activate the phosphatidylinositol 3-kinase (PI3K) pathway during cancer development by increasing levels of phosphatidylinositol-3,4,5-triphosphate (PIP3). 3-Phosphoinositide-dependent kinase 1 (PDK1) is the first node of the PI3K signal output and is required for activation of AKT. PIP3 recruits PDK1 and AKT to the cell membrane through interactions with their pleckstrin homology domains, allowing PDK1 to activate AKT by phosphorylating it at residue threonine-308. We show that total PDK1 protein and mRNA were overexpressed in a majority of human breast cancers and that 21% of tumors had five or more copies of the gene encoding PDK1, PDPK1. We found that increased PDPK1 copy number was associated with upstream pathway lesions (ERBB2 amplification, PTEN loss, or PIK3CA mutation), as well as patient survival. Examination of an independent set of breast cancers and tumor cell lines derived from multiple forms of human cancers also found increased PDK1 protein levels associated with such upstream pathway lesions. In human mammary cells, PDK1 enhanced the ability of upstream lesions to signal to AKT, stimulate cell growth and migration, and rendered cells more resistant to PDK1 and PI3K inhibition. After orthotopic transplantation, PDK1 overexpression was not oncogenic but dramatically enhanced the ability of ERBB2 to form tumors. Our studies argue that PDK1 overexpression and increased PDPK1 copy number are common occurrences in cancer that potentiate the oncogenic effect of upstream lesions on the PI3K pathway. Therefore, we conclude that alteration of PDK1 is a critical component of oncogenic PI3K signaling in breast cancer. [Cancer Res 2009;69(15):6299–306]

Bladder cancer is the fifth most common human malignancy and the second most frequently diagnosed genitourinary tumor after prostate cancer. The majority of malignant tumors arising in the urinary bladder are urothelial carcinomas. Clinically, superficial bladder tumors (stages Ta and Tis) account for 75% to 85% of neoplasms, while the remaining 15% to 25% are invasive (T1, T2–T4) or metastatic lesions at the time of initial presentation. Several studies have revealed that distinct genotypic and phenotypic patterns are associated with early vs. late stages of bladder cancer. Early superficial disease appears to segregate into 2 main pathways: (1) superficial papillary bladder tumors, which are characterized by gain-of-function mutations affecting oncogenes such as H-RAS, FGFR3, and PI3K, and deletions of the long arm of chromosome 9 (9q); (2) Carcinoma in situ, a “flat” high grade lesion considered to be a precursor of invasive cancer, is characterized by loss-of-function mutations affecting tumor suppressor genes, such as p53, RB, and PTEN. Based on these data, a model for bladder tumor progression has been proposed in which 2 separate genetic pathways characterize the evolution of early bladder neoplasms. Several molecular markers have been correlated with tumor stage, but the rationale for these 2 well-defined genetic pathways still remains unclear. Normal urothelium is a pseudo-stratified epithelium that coats the bladder, composed of 3 cell types: basal, intermediate, and superficial (“umbrella”) cells. We have identified a series of markers that are differently expressed in these distinct cells types, and postulated a novel model for urothelium development and configuration. Briefly, it is our working hypothesis that 2 distinct progenitor cells are responsible for basal/intermediate cells and “umbrella” cells, respectively. Basal and intermediate cells are characterized by a p63 positive phenotype, as well as expression of high molecular weight cytokeratins (CKs), such as CK5, CK10, and CK14. On the contrary, “umbrella” cells display a p63 negative phenotype and are characterized by expression of 2 specific low molecular weight CKs: CK18 and CK20. Neither urothelial stem cells nor bladder cancer stem cells have been identified to date. In this review, we will further expand on the issues discussed above.

To expand the available tools for investigating human sarcomas, we characterized the primary properties of 22 common, uncommon, and newly characterized sarcoma cell lines representing eight different histological subtypes. Throughout the characterization process we noticed that in vitro markers and assays are poor indicators of tumorigenicity and that generated xenografts often bear little resemblance to the original histopathology. In vitro properties examined included morphology, proliferation rate, cell cycle characteristics, invasiveness, and immunohistochemical expression of p53 and phospho-AKT. In vivo properties examined included days to tumor formation in NOD/SCID mice, xenograft morphology in several locations and immunohistochemical expression of Ki67, p53 and phospho-AKT. We believe that such an in depth comparison of a large cohort of sarcoma cell lines will be useful in both designing and interpreting experiments aimed at elucidating both the molecular biology and efficacy of therapeutic agents in sarcomas. However, that data generated also suggests a small set of sarcoma cell lines may be inappropriate for generalizations regarding biological behavior of specific sarcoma subtypes. Integration of functional genomics or other more sophisticated assays of cell lines may help bridge the differences in vitro and in vivo.

Lesions of ERBB2, PTEN, and PIK3CA activate the phosphatidylinositol 3-kinase (PI3K) pathway during cancer development by increasing levels of phosphatidylinositol-3,4,5-triphosphate (PIP3). 3-Phosphoinositide-dependent kinase 1 (PDK1) is the first node of the PI3K signal output and is required for activation of AKT. PIP3 recruits PDK1 and AKT to the cell membrane through interactions with their pleckstrin homology domains, allowing PDK1 to activate AKT by phosphorylating it at residue threonine-308. We show that total PDK1 protein and mRNA were overexpressed in a majority of human breast cancers and that 21% of tumors had five or more copies of the gene encoding PDK1, PDPK1. We found that increased PDPK1 copy number was associated with upstream pathway lesions (ERBB2 amplification, PTEN loss, or PIK3CA mutation), as well as patient survival. Examination of an independent set of breast cancers and tumor cell lines derived from multiple forms of human cancers also found increased PDK1 protein levels associated with such upstream pathway lesions. In human mammary cells, PDK1 enhanced the ability of upstream lesions to signal to AKT, stimulate cell growth and migration, and rendered cells more resistant to PDK1 and PI3K inhibition. After orthotopic transplantation, PDK1 overexpression was not oncogenic but dramatically enhanced the ability of ERBB2 to form tumors. Our studies argue that PDK1 overexpression and increased PDPK1 copy number are common occurrences in cancer that potentiate the oncogenic effect of upstream lesions on the PI3K pathway. Therefore, we conclude that alteration of PDK1 is a critical component of oncogenic PI3K signaling in breast cancer. [Cancer Res 2009;69(15):6299–306]

TREK-1 is a two-pore domain (K2P) potassium channel that carries a leak current that is time- and voltage-independent. Recently, potassium channels have been related to cell proliferation and some K2P family channels, such as TASK-3, have been shown to be overexpressed in specific neoplasms. In this study, we addressed the expression of TREK-1 in prostatic tissues and cell lines, and we have found that this potassium channel is highly expressed in prostate cancer but is not expressed in normal prostate nor in benign prostatic hyperplasia. Furthermore, expression of TREK-1 correlates strongly with the grade and the stage of the disease, suggesting a causal link between channel expression and abnormal cell proliferation. In vitro studies showed that TREK-1 is highly expressed in PC3 and LNCaP prostate cancer cell lines but is not detectable in normal prostate epithelial cells (NPE). In this report, we show that overexpression of TREK-1 in NPE and Chinese hamster ovary (CHO) cells leads to a significant increase in proliferation. Moreover, the increased cell proliferation rate of PC3 cells and TREK-1 overexpressing CHO cells could be reduced when TREK-1 current was reduced by overexpression of a dominant-negative TREK-1 mutant or when cells were exposed to a TREK-1 inhibitor. Taken together, these data suggest that TREK-1 expression is associated with abnormal cell proliferation and may be a novel marker for and a molecular target in prostate cancer. [Cancer Res 2008;68(4):1197–203]

Malignant fibrous histiocytoma (MFH), now termed high-grade undifferentiated pleomorphic sarcoma, is a commonly diagnosed mesenchymal tumor, yet both the underlying molecular mechanisms of tumorigenesis and cell of origin remain unidentified. We present evidence demonstrating that human mesenchymal stem cells (hMSCs) are the progenitors of MFH. DKK1, a Wnt inhibitor and mediator of hMSC proliferation, is overexpressed in MFH. Using recombinant proteins, antibody depletion, and siRNA knockdown strategies of specific Wnt elements, we show that DKK1 inhibits hMSC commitment to differentiation via Wnt2/beta-catenin canonical signaling and that Wnt5a/JNK noncanonical signaling regulates a viability checkpoint independent of Dkk1. Finally, we illustrate that hMSCs can be transformed via inhibition of Wnt signaling to form MFH-like tumors in nude mice, and conversely, MFH cells in which Wnt signaling is appropriately reestablished can differentiate along mature connective tissue lineages. Our results provide mechanistic insights regarding the cell of origin of MFH, establish what we believe is a novel tumor suppressor role for Wnt signaling, and identify a potential therapeutic differentiation strategy for sarcomas.

Urinary podocyte (podocyturia) has been studied as a diagnostic marker for preeclampsia. We sought to validate its use in preeclampsia and in differentiating it from other high risk pregnancy states. We studied an obstetric population at high risk to develop preeclampsia (study group) and uncomplicated pregnancies (control group) by analyzing their urine sediment for podocytes within 24 hours of delivery. Podocytes were identified by immunohistochemistry using the podocyte-specific protein synaptopodin. Of the 56 patients who were enrolled, 29 patients were diagnosed with preeclampsia, 9 patients had hypertensive conditions such as chronic and gestational hypertension, 6 patients had Type I/II and gestational diabetes mellitus, 3 patients were classified as others, and 9 patients exhibited uncomplicated pregnancies. Podocyturia was identified in 11 out of 29 (38%) of patients with preeclampsia/eclampsia, 3 out of 9 (33%) with gestational and chronic hypertension, and 3 out of 6 (50%) with Type I/II and gestational diabetes mellitus. None of the 9 patients (0%) with uncomplicated pregnancies demonstrated podocyturia. The sensitivity and specificity of podocyturia for preeclampsia were found to be 38% and 70%. Our study showed that podocyturia does not appear to be a sensitive nor a specific marker to diagnose preeclampsia.

Although bladder cancer represents a serious health problem worldwide, relevant mouse models for investigating disease progression or therapeutic targets have been lacking. We show that combined deletion of p53 and Pten in bladder epithelium leads to invasive cancer in a novel mouse model. Inactivation of p53 and PTEN promotes tumorigenesis in human bladder cells and is correlated with poor survival in human tumors. Furthermore, the synergistic effects of p53 and Pten deletion are mediated by deregulation of mammalian target of rapamycin (mTOR) signaling, consistent with the ability of rapamycin to block bladder tumorigenesis in preclinical studies. Our integrated analyses of mouse and human bladder cancer provide a rationale for investigating mTOR inhibition for treatment of patients with invasive disease.

Summary The Mre11 complex (Mre11, Rad50, and Nbs1) and Chk2 have been implicated in the DNA-damage response, an inducible process required for the suppression of malignancy. The Mre11 complex is predominantly required for repair and checkpoint activation in S phase, whereas Chk2 governs apoptosis. We examined the relationship between the Mre11 complex and Chk2 in the DNA-damage response via the establishment of Nbs1 ΔB/ΔB Chk2 −/− and Mre11 ATLD1/ATLD1 Chk2 −/− mice. Chk2 deficiency did not modify the checkpoint defects or chromosomal instability of Mre11 complex mutants; however, the double-mutant mice exhibited synergistic defects in DNA-damage-induced p53 regulation and apoptosis. Nbs1 ΔB/ΔB Chk2 −/− and Mre11 ATLD1/ATLD1 Chk2 −/− mice were also predisposed to tumors. In contrast, DNA-PKcs-deficient mice, in which G1-specific chromosome breaks are present, did not exhibit synergy with Chk2 −/− mutants. These data suggest that Chk2 suppresses the oncogenic potential of DNA damage arising during S and G2 phases of the cell cycle.

The tumor suppressor p53 is activated in response to many forms of cellular stress leading to cell cycle arrest, senescence or apoptosis. Appropriate sub-cellular localization is essential for modulating p53 function. We recently showed that p53 localizes to the nucleolus after proteasome inhibition with MG132 and this localization requires sequences within its carboxyl terminus. In the present study, we found that after treatment with MG132, p53 associates with a discrete sub-nucleolar component, the fibrillar center (FC), a region mainly enriched with RNA polymerase I. Moreover, we now demonstrate that this localization is an energy-dependent process as reduction of ATP levels prevents nucleolar localization. In addition, p53 sub-nucleolar accumulation is abolished when cells are subjected to various types of genotoxic stress. Furthermore, we show that monoubiquitination of p53, which causes it to localize to the cytoplasm and nucleoplasm, does not prevent the association of p53 with the nucleolus after MG132 treatment. Importantly, we demonstrate that p53 nucleolar association occurs in lung and bladder carcinomas.

The importance of adult stem cells in the development of neoplastic diseases is becoming increasingly well appreciated. We hypothesized that sarcomas of soft tissue could be categorized by their developmental/differentiation status from stem cell to mature tissue, similar to the hematological malignancies. We conducted gene expression analyses during in vitro differentiation of human mesenchymal stem cells into adipose tissue, as a representative mature connective tissue, and identified genes whose expression changed significantly during adipogenesis. Gene clustering and distance correlation analysis allowed the assignment of a unique time point during adipogenesis that strongly correlates to each of the four major liposarcoma subtypes. Using a novel gene expression strategy, in which liposarcomas are compared to their corresponding adipocytic maturing cells, we identified a group of genes overexpressed in liposarcomas that indicate the stage of differentiation arrest, ie, sharing a similar expression profile to adipocytic cells at a corresponding stage of differentiation, and a distinct set of genes overexpressed in liposarcomas that are not found in the corresponding stage of differentiation. We propose that the latter set is enriched for candidate transformation-associated genes. Our results indicate that a degree of developmental maturity can be quantitatively assigned to solid tumors, supporting the notion that transformation of a solid tumor stem cell can occur at distinct stages of maturation.