Kenneth L. Scott

SMAD4-dependent barrier constrains prostate cancer growth and metastatic progression

Effective clinical management of prostate cancer (PCA) has been challenged by significant intratumoural heterogeneity on the genomic and pathological levels and limited understanding of the genetic elements governing disease progression. Here, we exploited the experimental merits of the mouse to test the hypothesis that pathways constraining progression might be activated in indolent Pten-null mouse prostate tumours and that inactivation of such progression barriers in mice would engender a metastasis-prone condition. Comparative transcriptomic and canonical pathway analyses, followed by biochemical confirmation, of normal prostate epithelium versus poorly progressive Pten-null prostate cancers revealed robust activation of the TGFβ/BMP–SMAD4 signalling axis. The functional relevance of SMAD4 was further supported by emergence of invasive, metastatic and lethal prostate cancers with 100% penetrance upon genetic deletion of Smad4 in the Pten-null mouse prostate. Pathological and molecular analysis as well as transcriptomic knowledge-based pathway profiling of emerging tumours identified cell proliferation and invasion as two cardinal tumour biological features in the metastatic Smad4/Pten-null PCA model. Follow-on pathological and functional assessment confirmed cyclin D1 and SPP1 as key mediators of these biological processes, which together with PTEN and SMAD4, form a four-gene signature that is prognostic of prostate-specific antigen (PSA) biochemical recurrence and lethal metastasis in human PCA. This model-informed progression analysis, together with genetic, functional and translational studies, establishes SMAD4 as a key regulator of PCA progression in mice and humans.

GOLPH3 modulates mTOR signalling and rapamycin sensitivity in cancer

Genome-wide copy number analyses of human cancers identified a frequent 5p13 amplification in several solid tumour types, including lung (56%), ovarian (38%), breast (32%), prostate (37%) and melanoma (32%). Here, using integrative analysis of a genomic profile of the region, we identify a Golgi protein, GOLPH3, as a candidate targeted for amplification. Gain- and loss-of-function studies in vitro and in vivo validated GOLPH3 as a potent oncogene. Physically, GOLPH3 localizes to the trans-Golgi network and interacts with components of the retromer complex, which in yeast has been linked to target of rapamycin (TOR) signalling. Mechanistically, GOLPH3 regulates cell size, enhances growth-factor-induced mTOR (also known as FRAP1) signalling in human cancer cells, and alters the response to an mTOR inhibitor in vivo. Thus, genomic and genetic, biological, functional and biochemical data in yeast and humans establishes GOLPH3 as a new oncogene that is commonly targeted for amplification in human cancer, and is capable of modulating the response to rapamycin, a cancer drug in clinical use.

Intronic miR-211 Assumes the Tumor Suppressive Function of Its Host Gene in Melanoma

When it escapes early detection, malignant melanoma becomes a highly lethal and treatment-refractory cancer. Melastatin is greatly downregulated in metastatic melanomas and is widely believed to function as a melanoma tumor suppressor. Here we report that tumor suppressive activity is not mediated by melastatin but instead by a microRNA (miR-211) hosted within an intron of melastatin. Increasing expression of miR-211 but not melastatin reduced migration and invasion of malignant and highly invasive human melanomas characterized by low levels of melastatin and miR-211. An unbiased network analysis of melanoma-expressed genes filtered for their roles in metastasis identified three central node genes: IGF2R, TGFBR2, and NFAT5. Expression of these genes was reduced by miR-211, and knockdown of each gene phenocopied the effects of increased miR-211 on melanoma invasiveness. These data implicate miR-211 as a suppressor of melanoma invasion whose expression is silenced or selected against via suppression of the entire melastatin locus during human melanoma progression.

mTORC1-dependent and -independent regulation of stem cell renewal, differentiation, and mobilization

The Tuberous Sclerosis Complex component, TSC1, functions as a tumor suppressor via its regulation of diverse cellular processes, particularly cell growth. TSC1 exists in a complex with TSC2 and functions primarily as a key negative regulator of mammalian target of rapamycin complex 1 (mTORC1) signaling and protein synthesis, although the TSC1/TSC2 complex also shows mTORC1-independent outputs to other pathways. Here, we explored the role of TSC1 in various aspects of stem cell biology and dissected the extent to which TSC1 functions are executed via mTORC1-dependent versus mTORC1-independent pathways. Using hematopoietic stem cells (HSCs) as a model system, we demonstrate that somatic deletion of TSC1 produces striking stem cell and derivative effector cell phenotypes characterized by increased HSC cell cycling, mobilization, marked progressive depletion, defective long-term repopulating potential, and hematopoietic lineage developmental aberrations. On the mechanistic level, we further establish that TSC1 regulation of HSC quiescence and long-term repopulating potential and hematopoietic lineage development is mediated through mTORC1 signaling. In contrast, TSC1 regulation of HSC mobilization is effected in an mTORC1-independent manner, and gene profiling and functional analyses reveals the actin-bundling protein FSCN1 as a key TSC1/TSC2 target in the regulation of HSC mobilization. Thus, TSC1 is a critical regulator of HSC self-renewal, mobilization, and multilineage development and executes these actions via both mTORC1-dependent and -independent pathways.

The Genomic Landscape and Clinical Relevance of A-to-I RNA Editing in Human Cancers

Adenosine-to-inosine (A-to-I) RNA editing is a widespread post-transcriptional mechanism, but its genomic landscape and clinical relevance in cancer have not been investigated systematically. We characterized the global A-to-I RNA editing profiles of 6,236 patient samples of 17 cancer types from The Cancer Genome Atlas and revealed a striking diversity of altered RNA-editing patterns in tumors relative to normal tissues. We identified an appreciable number of clinically relevant editing events, many of which are in noncoding regions. We experimentally demonstrated the effects of several cross-tumor nonsynonymous RNA editing events on cell viability and provide the evidence that RNA editing could selectively affect drug sensitivity. These results highlight RNA editing as an exciting theme for investigating cancer mechanisms, biomarkers, and treatments.

Mig-6 controls EGFR trafficking and suppresses gliomagenesis

Glioblastoma multiforme (GBM) is the most common and lethal primary brain cancer that is driven by aberrant signaling of growth factor receptors, particularly the epidermal growth factor receptor (EGFR). EGFR signaling is tightly regulated by receptor endocytosis and lysosome-mediated degradation, although the molecular mechanisms governing such regulation, particularly in the context of cancer, remain poorly delineated. Here, high-resolution genomic profiles of GBM identified a highly recurrent focal 1p36 deletion encompassing the putative tumor suppressor gene, Mig-6. We show that Mig-6 quells the malignant potential of GBM cells and dampens EGFR signaling by driving EGFR into late endosomes and lysosome-mediated degradation upon ligand stimulation. Mechanistically, this effect is mediated by the binding of Mig-6 to a SNARE protein STX8, a protein known to be required for late endosome trafficking. Thus, Mig-6 functions to ensure recruitment of internalized receptor to late endosomes and subsequently the lysosomal degradation compartment through its ability to specifically link EGFR and STX8 during ligand-stimulated EGFR trafficking. In GBM, the highly frequent loss of Mig-6 would therefore serve to sustain aberrant EGFR-mediated oncogenic signaling. Together, these data uncover a unique tumor suppression mechanism involving the regulation of receptor trafficking.

Multiple Endocrine Neoplasia Type 1 Interacts with Forkhead Transcription Factor CHES1 in DNA Damage Response

Multiple endocrine neoplasia type 1 (MEN1) is a cancer susceptibility syndrome affecting several endocrine tissues. Investigations of the biochemical function of the MEN1 protein, menin, have suggested a role as a transcriptional comodulator. The mechanism by which MEN1 inactivation leads to tumor formation is not fully understood. MEN1 was implicated to function in both regulation of cell proliferation and maintenance of genomic integrity. Here, we investigate the mechanism by which MEN1 affects DNA damage response. We found that Drosophila larval tissue and mouse embryonic fibroblasts mutant for the MEN1 homologue were deficient for a DNA damage-activated S-phase checkpoint. The forkhead transcription factor CHES1 (FOXN3) was identified as an interacting protein by a genetic screen, and overexpression of CHES1 restored both cell cycle arrest and viability of MEN1 mutant flies after ionizing radiation exposure. We showed a biochemical interaction between human menin and CHES1 and showed that the COOH terminus of menin, which is frequently mutated in MEN1 patients, is necessary for this interaction. Our data indicate that menin is involved in the activation of S-phase arrest in response to ionizing radiation. CHES1 is a component of a transcriptional repressor complex, that includes mSin3a, histone deacetylase (HDAC) 1, and HDAC2, and it interacts with menin in an S-phase checkpoint pathway related to DNA damage response.

Signaling from the Golgi: mechanisms and models for Golgi phosphoprotein 3-mediated oncogenesis.

Golgi phosphoprotein 3 (GOLPH3; also known as GPP34/GMx33/MIDAS) represents an exciting new class of oncoproteins involved in vesicular trafficking. Encoded by a gene residing on human chromosome 5p13, which is frequently amplified in multiple solid tumor types, GOLPH3 was initially discovered as a phosphorylated protein localized to the Golgi apparatus. Recent functional, cell biological, and biochemical analyses show that GOLPH3 can function as an oncoprotein to promote cell transformation and tumor growth by enhancing activity of the mammalian target of rapamycin, a serine/threonine protein kinase known to regulate cell growth, proliferation, and survival. Although its precise mode of action in cancer remains to be elucidated, the fact that GOLPH3 has been implicated in protein trafficking, receptor recycling, and glycosylation points to potential links of these cellular processes to tumorigenesis. Understanding how these processes may be deregulated and contribute to cancer pathogenesis and drug response will uncover new avenues for therapeutic intervention. Clin Cancer Res; 16(8); 2229–34. ©2010 AACR.

Loss of Sin3/Rpd3 Histone Deacetylase Restores the DNA Damage Response in Checkpoint-Deficient Strains of Saccharomyces cerevisiae

ABSTRACT We previously reported that expression of the human forkhead/winged helix transcription factor, CHES1 (checkpoint suppressor 1; FOXN3), suppresses sensitivity to DNA damage and restores damage-induced G2/M arrest in checkpoint-deficient strains of Saccharomyces cerevisiae. We find that a functional glutathione S-transferase-Ches1 fusion protein binds in vivo to Sin3, a component of the S. cerevisiae Sin3/Rpd3 histone deacetylase complex. Checkpoint mutant strains with SIN3 deleted show increased resistance to UV irradiation, which is not further enhanced by CHES1 expression. Conversely, overexpression of SIN3 blocks the Ches1-mediated G2/M delay in response to DNA damage, which is consistent with Ches1 acting by inhibiting the Sin3/Rpd3 complex. Deletion of either SIN3 or RPD3 in rad9 or mec1 checkpoint mutant strains suppresses sensitivity to replication blocks and DNA damage resulting from Cdc9 ligase deficiency and UV irradiation. SIN3 or RPD3 deletions also restored G2/M arrest after DNA damage without concomitant Rad53 phosphorylation in mec1 mutant strains. This DNA damage response is absent in mad1 spindle checkpoint mutants. These data suggest that modulation of chromatin structure may regulate checkpoint responses in S. cerevisiae. Inhibition of histone deacetylation results in a DNA damage checkpoint response mediated by the spindle checkpoint pathway that compensates for loss of the primary DNA damage checkpoint pathway.

Lysyl hydroxylase 2 induces a collagen cross-link switch in tumor stroma

Epithelial tumor metastasis is preceded by an accumulation of collagen cross-links that heighten stromal stiffness and stimulate the invasive properties of tumor cells. However, the biochemical nature of collagen cross-links in cancer is still unclear. Here, we postulated that epithelial tumorigenesis is accompanied by changes in the biochemical type of collagen cross-links. Utilizing resected human lung cancer tissues and a p21CIP1/WAF1-deficient, K-rasG12D-expressing murine metastatic lung cancer model, we showed that, relative to normal lung tissues, tumor stroma contains higher levels of hydroxylysine aldehyde-derived collagen cross-links (HLCCs) and lower levels of lysine aldehyde-derived cross-links (LCCs), which are the predominant types of collagen cross-links in skeletal tissues and soft tissues, respectively. Gain- and loss-of-function studies in tumor cells showed that lysyl hydroxylase 2 (LH2), which hydroxylates telopeptidyl lysine residues on collagen, shifted the tumor stroma toward a high-HLCC, low-LCC state, increased tumor stiffness, and enhanced tumor cell invasion and metastasis. Together, our data indicate that LH2 enhances the metastatic properties of tumor cells and functions as a regulatory switch that controls the relative abundance of biochemically distinct types of collagen cross-links in the tumor stroma.