Norman Sharpless - Academia.edu (original) (raw)

Papers by Norman Sharpless

Cancer Cell

Ink4a/Arf inactivation and epidermal growth factor receptor (EGFR) activation are signature lesio... more Ink4a/Arf inactivation and epidermal growth factor receptor (EGFR) activation are signature lesions in high-grade gliomas. How these mutations mediate the biological features of these tumors is poorly understood. Here, we demonstrate that combined loss of p16(INK4a) and p19(ARF), but not of p53, p16(INK4a), or p19(ARF), enables astrocyte dedifferentiation in response to EGFR activation. Moreover, transduction of Ink4a/Arf(-/-) neural stem cells (NSCs) or astrocytes with constitutively active EGFR induces a common high-grade glioma phenotype. These findings identify NSCs and astrocytes as equally permissive compartments for gliomagenesis and provide evidence that p16(INK4a) and p19(ARF) synergize to maintain terminal astrocyte differentiation. These data support the view that dysregulation of specific genetic pathways, rather than cell-of-origin, dictates the emergence and phenotype of high-grade gliomas.

PLOS ONE, 2015

The recent FDA approval of the MiSeqDx platform provides a unique opportunity to develop targeted... more The recent FDA approval of the MiSeqDx platform provides a unique opportunity to develop targeted next generation sequencing (NGS) panels for human disease, including cancer. We have developed a scalable, targeted panel-based assay termed UNCseq, which involves a NGS panel of over 200 cancer-associated genes and a standardized downstream bioinformatics pipeline for detection of single nucleotide variations (SNV) as well as small insertions and deletions (indel). In addition, we developed a novel algorithm, NGScopy, designed for samples with sparse sequencing coverage to detect large-scale copy number variations (CNV), similar to human SNP Array 6.0 as well as small-scale intragenic CNV. Overall, we applied this assay to 100 snap-frozen lung cancer specimens lacking same-patient germline DNA (07-0120 tissue cohort) and validated our results against Sanger sequencing, SNP Array, and our recently published integrated DNA-seq/RNA-seq assay, UNCqeR, where RNA-seq of same-patient tumor specimens confirmed SNV detected by DNA-seq, if RNA-seq coverage depth was adequate. In addition, we applied the UNCseq assay on an independent lung cancer tumor tissue collection with available same-patient germline DNA (11-1115 tissue cohort) and confirmed mutations using assays performed in a CLIA-certified laboratory. We conclude that UNCseq can identify SNV, indel, and CNV in tumor specimens lacking germline DNA in a cost-efficient fashion.

Medical oncology (Northwood, London, England), 2010

We report two cases of stage IV malignant melanoma arising in patients treated with azathioprine ... more We report two cases of stage IV malignant melanoma arising in patients treated with azathioprine for myasthenia gravis. In both cases, the melanoma metastases regressed upon withdrawal of immunosuppression. One patient remains melanoma free at 10 years, and the second patient experienced an 18-month disease free period. There is one prior case report in the medical literature to support full immune reconstitution for treatment in advanced immunosuppression-related melanoma, and one case series suggesting that transplant patients developing melanoma may benefit from a switch to sirolimus. Virtually, no data exist for the medical management of early stage melanoma in the immunosuppressed patients. We review the limited preclinical data in support of immune reconstitution and the data on immunosuppression as a risk factor for melanoma. We conclude that reduction or withdrawal of immunosuppression may be beneficial in patients with advanced stage melanoma and warrants further considerat...

Nature, Jan 6, 2001

The cyclin-dependent kinase inhibitor p16INK4a can induce senescence of human cells, and its loss... more The cyclin-dependent kinase inhibitor p16INK4a can induce senescence of human cells, and its loss by deletion, mutation or epigenetic silencing is among the most frequently observed molecular lesions in human cancer. Overlapping reading frames in the INK4A/ARF gene encode p16INK4a and a distinct tumour-suppressor protein, p19ARF (ref. 3). Here we describe the generation and characterization of a p16Ink4a-specific knockout mouse that retains normal p19Arf function. Mice lacking p16Ink4a were born with the expected mendelian distribution and exhibited normal development except for thymic hyperplasia. T cells deficient in p16Ink4a exhibited enhanced mitogenic responsiveness, consistent with the established role of p16Ink4a in constraining cellular proliferation. In contrast to mouse embryo fibroblasts (MEFs) deficient in p19Arf (ref. 4), p16Ink4a-null MEFs possessed normal growth characteristics and remained susceptible to Ras-induced senescence. Compared with wild-type MEFs, p16Ink4a-...

Cancer research, Jan 15, 2002

The combined impact of mutations in p16(INK4a) and p53 was examined in cellular growth,transforma... more The combined impact of mutations in p16(INK4a) and p53 was examined in cellular growth,transformation, and tumor formation. In cultured cells, p16(INK4a) loss enhanced growth at high density and conferred susceptibility to oncogene-induced transformation. In vivo, mice doubly deficient for p16(INK4a) and p53 showed an increased rate of tumor formation with particular susceptibility to aggressive angiosarcomas. Furthermore, p16(INK4a) silencing by promoter methylation was detected in tumors derived from p16(INK4a+/-) and (+/+) mice, independent of p53 status. These data suggest at least one general feature of malignancy, resistance to density-mediated growth arrest depends on p16(INK4a) rather than p53. This cooperation between p16(INK4a) and p53 loss in tumorigenesis is consistent with the view that these genes function in distinct anticancer pathways.

Molecular Cell, 2001

Although nonhomologous end-joining (NHEJ) deficiency has been shown to accelerate lymphoma format... more Although nonhomologous end-joining (NHEJ) deficiency has been shown to accelerate lymphoma formation in mice, its role in suppressing tumors in cells that do not undergo V(D)J recombination is unclear. Utilizing a tumor-prone mouse strain (ink4a/arf(-/-)), we examined the impact of haploinsufficiency of a NHEJ component, DNA ligase IV (Lig4), on murine tumorigenesis. We demonstrate that lig4 heterozygosity promotes the development of soft-tissue sarcomas that possess clonal amplifications, deletions, and translocations. That these genomic alterations are relevant in tumorigenesis is supported by the finding of frequent mdm2 amplification, a known oncogene in human sarcoma. Together, these findings support the view that loss of a single lig4 allele results in NHEJ activity being sufficiently reduced to engender chromosomal aberrations that drive non-lymphoid tumorigenesis.

Cell cycle progression from G1 to S phase depends on phosphorylation of pRb by complexes containi... more Cell cycle progression from G1 to S phase depends on phosphorylation of pRb by complexes containing a cyclin (D type or E type) and cyclin-dependent kinase (e.g., cdk2, cdk4, or cdk6). Ink4 proteins function to oppose the action of cdk4/ 6-cyclin D complexes by inhibiting cdk4/6. We employed genetic and pharmacologic approaches to study the interplay among Ink4 proteins and

Proceedings of the National Academy of Sciences, 2002

Telomerase is up-regulated in the vast majority of human cancers and serves to halt the progressi... more Telomerase is up-regulated in the vast majority of human cancers and serves to halt the progressive telomere shortening that ultimately blocks would-be cancer cells from achieving a full malignant phenotype. In contrast to humans, the laboratory mouse possesses long telomeres and, even in early generation telomerase-deficient mice, the level of telomere reserve is sufficient to avert telomere-based checkpoint responses and to permit full malignant progression. These features in the mouse provide an opportunity to determine whether enforced high-level telomerase activity can serve functions that extend beyond its ability to sustain telomere length and function. Here, we report the generation and characterization of transgenic mice that express the catalytic subunit of telomerase (mTERT) at high levels in a broad variety of tissues. Expression of mTERT conferred increased telomerase enzymatic activity in several tissues, including mammary gland, splenocytes, and cultured mouse embryonic fibroblasts. In mouse embryonic fibroblasts, mTERT overexpression extended telomere lengths but did not prevent culture-induced replicative arrest, thus reinforcing the view that this phenomenon is not related to occult telomere shortening. Robust telomerase activity, however, was associated with the spontaneous development of mammary intraepithelial neoplasia and invasive mammary carcinomas in a significant proportion of aged females. These data indicate that enforced mTERT expression can promote the development of spontaneous cancers even in the setting of ample telomere reserve.

The Journal of Cell Biology, 2014

Somatic inactivation of the serine/threonine kinase gene STK11/LKB1/PAR-4 occurs in a variety of ... more Somatic inactivation of the serine/threonine kinase gene STK11/LKB1/PAR-4 occurs in a variety of cancers, including ∼10% of melanoma. However, how the loss of LKB1 activity facilitates melanoma invasion and metastasis remains poorly understood. In LKB1-null cells derived from an autochthonous murine model of melanoma with activated Kras and Lkb1 loss and matched reconstituted controls, we have investigated the mechanism by which LKB1 loss increases melanoma invasive motility. Using a microfluidic gradient chamber system and time-lapse microscopy, in this paper, we uncover a new function for LKB1 as a directional migration sensor of gradients of extracellular matrix (haptotaxis) but not soluble growth factor cues (chemotaxis). Systematic perturbation of known LKB1 effectors demonstrated that this response does not require canonical adenosine monophosphate-activated protein kinase (AMPK) activity but instead requires the activity of the AMPK-related microtubule affinity-regulating kinase (MARK)/PAR-1 family kinases. Inhibition of the LKB1-MARK pathway facilitated invasive motility, suggesting that loss of the ability to sense inhibitory matrix cues may promote melanoma invasion.

The Journal of Cell Biology, 2013

The Cancer Journal, 2002

Advertisement. Close Window. Close Window. Thank you for choosing to subscribe to the eTOC for Th... more Advertisement. Close Window. Close Window. Thank you for choosing to subscribe to the eTOC for The Cancer Journal. Enter your Email address: Wolters Kluwer Health may email you for journal alerts and information, but is ...

Proceedings of the National Academy of Sciences, 2007

Inactivation of the p53 pathway represents the most common molecular defect of human cancer. But ... more Inactivation of the p53 pathway represents the most common molecular defect of human cancer. But in the setting of melanoma, a highly aggressive and invariably fatal malignancy in its advanced disseminated form, mutation/deletion of p53 is relatively rare, whereas its positive regulator ARF is often lost. Here, we show that genetic deficiency in Arf but not p53 facilitates rapid development of melanoma in a genetically engineered mouse model. This difference is accounted for, at least in part, by the unanticipated observation that, unlike fibroblasts, senescence control in melanocytes is strongly regulated by Arf and not p53. Moreover, oncogenic NRAS collaborates with deficiency in Arf, but not p53, to fully transform melanocytes. Our data demonstrate that ARF and p53, although linked in a common pathway, suppress tumorigenesis through distinct, lineage-dependent mechanisms and suggest that ARF helps restrict melanoma progression by executing the oncogene-induced senescence program in benign nevi. Thus, therapeutics designed to restore wild-type p53 function may be insufficient to counter melanoma and other malignancies in which ARF holds p53-independent tumor suppressor activity.

Proceedings of the National Academy of Sciences, 2003

Epidemiological studies support a link between melanoma risk and UV exposure early in life, yet t... more Epidemiological studies support a link between melanoma risk and UV exposure early in life, yet the molecular targets of UV&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;s mutagenic actions are not known. By using well characterized murine models of melanoma, we provide genetic and molecular evidence that identifies components of the Rb pathway as the principal targets of UV mutagenesis in murine melanoma development. In a melanoma model driven by H-RAS activation and loss of p19(ARF) function, UV exposure resulted in a marked acceleration in melanoma genesis, with nearly half of these tumors harboring amplification of cyclin-dependent kinase (cdk) 6, whereas none of the melanomas arising in the absence of UV treatment possessed cdk6 amplification. Moreover, UV-induced melanomas showed a strict reciprocal relationship between cdk6 amplification and p16(INK4a) loss, which is consistent with the actions of UV along the Rb pathway. Most significantly, UV exposure had no impact on the kinetics of melanoma driven by H-RAS activation and p16(INK4a) deficiency. Together, these molecular and genetic data identify components of the Rb pathway as critical biological targets of UV-induced mutagenesis in the development of murine melanoma in vivo.

Proceedings of the National Academy of Sciences, 2001

p19ARF suppresses the growth of cells lacking p53 through an unknown mechanism. p19ARF was found ... more p19ARF suppresses the growth of cells lacking p53 through an unknown mechanism. p19ARF was found to complex with transcription factors E2F1, -2, and -3. Levels of endogenous or ectopically expressed E2F1, -2, and -3, but not E2F6, were reduced after synthesis of p19ARF, through a mechanism involving increased turnover. p19ARF-induced degradation of E2F1 depended on a functional proteasome, and E2F1 was relocalized to nucleoli when coexpressed with p19ARF. Consistent with reduced levels of E2F1 and E2F3, the proliferation of cells defective for p53 function was suppressed by p19ARF, and the effect was partially reversed by ectopic overexpression of E2F1. These results suggest a broader role for p19ARF as a tumor suppressor, in which targeting of certain E2F species may cooperate with stimulation of the p53 pathway to counteract oncogenic growth signals.

Oncogene, 2003

Deletion of the INK4a/ARF locus at 9p21 is detected with high frequency in human melanoma. Within... more Deletion of the INK4a/ARF locus at 9p21 is detected with high frequency in human melanoma. Within a short genomic distance, this locus encodes several proteins with established tumor-suppressor roles in a broad spectrum of cancer types. Several lines of evidence support the view that p16INK4a and p19ARF exert the tumor-suppressor activities of this locus, although their relative importance in specific cancer types such as melanoma has been less rigorously documented on the genetic level. Here, we exploit a well-defined mouse model of RAS-induced melanomas to examine the impact of germline p16INK4a or p19ARF nullizygosity on melanoma formation. We demonstrate that loss of either Ink4a/Arf product can cooperate with RAS activation to produce clinically indistinguishable melanomas. In line with the common phenotypic end point, we further show that RAS+ p16INK4a-/- melanomas sustain somatic inactivation of p19ARF-p53 and, correspondingly, that RAS+ p19ARF-/- melanomas experience high-frequency loss of p16INK4a. These genetic studies provide definitive proof that p16INK4a and p19ARF cooperate to suppress the development of melanoma in vivo.

Oncogene, 2004

Mounting genetic evidence suggests that each product of the Ink4a/Arf locus, p16(INK4a) and p19(A... more Mounting genetic evidence suggests that each product of the Ink4a/Arf locus, p16(INK4a) and p19(ARF), possesses tumor-suppressor activity (Kamijo et al., 1997; Krimpenfort et al., 2001; Sharpless et al., 2001a). We report the generation and characterization of a p19(ARF)-specific knockout allele (p19(ARF)-/-) and direct comparison with mice and derivative cells deficient for p16(INK4a), both p16(INK4a) and p19(ARF), and p53. Like Ink4a/Arf-/- murine embryo fibroblasts (MEFs), p19(ARF)-/- MEFs were highly susceptible to oncogenic transformation, exhibited enhanced subcloning efficiency at low density, and resisted both RAS- and culture-induced growth arrest. In contrast, the biological profile of p16(INK4a)-/- MEFs in these assays more closely resembled that of wild-type cells. In vivo, however, both p19(ARF)-/- and p16(INK4a)-/- animals were significantly more tumor prone than wild-type animals, but each less so than p53-/- or Ink4a/Arf-/- animals, and with differing tumor spectra. These data confirm the predominant role of p19(ARF) over p16(INK4a) in cell culture-based assays of MEFs, yet also underscore the importance of the analysis of tumor suppressors across many cell types within the organism. The cancer-prone conditions of mice singly deficient for either p16(INK4a) or p19(ARF) agree with data derived from human cancer genetics, and reinforce the view that both gene products play significant and nonredundant roles in suppressing malignant transformation in vivo.

Nature Cell Biology, 2006

Articles and research papers on cell division, cell structure, animal and plant cell biology and ... more Articles and research papers on cell division, cell structure, animal and plant cell biology and cell cycles.

Nature Biotechnology, 2014

Circular RNA transcripts were first identified in the early 1990s but knowledge of these species ... more Circular RNA transcripts were first identified in the early 1990s but knowledge of these species has remained limited, as their study through traditional methods of RNA analysis has been difficult. Now, novel bioinformatic approaches coupled with biochemical enrichment strategies and deep sequencing have allowed comprehensive studies of circular RNA species. Recent studies have revealed thousands of endogenous circular RNAs in mammalian cells, some of which are highly abundant and evolutionarily conserved. Evidence is emerging that some circRNAs might regulate microRNA (miRNA) function, and roles in transcriptional control have also been suggested. Therefore, study of this class of noncoding RNAs has potential implications for therapeutic and research applications. We believe the key future challenge for the field will be to understand the regulation and function of these unusual molecules.