Publications by authors named "Verline Justilien"

25 Publications

  • Page 1 of 1

Protein kinase C and SRC signaling define reciprocally related subgroups of glioblastoma with distinct therapeutic vulnerabilities.

Cell Rep 2021 Nov;37(8):110054

Department of Cancer Biology, Mayo Clinic, Jacksonville, FL 32224, USA. Electronic address:

We report that atypical protein kinase Cι (PKCι) is an oncogenic driver of glioblastoma (GBM). Deletion or inhibition of PKCι significantly impairs tumor growth and prolongs survival in murine GBM models. GBM cells expressing elevated PKCι signaling are sensitive to PKCι inhibitors, whereas those expressing low PKCι signaling exhibit active SRC signaling and sensitivity to SRC inhibitors. Resistance to the PKCι inhibitor auranofin is associated with activated SRC signaling and response to a SRC inhibitor, whereas resistance to a SRC inhibitor is associated with activated PKCι signaling and sensitivity to auranofin. Interestingly, PKCι- and SRC-dependent cells often co-exist in individual GBM tumors, and treatment of GBM-bearing mice with combined auranofin and SRC inhibitor prolongs survival beyond either drug alone. Thus, we identify PKCι and SRC signaling as distinct therapeutic vulnerabilities that are directly translatable into an improved treatment for GBM.
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http://dx.doi.org/10.1016/j.celrep.2021.110054DOI Listing
November 2021

Aberrant expression and subcellular localization of ECT2 drives colorectal cancer progression and growth.

Cancer Res 2021 Nov 4. Epub 2021 Nov 4.

Department of Pharmacology, University of North Carolina at Chapel Hill

ECT2 is an activator of RHO GTPases that is essential for cytokinesis. Additionally, ECT2 was identified as an oncoprotein when expressed ectopically in NIH/3T3 fibroblasts. However, oncogenic activation of ECT2 resulted from N-terminal truncation, and such truncated ECT2 proteins have not been found in cancer patients. In this study, we observed elevated expression of full-length ECT2 protein in preneoplastic colon adenomas, driven by increased ECT2 mRNA abundance and associated with APC tumor suppressor loss. Elevated ECT2 levels were detected in the cytoplasm and nucleus of colorectal cancer (CRC) tissue, suggesting cytoplasmic mislocalization as one mechanism of early oncogenic ECT2 activation. Importantly, elevated nuclear ECT2 correlated with poorly differentiated tumors, and a low cytoplasmic:nuclear ratio of ECT2 protein correlated with poor patient survival, suggesting that nuclear and cytoplasmic ECT2 play distinct roles in CRC. Depletion of ECT2 reduced anchorage-independent cancer cell growth and invasion independent of its function in cytokinesis, and loss of Ect2 extended survival in a KrasG12D Apc-null colon cancer mouse model. Expression of ECT2 variants with impaired nuclear localization or guanine nucleotide exchange catalytic activity failed to restore cancer cell growth or invasion, indicating that active, nuclear ECT2 is required to support tumor progression. Nuclear ECT2 promoted ribosomal DNA transcription and ribosome biogenesis in CRC. These results support a driver role for both cytoplasmic and nuclear ECT2 overexpression in CRC and emphasize the critical role of precise subcellular localization in dictating ECT2 function in neoplastic cells.
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http://dx.doi.org/10.1158/0008-5472.CAN-20-4218DOI Listing
November 2021

Recurrent copy number gains drive PKCι expression and PKCι-dependent oncogenic signaling in human cancers.

Adv Biol Regul 2020 12 12;78:100754. Epub 2020 Sep 12.

Department of Cancer Cell Biology, Mayo Clinic Florida, Jacksonville, FL, 32224, USA. Electronic address:

PRKCI is frequently overexpressed in multiple human cancers, and PKCι expression is often prognostic for poor patient survival, indicating that elevated PKCι broadly plays an oncogenic role in the cancer phenotype. PKCι drives multiple oncogenic signaling pathways involved in transformed growth, and transgenic mouse models have revealed that PKCι is a critical oncogenic driver in both lung and ovarian cancers. We now report that recurrent 3q26 copy number gain (CNG) is the predominant genetic driver of PRKCI mRNA expression in all major human cancer types exhibiting such CNGs. In addition to PRKCI, CNG at 3q26 leads to coordinate CNGs of ECT2 and SOX2, two additional 3q26 genes that collaborate with PRKCI to drive oncogenic signaling and tumor initiation in lung squamous cell carcinoma. Interestingly however, whereas 3q26 CNG is a strong driver of PRKCI mRNA expression across all tumor types examined, it has differential effects on ECT2 and SOX2 mRNA expression. In some tumors types, particularly those with squamous histology, all three 3q26 oncogenes are coordinately overexpressed as a consequence of 3q26 CNG, whereas in other cancers only PRKCI and ECT2 mRNA are coordinately overexpressed. This distinct pattern of expression of 3q26 genes corresponds to differences in genomic signatures reflective of activation of specific PKCι oncogenic signaling pathways. In addition to highly prevalent CNG, some tumor types exhibit monoallelic loss of PRKCI. Interestingly, many tumors harboring monoallelic loss of PRKCI express significantly lower PRKCI mRNA and exhibit evidence of WNT/β-catenin signaling pathway activation, which we previously characterized as a major oncogenic pathway in a newly described, PKCι-independent molecular subtype of lung adenocarcinoma. Finally, we show that CNG-driven activation of PKCι oncogenic signaling predicts poor patient survival in many major cancer types. We conclude that CNG and monoallelic loss are the major determinants of tumor PRKCI mRNA expression across virtually all tumor types, but that tumor-type specific mechanisms determine whether these copy number alterations also drive expression of the collaborating 3q26 oncogenes ECT2 and SOX2, and the oncogenic PKCι signaling pathways activated through the collaborative action of these genes. Our analysis may be useful in identifying tumor-specific predictive biomarkers and effective PKCι-targeted therapeutic strategies in the multitude of human cancers harboring genetic activation of PRKCI.
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http://dx.doi.org/10.1016/j.jbior.2020.100754DOI Listing
December 2020

Protein kinase Cι promotes UBF1-ECT2 binding on ribosomal DNA to drive rRNA synthesis and transformed growth of non-small-cell lung cancer cells.

J Biol Chem 2020 06 29;295(24):8214-8226. Epub 2020 Apr 29.

Department of Cancer Biology, Mayo Clinic Comprehensive Cancer Center, Jacksonville, Florida, USA

Epithelial cell-transforming sequence 2 (ECT2) is a guanine nucleotide exchange factor for Rho GTPases that is overexpressed in many cancers and involved in signal transduction pathways that promote cancer cell proliferation, invasion, and tumorigenesis. Recently, we demonstrated that a significant pool of ECT2 localizes to the nucleolus of non-small-cell lung cancer (NSCLC) cells, where it binds the transcription factor upstream binding factor 1 (UBF1) on the promoter regions of ribosomal DNA (rDNA) and activates rDNA transcription, transformed cell growth, and tumor formation. Here, we investigated the mechanism by which ECT2 engages UBF1 on rDNA promoters. Results from ECT2 mutagenesis indicated that the tandem BRCT domain of ECT2 mediates binding to UBF1. Biochemical and MS-based analyses revealed that protein kinase Cι (PKCι) directly phosphorylates UBF1 at Ser-412, thereby generating a phosphopeptide-binding epitope that binds the ECT2 BRCT domain. Lentiviral shRNA knockdown and reconstitution experiments revealed that both a functional ECT2 BRCT domain and the UBF1 Ser-412 phosphorylation site are required for UBF1-mediated ECT2 recruitment to rDNA, elevated rRNA synthesis, and transformed growth. Our findings provide critical molecular insight into ECT2-mediated regulation of rDNA transcription in cancer cells and offer a rationale for therapeutic targeting of UBF1- and ECT2-stimulated rDNA transcription for the management of NSCLC.
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http://dx.doi.org/10.1074/jbc.RA120.013175DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7294082PMC
June 2020

Chromosome 3q26 Gain Is an Early Event Driving Coordinated Overexpression of the PRKCI, SOX2, and ECT2 Oncogenes in Lung Squamous Cell Carcinoma.

Cell Rep 2020 01;30(3):771-782.e6

Department of Cancer Biology, Mayo Clinic Florida, Jacksonville, FL 32224, USA. Electronic address:

Lung squamous cell carcinoma (LSCC) is a prevalent form of lung cancer exhibiting distinctive histological and genetic characteristics. Chromosome 3q26 copy number gain (CNG) is a genetic hallmark of LSCC present in >90% of tumors. We report that 3q26 CNGs occur early in LSCC tumorigenesis, persist during tumor progression, and drive coordinate overexpression of PRKCI, SOX2, and ECT2. Overexpression of PRKCI, SOX2, and ECT2 in the context of Trp53 loss is sufficient to transform mouse lung basal stem cells into tumors with histological and genomic features of LSCC. Functionally, PRKCI and SOX2 collaborate to activate an extensive transcriptional program that enforces a lineage-restricted LSCC phenotype, whereas PRKCI and ECT2 collaborate to promote oncogenic growth. Gene signatures indicative of PKCι-SOX2 and PKCι-ECT2 signaling activity are enriched in the classical subtype of human LSCC and predict distinct therapeutic vulnerabilities. Thus, the PRKCI, SOX2, and ECT2 oncogenes represent a multigenic driver of LSCC.
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http://dx.doi.org/10.1016/j.celrep.2019.12.071DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7238436PMC
January 2020

Protein Kinase Cι and Wnt/β-Catenin Signaling: Alternative Pathways to Kras/Trp53-Driven Lung Adenocarcinoma.

Cancer Cell 2019 08 1;36(2):156-167.e7. Epub 2019 Aug 1.

Department of Cancer Biology, Mayo Clinic Florida, 4500 San Pablo Road, Griffin Cancer Research Building, Room 212, Jacksonville, FL 32224, USA. Electronic address:

We report that mouse LSL-Kras;Trp53 (KP)-mediated lung adenocarcinoma (LADC) tumorigenesis can proceed through both PKCι-dependent and PKCι-independent pathways. The predominant pathway involves PKCι-dependent transformation of bronchoalveolar stem cells (BASCs). However, KP mice harboring conditional knock out Prkci alleles (KPI mice) develop LADC tumors through PKCι-independent transformation of Axin2 alveolar type 2 (AT2) stem cells. Transformed growth of KPI, but not KP, tumors is blocked by Wnt pathway inhibition in vitro and in vivo. Furthermore, a KPI-derived genomic signature predicts sensitivity of human LADC cells to Wnt inhibition, and identifies a distinct subset of primary LADC tumors exhibiting a KPI-like genotype. Thus, LADC can develop through both PKCι-dependent and PKCι-independent pathways, resulting in tumors exhibiting distinct oncogenic signaling and pharmacologic vulnerabilities.
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http://dx.doi.org/10.1016/j.ccell.2019.07.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6693680PMC
August 2019

Protein kinase C: A versatile oncogene in the lung.

Mol Cell Oncol 2018 10;5(5):e1190886. Epub 2018 May 10.

Department of Cancer Biology, Mayo Clinic Florida Jacksonville, Florida.

We have recently demonstrated that protein kinase C (PKC) promotes a stem-like, tumor-initiating cell phenotype in -driven lung adenocarcinoma by activating a novel ELF3-NOTCH3 signaling axis. Combined PKC and NOTCH inhibition was identified as a novel strategy for the treatment of -driven lung adenocarcinoma.
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http://dx.doi.org/10.1080/23723556.2016.1190886DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6154840PMC
May 2018

Oncogenic Ect2 signaling regulates rRNA synthesis in NSCLC.

Small GTPases 2019 09 5;10(5):388-394. Epub 2017 Jul 5.

a Department of Cancer Biology, Mayo Clinic Comprehensive Cancer Center , Jacksonville , FL , USA.

The Rho GTPase family members Rac1, Cdc42 and RhoA play key contributory roles in the transformed phenotype of human cancers. Epithelial Cell Transforming Sequence 2 (Ect2), a guanine nucleotide exchange factor (GEF) for these Rho GTPases, has also been implicated in a variety of human cancers. We have shown that Ect2 is frequently overexpressed in both major forms of non-small cell lung cancer (NSCLC), lung adenocarcinoma (LADC) and lung squamous cell carcinoma (LSCC), which together make up approximately 70% of all lung cancer diagnoses. Furthermore, we have found that Ect2 is required for multiple aspects of the transformed phenotype of NSCLC cells including transformed growth and invasion and tumorigenesis . More recently, we showed that a major mechanism by which Ect2 drives -mediated LADC transformation is by regulating rRNA (rRNA) synthesis. However, it remains unclear whether Ect2 plays a similar role in ribosome biogenesis in LSCC. Here we demonstrate that Ect2 expression correlates positively with expression of ribosome biogenesis genes and with pre-ribosomal 45S RNA abundance in primary LSCC tumors. Furthermore, we demonstrate that Ect2 functionally regulates rRNA synthesis in LSCC cells. Based on these data, we propose that inhibition of Ect2-mediated nucleolar signaling holds promise as a potential therapeutic strategy for improved treatment of both LADC and LSCC.
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http://dx.doi.org/10.1080/21541248.2017.1335274DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6748369PMC
September 2019

Ect2-Dependent rRNA Synthesis Is Required for KRAS-TRP53-Driven Lung Adenocarcinoma.

Cancer Cell 2017 02 19;31(2):256-269. Epub 2017 Jan 19.

Department of Cancer Biology, Mayo Clinic Comprehensive Cancer Center, Griffin Cancer Research Building, Room 212, 4500 San Pablo Road, Jacksonville, FL 32224, USA. Electronic address:

The guanine nucleotide exchange factor (GEF) epithelial cell transforming sequence 2 (Ect2) has been implicated in cancer. However, it is not clear how Ect2 causes transformation and whether Ect2 is necessary for tumorigenesis in vivo. Here, we demonstrate that nuclear Ect2 GEF activity is required for Kras-Trp53 lung tumorigenesis in vivo and that Ect2-mediated transformation requires Ect2-dependent rDNA transcription. Ect2 activates rRNA synthesis by binding the nucleolar transcription factor upstream binding factor 1 (UBF1) on rDNA promoters and recruiting Rac1 and its downstream effector nucleophosmin (NPM) to rDNA. Protein kinase Cι (PKCι)-mediated Ect2 phosphorylation stimulates Ect2-dependent rDNA transcription. Thus, Ect2 regulates rRNA synthesis through a PKCι-Ect2-Rac1-NPM signaling axis that is required for lung tumorigenesis.
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http://dx.doi.org/10.1016/j.ccell.2016.12.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5310966PMC
February 2017

SOX2 Determines Lineage Restriction: Modeling Lung Squamous Cell Carcinoma in the Mouse.

Cancer Cell 2016 10;30(4):505-507

Department of Cancer Biology, Mayo Clinic College of Medicine, Jacksonville, FL 32224, USA. Electronic address:

In this issue of Cancer Cell, Ferone et al. demonstrate that SOX2 not only drives lung tumor formation but also restricts tumor lineage to squamous cell carcinoma (LSCC), regardless of cell of origin. This novel LSCC model should facilitate identification of key oncogenic drivers and treatment strategies for this lung cancer subtype.
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http://dx.doi.org/10.1016/j.ccell.2016.09.012DOI Listing
October 2016

Targeting oncogenic protein kinase Cι for treatment of mutant KRAS LADC.

Small GTPases 2017 01 31;8(1):58-64. Epub 2016 May 31.

a Department of Cancer Biology , Mayo Clinic , Jacksonville , FL , USA.

Lung cancer is the leading cause of cancer death in the US with ∼124,000 new cases annually, and a 5 y survival rate of ∼16%. Mutant KRAS-driven lung adenocarcinoma (KRAS LADC) is a particularly prevalent and deadly form of lung cancer. Protein kinase Cι (PKCι) is an oncogenic effector of KRAS that activates multiple signaling pathways that stimulate transformed growth and invasion, and maintain a KRAS LADC tumor-initiating cell (TIC) phenotype. PKCι inhibitors used alone and in strategic combination show promise as new therapeutic approaches to treatment of KRAS LADC. These novel drug combinations may improve clinical management of KRAS LADC.
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http://dx.doi.org/10.1080/21541248.2016.1194953DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5331898PMC
January 2017

Protein Kinase Cι Drives a NOTCH3-dependent Stem-like Phenotype in Mutant KRAS Lung Adenocarcinoma.

Cancer Cell 2016 Mar;29(3):367-378

Department of Cancer Biology, Mayo Clinic Cancer Center, Jacksonville, FL 32224, USA. Electronic address:

We report that the protein kinase Cι (PKCι) oncogene controls expression of NOTCH3, a key driver of stemness, in KRAS-mediated lung adenocarcinoma (LADC). PKCι activates NOTCH3 expression by phosphorylating the ELF3 transcription factor and driving ELF3 occupancy on the NOTCH3 promoter. PKCι-ELF3-NOTCH3 signaling controls the tumor-initiating cell phenotype by regulating asymmetric cell division, a process necessary for tumor initiation and maintenance. Primary LADC tumors exhibit PKCι-ELF3-NOTCH3 signaling, and combined pharmacologic blockade of PKCι and NOTCH synergistically inhibits tumorigenic behavior in vitro and LADC growth in vivo demonstrating the therapeutic potential of PKCι-ELF3-NOTCH3 signal inhibition to more effectively treat KRAS LADC.
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http://dx.doi.org/10.1016/j.ccell.2016.02.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4795153PMC
March 2016

Parkinson Disease-Mediated Gastrointestinal Disorders and Rational for Combinatorial Therapies.

Med Sci (Basel) 2016 Jan 20;4(1). Epub 2016 Jan 20.

Department of Cancer Biology, Mayo Clinic Cancer Center, Jacksonville, FL 32224, USA.

A gradual loss of dopamine-producing nerve cells gives rise to a common neurodegenerative Parkinson's disease (PD). This disease causes a neurotransmitter imbalance in the brain and initiates a cascade of complications in the rest of the body that appears as distressing symptoms which include gait problems, tremor, gastrointestinal (GI) disorders and cognitive decline. To aid dopamine deficiency, treatment in PD patients includes oral medications, in addition to other methods such as deep brain stimulation and surgical lesioning. Scientists are extensively studying molecular and signaling mechanisms, particularly those involving phenotypic transcription factors and their co-regulatory proteins that are associated with neuronal stem cell (SC) fate determination, maintenance and disease state, and their role in the pathogenesis of PD. Advancement in scientific research and "personalized medicine" to augment current therapeutic intervention and minimize the side effects of chemotherapy may lead to the development of more effective therapeutic strategies in the near future. This review focuses on PD and associated GI complications and summarizes the current therapeutic modalities that include stem cell studies and combinatorial drug treatment.
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http://dx.doi.org/10.3390/medsci4010001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5635767PMC
January 2016

The chromosome 3q26 OncCassette: A multigenic driver of human cancer.

Adv Biol Regul 2016 Jan 23;60:47-63. Epub 2015 Dec 23.

Department of Cancer Biology, Mayo Clinic Comprehensive Cancer Center, Jacksonville, FL, United States.

Recurrent copy number variations (CNVs) are genetic alterations commonly observed in human tumors. One of the most frequent CNVs in human tumors involves copy number gains (CNGs) at chromosome 3q26, which is estimated to occur in >20% of human tumors. The high prevalence and frequent occurrence of 3q26 CNG suggest that it drives the biology of tumors harboring this genetic alteration. The chromosomal region subject to CNG (the 3q26 amplicon) spans from chromosome 3q26 to q29, a region containing ∼200 protein-encoding genes. The large number of genes within the amplicon makes it difficult to identify relevant oncogenic target(s). Whereas a number of genes in this region have been linked to the transformed phenotype, recent studies indicate a high level of cooperativity among a subset of frequently amplified 3q26 genes. Here we use a novel bioinformatics approach to identify potential driver genes within the recurrent 3q26 amplicon in lung squamous cell carcinoma (LSCC). Our analysis reveals a set of 35 3q26 amplicon genes that are coordinately amplified and overexpressed in human LSCC tumors, and that also map to a major LSCC susceptibility locus identified on mouse chromosome 3 that is syntenic with human chromosome 3q26. Pathway analysis reveals that 21 of these genes exist within a single predicted network module. Four 3q26 genes, SOX2, ECT2, PRKCI and PI3KCA occupy the hub of this network module and serve as nodal genes around which the network is organized. Integration of available genetic, genomic, biochemical and functional data demonstrates that SOX2, ECT2, PRKCI and PIK3CA are cooperating oncogenes that function within an integrated cell signaling network that drives a highly aggressive, stem-like phenotype in LSCC tumors harboring 3q26 amplification. Based on the high level of genomic, genetic, biochemical and functional integration amongst these 4 3q26 nodal genes, we propose that they are the key oncogenic targets of the 3q26 amplicon and together define a "3q26 OncCassette" that mediates 3q26 CNG-driven tumorigenesis. Genomic analysis indicates that the 3q26 OncCassette also operates in other major tumor types that exhibit frequent 3q26 CNGs, including head and neck squamous cell carcinoma (HNSCC), ovarian serous cancer and cervical cancer. Finally, we discuss how the 3q26 OncCassette represents a tractable target for development of novel therapeutic intervention strategies that hold promise for improving treatment of 3q26-driven cancers.
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http://dx.doi.org/10.1016/j.jbior.2015.10.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4729592PMC
January 2016

Molecular pathways: novel approaches for improved therapeutic targeting of Hedgehog signaling in cancer stem cells.

Clin Cancer Res 2015 Feb;21(3):505-13

Department of Cancer Biology, Mayo Clinic Comprehensive Cancer Center, Jacksonville, Florida.

The Hedgehog (Hh) signaling pathway is critical for embryonic development. In adult tissues, Hh signaling is relatively quiescent with the exception of roles in tissue maintenance and repair. Aberrant activation of Hh signaling is implicated in multiple aspects of transformation, including the maintenance of the cancer stem cell (CSC) phenotype. Preclinical studies indicate that CSCs from many tumor types are sensitive to Hh pathway inhibition and that Hh-targeted therapeutics block many aspects of transformation attributed to CSCs, including drug resistance, relapse, and metastasis. However, to date, Hh inhibitors, specifically those targeting Smoothened [such as vismodegib, BMS-833923, saridegib (IPI-926), sonidegib/erismodegib (LDE225), PF-04449913, LY2940680, LEQ 506, and TAK-441], have demonstrated good efficacy as monotherapy in patients with basal cell carcinoma and medulloblastoma, but have shown limited activity in other tumor types. This lack of success is likely due to many factors, including a lack of patient stratification in early trials, cross-talk between Hh and other oncogenic signaling pathways that can modulate therapeutic response, and a limited knowledge of Hh pathway activation mechanisms in CSCs from most tumor types. Here, we discuss Hh signaling mechanisms in the context of human cancer, particularly in the maintenance of the CSC phenotype, and consider new therapeutic strategies that hold the potential to expand considerably the scope and therapeutic efficacy of Hh-directed anticancer therapy.
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http://dx.doi.org/10.1158/1078-0432.CCR-14-0507DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4316382PMC
February 2015

The PRKCI and SOX2 oncogenes are coamplified and cooperate to activate Hedgehog signaling in lung squamous cell carcinoma.

Cancer Cell 2014 Feb;25(2):139-51

Department of Cancer Biology, Mayo Clinic Cancer Center, Jacksonville, FL 32224, USA. Electronic address:

We report that two oncogenes coamplified on chromosome 3q26, PRKCI and SOX2, cooperate to drive a stem-like phenotype in lung squamous cell carcinoma (LSCC). Protein kinase Cι (PKCι) phosphorylates SOX2, a master transcriptional regulator of stemness, and recruits it to the promoter of Hedgehog (Hh) acyltransferase (HHAT) that catalyzes the rate-limiting step in Hh ligand production. PKCι-mediated SOX2 phosphorylation is required for HHAT promoter occupancy, HHAT expression, and maintenance of a stem-like phenotype. Primary LSCC tumors coordinately overexpress PKCι, SOX2, and HHAT and require PKCι-SOX2-HHAT signaling to maintain a stem-like phenotype. Thus, PKCι and SOX2 are genetically, biochemically, and functionally linked in LSCC, and together they drive tumorigenesis by establishing a cell-autonomous Hh signaling axis.
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http://dx.doi.org/10.1016/j.ccr.2014.01.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3949484PMC
February 2014

Utility and applications of orthotopic models of human non-small cell lung cancer (NSCLC) for the evaluation of novel and emerging cancer therapeutics.

Curr Protoc Pharmacol 2013 Oct 8;62:14.27.1-14.27.17. Epub 2013 Oct 8.

Department of Cancer Biology, Mayo Clinic College of Medicine, Jacksonville, Florida.

Lung cancer is a leading cause of cancer deaths worldwide. Despite advances in chemotherapy, radiation therapy, and surgery, lung cancer continues to have a low 5-year survival rate, highlighting a dire need for more effective means of prevention, diagnosis, prognosis, and treatment. Mouse models that recapitulate the clinical features of advanced human lung cancer are critical for testing novel therapeutic approaches. This unit describes a highly reproducible, easy-to-establish orthotopic murine model of lung cancer, provides methods for in vivo imaging and monitoring of tumor growth, and discusses the usefulness of this model for translational lung cancer research and the development of therapeutic strategies.
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http://dx.doi.org/10.1002/0471141755.ph1427s62DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4362678PMC
October 2013

Atypical protein kinase Cι as a human oncogene and therapeutic target.

Biochem Pharmacol 2014 Mar 11;88(1):1-11. Epub 2013 Nov 11.

Department of Cancer Biology, Mayo Clinic Comprehensive Cancer Center, 45400 San Pablo Road, Jacksonville, FL 32224, USA. Electronic address:

Protein kinase inhibitors represent a major class of targeted therapeutics that has made a positive impact on treatment of cancer and other disease indications. Among the promising kinase targets for further therapeutic development are members of the Protein Kinase C (PKC) family. The PKCs are central components of many signaling pathways that regulate diverse cellular functions including proliferation, cell cycle, differentiation, survival, cell migration, and polarity. Genetic manipulation of individual PKC isozymes has demonstrated that they often fulfill distinct, nonredundant cellular functions. Participation of PKC members in different intracellular signaling pathways reflects responses to varying extracellular stimuli, intracellular localization, tissue distribution, phosphorylation status, and intermolecular interactions. PKC activity, localization, phosphorylation, and/or expression are often altered in human tumors, and PKC isozymes have been implicated in various aspects of transformation, including uncontrolled proliferation, migration, invasion, metastasis, angiogenesis, and resistance to apoptosis. Despite the strong relationship between PKC isozymes and cancer, to date only atypical PKCiota has been shown to function as a bona fide oncogene, and as such is a particularly attractive therapeutic target for cancer treatment. In this review, we discuss the role of PKCiota in transformation and describe mechanism-based approaches to therapeutically target oncogenic PKCiota signaling in cancer.
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http://dx.doi.org/10.1016/j.bcp.2013.10.023DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3944347PMC
March 2014

Matrix metalloproteinase-10 is required for lung cancer stem cell maintenance, tumor initiation and metastatic potential.

PLoS One 2012 24;7(4):e35040. Epub 2012 Apr 24.

Department of Cancer Biology, Mayo Clinic College of Medicine, Jacksonville, Florida, United States of America.

Matrix metalloproteinases (Mmps) stimulate tumor invasion and metastasis by degrading the extracellular matrix. Here we reveal an unexpected role for Mmp10 (stromelysin 2) in the maintenance and tumorigenicity of mouse lung cancer stem-like cells (CSC). Mmp10 is highly expressed in oncosphere cultures enriched in CSCs and RNAi-mediated knockdown of Mmp10 leads to a loss of stem cell marker gene expression and inhibition of oncosphere growth, clonal expansion, and transformed growth in vitro. Interestingly, clonal expansion of Mmp10 deficient oncospheres can be restored by addition of exogenous Mmp10 protein to the culture medium, demonstrating a direct role for Mmp10 in the proliferation of these cells. Oncospheres exhibit enhanced tumor-initiating and metastatic activity when injected orthotopically into syngeneic mice, whereas Mmp10-deficient cultures show a severe defect in tumor initiation. Conversely, oncospheres implanted into syngeneic non-transgenic or Mmp10(-/-) mice show no significant difference in tumor initiation, growth or metastasis, demonstrating the importance of Mmp10 produced by cancer cells rather than the tumor microenvironment in lung tumor initiation and maintenance. Analysis of gene expression data from human cancers reveals a strong positive correlation between tumor Mmp10 expression and metastatic behavior in many human tumor types. Thus, Mmp10 is required for maintenance of a highly tumorigenic, cancer-initiating, metastatic stem-like cell population in lung cancer. Our data demonstrate for the first time that Mmp10 is a critical lung cancer stem cell gene and novel therapeutic target for lung cancer stem cells.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0035040PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3335833PMC
August 2012

Protein kinase Cα promotes cell migration through a PDZ-dependent interaction with its novel substrate discs large homolog 1 (DLG1).

J Biol Chem 2011 Dec 25;286(50):43559-68. Epub 2011 Oct 25.

Department of Pharmacology, University of California, San Diego, La Jolla, California 92093, USA.

Protein scaffolds maintain precision in kinase signaling by coordinating kinases with components of specific signaling pathways. Such spatial segregation is particularly important in allowing specificity of signaling mediated by the 10-member family of protein kinase C (PKC) isozymes. Here we identified a novel interaction between PKCα and the Discs large homolog (DLG) family of scaffolds that is mediated by a class I C-terminal PDZ (PSD-95, disheveled, and ZO1) ligand unique to this PKC isozyme. Specifically, use of a proteomic array containing 96 purified PDZ domains identified the third PDZ domains of DLG1/SAP97 and DLG4/PSD95 as interaction partners for the PDZ binding motif of PKCα. Co-immunoprecipitation experiments verified that PKCα and DLG1 interact in cells by a mechanism dependent on an intact PDZ ligand. Functional assays revealed that the interaction of PKCα with DLG1 promotes wound healing; scratch assays using cells depleted of PKCα and/or DLG1 have impaired cellular migration that is no longer sensitive to PKC inhibition, and the ability of exogenous PKCα to rescue cellular migration is dependent on the presence of its PDZ ligand. Furthermore, we identified Thr-656 as a novel phosphorylation site in the SH3-Hook region of DLG1 that acts as a marker for PKCα activity at this scaffold. Increased phosphorylation of Thr-656 is correlated with increased invasiveness in non-small cell lung cancer lines from the NCI-60, consistent with this phosphorylation site serving as a marker of PKCα-mediated invasion. Taken together, these data establish the requirement of scaffolding to DLG1 for PKCα to promote cellular migration.
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http://dx.doi.org/10.1074/jbc.M111.294603DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3234831PMC
December 2011

Matrix metalloproteinase-10 promotes Kras-mediated bronchio-alveolar stem cell expansion and lung cancer formation.

PLoS One 2011 17;6(10):e26439. Epub 2011 Oct 17.

Department of Cancer Biology, Mayo Clinic College of Medicine, Jacksonville, Florida, United States of America.

Matrix metalloproteinase 10 (MMP-10; stromelysin 2) is a member of a large family of structurally related matrix metalloproteinases, many of which have been implicated in tumor progression, invasion and metastasis. We recently identified Mmp10 as a gene that is highly induced in tumor-initiating lung bronchioalveolar stem cells (BASCs) upon activation of oncogenic Kras in a mouse model of lung adenocarcinoma. However, the potential role of Mmp10 in lung tumorigenesis has not been addressed. Here, we demonstrate that Mmp10 is overexpressed in lung tumors induced by either the smoke carcinogen urethane or oncogenic Kras. In addition, we report a significant reduction in lung tumor number and size after urethane exposure or genetic activation of oncogenic Kras in Mmp10 null (Mmp10(-/-)) mice. This inhibitory effect is reflected in a defect in the ability of Mmp10-deficient BASCs to expand and undergo transformation in response to urethane or oncogenic Kras in vivo and in vitro, demonstrating a role for Mmp10 in the tumor-initiating activity of Kras-transformed lung stem cells. To determine the potential relevance of MMP10 in human cancer we analyzed Mmp10 expression in publicly-available gene expression profiles of human cancers. Our analysis reveals that MMP10 is highly overexpressed in human lung tumors. Gene set enhancement analysis (GSEA) demonstrates that elevated MMP10 expression correlates with both cancer stem cell and tumor metastasis genomic signatures in human lung cancer. Finally, Mmp10 is elevated in many human tumor types suggesting a widespread role for Mmp10 in human malignancy. We conclude that Mmp10 plays an important role in lung tumor initiation via maintenance of a highly tumorigenic, cancer-initiating, stem-like cell population, and that Mmp10 expression is associated with stem-like, highly metastatic genotypes in human lung cancers. These results indicate that Mmp10 may represent a novel therapeutic approach to target lung cancer stem cells.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0026439PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3195727PMC
February 2012

Oncogenic activity of Ect2 is regulated through protein kinase C iota-mediated phosphorylation.

J Biol Chem 2011 Mar 28;286(10):8149-8157. Epub 2010 Dec 28.

From the Department of Cancer Biology, Mayo Clinic College of Medicine, Jacksonville, Florida 32224 and. Electronic address:

The Rho GTPase guanine nucleotide exchange factor Ect2 is genetically and biochemically linked to the PKCι oncogene in non-small cell lung cancer (NSCLC). Ect2 is overexpressed and mislocalized to the cytoplasm of NSCLC cells where it binds the oncogenic PKCι-Par6 complex, leading to activation of the Rac1 small GTPase. Here, we identify a previously uncharacterized phosphorylation site on Ect2, threonine 328, that serves to regulate the oncogenic activity of Ect2 in NSCLC cells. PKCι directly phosphorylates Ect2 at Thr-328 in vitro, and RNAi-mediated knockdown of either PKCι or Par6 leads to a decrease in phospho-Thr-328 Ect2, indicating that PKCι regulates Thr-328 Ect2 phosphorylation in NSCLC cells. Both wild-type Ect2 and a phosphomimetic T328D Ect2 mutant bind the PKCι-Par6 complex, activate Rac1, and restore transformed growth and invasion when expressed in NSCLC cells made deficient in endogenous Ect2 by RNAi-mediated knockdown. In contrast, a phosphorylation-deficient T328A Ect2 mutant fails to bind the PKCι-Par6 complex, activate Rac1, or restore transformation. Our data support a model in which PKCι-mediated phosphorylation regulates Ect2 binding to the oncogenic PKCι-Par6 complex thereby activating Rac1 activity and driving transformed growth and invasion.
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http://dx.doi.org/10.1074/jbc.M110.196113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3048701PMC
March 2011

The guanine nucleotide exchange factor (GEF) Ect2 is an oncogene in human cancer.

Adv Enzyme Regul 2010 6;50(1):190-200. Epub 2009 Nov 6.

Department of Cancer Biology, Mayo Clinic Comprehensive Cancer Center, Jacksonville, FL, USA.

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http://dx.doi.org/10.1016/j.advenzreg.2009.10.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2863999PMC
August 2010

SOD2 knockdown mouse model of early AMD.

Invest Ophthalmol Vis Sci 2007 Oct;48(10):4407-20

Department of Molecular Genetics, University of Florida, Gainesville, Florida, USA.

Purpose: To test the hypothesis that oxidative injury to the retinal pigment epithelium (RPE) may lead to retinal damage similar to that associated with the early stages of age-related macular degeneration (AMD).

Methods: A ribozyme that targets the protective enzyme manganese superoxide dismutase (MnSOD) was expressed in RPE-J cells, and adeno-associated virus (AAV) expressing the ribozyme gene was injected beneath the retinas of adult C57BL/6 mice. The RPE/choroid complex was examined for SOD2 protein levels and protein markers of oxidative damage using immunoblot analysis and LC MS/MS-identification of proteins and nitration sites. Lipids were extracted from retinal tissue and analyzed for the bis-retinoid compounds A2E and iso-A2E. The mice were analyzed by full-field electroretinography (ERG) for light response. Light and electron microscopy were used to measure cytological changes in the retinas.

Results: The treatment of RPE-J cells with Rz432 resulted in decreased MnSOD mRNA and protein as well as increased levels of superoxide anion and apoptotic cell death. When delivered by AAV, Rz432 reduced MnSOD protein and increased markers of oxidative damage, including nitrated and carboxyethylpyrrole-modified proteins in the RPE-choroid of mice. Ribozyme delivery caused a progressive loss of electroretinograph response, vacuolization, degeneration of the RPE, thickening of Bruch's membrane, and shortening and disorganization of the photoreceptor outer and inner segments. Progressive thinning of the photoreceptor outer nuclear layer resulted from apoptotic cell death. Similar to the eyes of patients with AMD, ribozyme-treated eyes exhibited increased autofluorescence and elevated levels of A2E and iso-A2E, major bis-retinoid pigments of lipofuscin.

Conclusions: These results support the hypothesis that oxidative damage to the RPE may play a role in some of the key features of AMD.
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http://dx.doi.org/10.1167/iovs.07-0432DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6549721PMC
October 2007
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