Central xanthoma of the jaw in association with Noonan syndrome.

Xanthomas are histiocytic lesions of the skin, soft tissue, and bone and are generally considered to be reactive in nature. When they arise in the bones of the jaw, they are referred to as central xanthomas. New evidence supports the hypothesis that central xanthomas are a separate and distinct entity from their extragnathic counterparts. Noonan syndrome (NS) is an autosomal dominant disorder that has been associated with giant cell lesions, which also commonly occur in the jaw. We present a case of a 15-year-old boy with NS who presented with a radiolucent lesion of the mandible that on excision was found to be a central xanthoma. Although giant cell lesions have been well described in NS, xanthomas of the jaw have not been reported. We will also discuss the entities that must be excluded before making a diagnosis of central xanthoma, as this can affect both treatment and follow-up.

Deubiquitinase USP18 Loss Mislocalizes and Destabilizes KRAS in Lung Cancer.

KRAS is frequently mutated in lung cancers and is associated with aggressive biology and chemotherapy resistance. Therefore, innovative approaches are needed to treat these lung cancers. Prior work implicated the IFN-stimulated gene 15 (ISG15) deubiquitinase (DUB) USP18 as having antineoplastic activity by regulating lung cancer growth and oncoprotein stability. This study demonstrates that USP18 affects the stability of the KRAS oncoprotein. Interestingly, loss of USP18 reduced KRAS expression, and engineered gain of USP18 expression increased KRAS protein levels in lung cancer cells. Using the protein synthesis inhibitor cycloheximide, USP18 knockdown significantly reduced the half-life of KRAS, but gain of USP18 expression significantly increased its stability. Intriguingly, loss of USP18 altered KRAS subcellular localization by mislocalizing KRAS from the plasma membrane. To explore the biologic consequences, immunohistochemical (IHC) expression profiles of USP18 were compared in lung cancers of KrasLA2/+ versus cyclin E engineered mouse models. USP18 expression was higher in Kras-driven murine lung cancers, indicating a link between KRAS and USP18 expression in vivo To solidify this association, loss of Usp18 in KrasLA2/+ /Usp18-/- mice was found to significantly reduce lung cancers as compared with parental KrasLA2/+ mice. Finally, translational relevance was confirmed in a human lung cancer panel by showing that USP18 IHC expression was significantly higher in KRAS-mutant versus wild-type lung adenocarcinomas.Implications: Taken together, this study highlights a new way to combat the oncogenic consequences of activated KRAS in lung cancer by inhibiting the DUB USP18. Mol Cancer Res; 15(7); 905-14. ©2017 AACR.

Clinical Genotyping of Non-Small Cell Lung Cancers Using Targeted Next-Generation Sequencing: Utility of Identifying Rare and Co-mutations in Oncogenic Driver Genes.

Detection of somatic mutations in non-small cell lung cancers (NSCLCs), especially adenocarcinomas, is important for directing patient care when targeted therapy is available. Here, we present our experience with genotyping NSCLC using the Ion Torrent Personal Genome Machine (PGM) and the AmpliSeq Cancer Hotspot Panel v2. We tested 453 NSCLC samples from 407 individual patients using the 50 gene AmpliSeq Cancer Hotspot Panel v2 from May 2013 to July 2015. Using 10 ng of DNA, up to 11 samples were simultaneously sequenced on the Ion Torrent PGM (316 and 318 chips). We identified variants with the Ion Torrent Variant Caller Plugin, and Golden Helix's SVS software was used for annotation and prediction of the significance of the variants. Three hundred ninety-eight samples were successfully sequenced (12.1% failure rate). In all, 633 variants in 41 genes were detected with a median of 2 (range of 0 to 7) variants per sample. Mutations detected in BRAF, EGFR, ERBB2, KRAS, NRAS, and PIK3CA were considered potentially actionable and were identified in 237 samples, most commonly in KRAS (37.9%), EGFR (11.1%), BRAF (4.8%), and PIK3CA (4.3%). In our patient population, all mutations in EGFR, KRAS, and BRAF were mutually exclusive. The Ion Torrent Ampliseq technology can be utilized on small biopsy and cytology specimens, requires very little input DNA, and can be applied in clinical laboratories for genotyping of NSCLC. This targeted next-generation sequencing approach allows for detection of common and also rare mutations that are clinically actionable in multiple patients simultaneously.

Mice null for the deubiquitinase USP18 spontaneously develop leiomyosarcomas.

BACKGROUND: USP18 (ubiquitin-specific protease 18) removes ubiquitin-like modifier interferon stimulated gene 15 (ISG15) from conjugated proteins. USP18 null mice in a FVB/N background develop tumors as early as 2 months of age. These tumors are leiomyosarcomas and thus represent a new murine model for this disease. METHODS: Heterozygous USP18 +/- FVB/N mice were bred to generate wild-type, heterozygous and homozygous cohorts. Tumors were characterized immunohistochemically and two cell lines were derived from independent tumors. Cell lines were karyotyped and their responses to restoration of USP18 activity assessed. Drug testing and tumorigenic assays were also performed. USP18 immunohistochemical staining in a large series of human leiomyosacomas was examined. RESULTS: USP18 -/- FVB/N mice spontaneously develop tumors predominantly on the back of the neck with most tumors evident between 6-12 months (80 % penetrance). Immunohistochemical characterization of the tumors confirmed they were leiomyosarcomas, which originate from smooth muscle. Restoration of USP18 activity in sarcoma-derived cell lines did not reduce anchorage dependent or independent growth or xenograft tumor formation demonstrating that these cells no longer require USP18 suppression for tumorigenesis. Karyotyping revealed that both tumor-derived cell lines were aneuploid with extra copies of chromosomes 3 and 15. Chromosome 15 contains the Myc locus and MYC is also amplified in human leiomyosarcomas. MYC protein levels were elevated in both murine leiomyosarcoma cell lines. Stabilized P53 protein was detected in a subset of these murine tumors, another feature of human leiomyosarcomas. Immunohistochemical analyses of USP18 in human leiomyosarcomas revealed a range of staining intensities with the highest USP18 expression in normal vascular smooth muscle. USP18 tissue array analysis of primary leiomyosarcomas from 89 patients with a clinical database revealed cases with reduced USP18 levels had a significantly decreased time to metastasis (P = 0.0441). CONCLUSIONS: USP18 null mice develop leiomyosarcoma recapitulating key features of clinical leiomyosarcomas and patients with reduced-USP18 tumor levels have an unfavorable outcome. USP18 null mice and the derived cell lines represent clinically-relevant models of leiomyosarcoma and can provide insights into both leiomyosarcoma biology and therapy.

Effective capture of circulating tumor cells from a transgenic mouse lung cancer model using dendrimer surfaces immobilized with anti-EGFR.

The lack of an effective detection method for lung circulating tumor cells (CTCs) presents a substantial challenge to elucidate the value of CTCs as a diagnostic or prognostic indicator in lung cancer, particularly in nonsmall cell lung cancer (NSCLC). In this study, we prepared a capture surface exploiting strong multivalent binding mediated by poly(amidoamine) (PAMAM) dendrimers to capture CTCs originating from lung cancers. Given that 85% of the tumor cells from NSCLC patients overexpress epidermal growth factor receptor (EGFR), anti-EGFR was chosen as a capture agent. Following in vitro confirmation using the murine lung cancer cell lines (ED-1 and ED1-SC), cyclin E-overexpressing (CEO) transgenic mice were employed as an in vivo lung tumor model to assess specificity and sensitivity of the capture surface. The numbers of CTCs in blood from the CEO transgenic mice were significantly higher than those from the healthy controls (on average 75.3 ± 14.9 vs 4.4 ± 1.2 CTCs/100 µL of blood, p < 0.005), indicating the high sensitivity and specificity of our surface. Furthermore, we found that the capture surface also offers a simple, effective method for monitoring treatment responses, as observed by the significant decrease in the CTC numbers from the CEO mice upon a treatment using a novel anti-miR-31 locked nucleic acid (LNA), compared to a vehicle treatment and a control-LNA treatment (p < 0.05). This in vivo evaluation study confirms that our capture surface is highly efficient in detecting in vivo CTCs and thus has translational potential as a diagnostic and prognostic tool for lung cancer.

Impact of the 2013 ASCO/CAP HER2 Guideline Updates at an Academic Medical Center That Performs Primary HER2 FISH Testing: Increase in Equivocal Results and Utility of Reflex Immunohistochemistry.

OBJECTIVES: The 2013 American Society of Clinical Oncology (ASCO)/College of American Pathologists (CAP) guideline updates lowered the threshold for HER2 positivity and altered the equivocal category. The goal of this study was to evaluate the impact of these changes on the distribution of HER2 fluorescence in situ hybridization (FISH) status. The utility of reflex HER2 immunohistochemistry (IHC) for FISH equivocal cases was also examined. METHODS: We retrospectively reviewed all invasive breast cancers analyzed for HER2 via dual-probe FISH (PathVysion; Abbott Laboratories. Abbott Park, IL) 12 months before and after the HER2 guidelines updates were implemented. Reflex HER2 IHC results were recorded for HER2 FISH equivocal cases. RESULTS: There was a significant increase in the number of HER2 FISH equivocal results after the guideline updates (4.9% vs 1.4%, P = .0087) that was independent of specimen type (core vs surgical, P = .6). All 17 FISH equivocal cases after the updates had reflex HER2 IHC: two (12%) of 17 were positive, 12 (71%) of 17 remained equivocal, and three (18%) of 17 were negative. CONCLUSIONS: Implementation of the 2013 ASCO/CAP HER2 guideline updates resulted in an increase in HER2 FISH equivocal results, which can be attributed to HER2 copy number, regardless of the HER2/CEP17 ratio. Reflex IHC for FISH equivocal cases is of limited utility; however, IHC does assign HER2 positivity or negativity in a small percentage of cases.

Implementation of a Molecular Tumor Board: The Impact on Treatment Decisions for 35 Patients Evaluated at Dartmouth-Hitchcock Medical Center.

BACKGROUND: Although genetic profiling of tumors is a potentially powerful tool to predict drug sensitivity and resistance, its routine use has been limited because clinicians are often unfamiliar with interpretation and incorporation of the information into practice. We established a Molecular Tumor Board (MTB) to interpret individual patients' tumor genetic profiles and provide treatment recommendations. PATIENTS AND METHODS: DNA from tumor specimens was sequenced in a Clinical Laboratory Improvement Amendments-certified laboratory to identify coding mutations in a 50-gene panel (n = 34) or a 255-gene panel (n = 1). Cases were evaluated by a multidisciplinary MTB that included pathologists, oncologists, hematologists, basic scientists, and genetic counselors. RESULTS: During the first year, 35 cases were evaluated by the MTB, with 32 presented for recommendations on targeted therapies, and 3 referred for potential germline mutations. In 56.3% of cases, MTB recommended treatment with a targeted agent based on evaluation of tumor genetic profile and treatment history. Four patients (12.5%) were subsequently treated with a MTB-recommended targeted therapy; 3 of the 4 patients remain on therapy, 2 of whom experienced clinical benefit lasting >10 months. CONCLUSION: For the majority of cases evaluated, the MTB was able to provide treatment recommendations based on targetable genetic alterations. The most common reasons that MTB-recommended therapy was not administered stemmed from patient preferences and genetic profiling at either very early or very late stages of disease; lack of drug access was rarely encountered. Increasing awareness of molecular profiling and targeted therapies by both clinicians and patients will improve acceptance and adherence to treatments that could significantly improve outcomes. IMPLICATIONS FOR PRACTICE: Case evaluation by a multidisciplinary Molecular Tumor Board (MTB) is critical to benefit from individualized genetic data and maximize clinical impact. MTB recommendations shaped treatment options for the majority of cases evaluated. In the few patients treated with MTB-recommended therapy, disease outcomes were positive and support genetically informed treatment.

Cell cycle and apoptosis regulatory proteins, proliferative markers, cell signaling molecules, CD209, and decorin immunoreactivity in low-grade myxofibrosarcoma and myxoma.

The histologic differential diagnosis between intramuscular myxoma and low-grade myxofibrosarcoma can be quite difficult in some cases. To identify a diagnostic immunohistochemical marker, we compared the staining profiles of 19 different antigens, including cell cycle proteins, apoptosis proteins, and proliferative markers, and selected other signaling and structural proteins in these two tumors. Ten cases each of intramuscular myxoma and low-grade myxofibrosarcoma were stained with antibodies directed against apoptosis regulatory proteins (Bcl2, activated caspase-3, phospho-H2A.X, and cleaved PARP), cell cycle regulatory proteins (Rb1, Cyclin-A, CDKN1B, and Cdt1), proliferative markers (KI67, MCM2, phospho-histone H3, and geminin), cell signalling molecules (c-Myc, EGF, EGFR, PLA2G4A, and HSP90), a dendritic cell marker (CD209), and the extracellular matrix proteoglycan decorin. Staining patterns of myxoma and myxofibrosarcoma were compared using Fisher's exact test and the Mann-Whitney test. For each potential diagnostic marker studied, the proportions of cases scored as positive on both dichotomous or ordinal scales were not significantly different between myxoma and myxofibrosarcoma. Myxoma and myxofibrosarcoma share a common immunophenotype for each of the markers studied. Distinction between these tumors is still predominantly based on morphologic criteria.

CDK2 Inhibition Causes Anaphase Catastrophe in Lung Cancer through the Centrosomal Protein CP110.

Aneuploidy is frequently detected in human cancers and is implicated in carcinogenesis. Pharmacologic targeting of aneuploidy is an attractive therapeutic strategy, as this would preferentially eliminate malignant over normal cells. We previously discovered that CDK2 inhibition causes lung cancer cells with more than two centrosomes to undergo multipolar cell division leading to apoptosis, defined as anaphase catastrophe. Cells with activating KRAS mutations were especially sensitive to CDK2 inhibition. Mechanisms of CDK2-mediated anaphase catastrophe and how activated KRAS enhances this effect were investigated. Live-cell imaging provided direct evidence that following CDK2 inhibition, lung cancer cells develop multipolar anaphase and undergo multipolar cell division with the resulting progeny apoptotic. The siRNA-mediated repression of the CDK2 target and centrosome protein CP110 induced anaphase catastrophe of lung cancer cells. In contrast, CP110 overexpression antagonized CDK2 inhibitor-mediated anaphase catastrophe. Furthermore, activated KRAS mutations sensitized lung cancer cells to CDK2 inhibition by deregulating CP110 expression. Thus, CP110 is a critical mediator of CDK2 inhibition-driven anaphase catastrophe. Independent examination of murine and human paired normal-malignant lung tissues revealed marked upregulation of CP110 in malignant versus normal lung. Human lung cancers with KRAS mutations had significantly lower CP110 expression as compared with KRAS wild-type cancers. Thus, a direct link was found between CP110 and CDK2 inhibitor antineoplastic response. CP110 plays a mechanistic role in response of lung cancer cells to CDK2 inhibition, especially in the presence of activated KRAS mutations.

Small cell neuroendocrine carcinomas of the lung do not harbor high-risk human papillomavirus.

High-risk subtypes of the human papillomavirus (HPV) are known to drive the pathogenesis of cervical, anogenital, and oropharyngeal squamous cell carcinomas. Recent reports have shown that HPV is also associated with small cell neuroendocrine carcinomas of the cervix and oropharynx. Little is known about HPV as a driver of neuroendocrine tumors at other sites, in particular, small cell lung cancer (SCLC). The aim of this study was to evaluate SCLC for the presence of high-risk HPV to further elucidate the role of HPV in SCLC. Archived formalin-fixed, paraffin-embedded surgical resection specimens from 20 primary SCLC from 19 patients were identified from 2004 to 2013. Two cervical small cell carcinomas were included as controls. Small cell neuroendocrine phenotype was confirmed by review of morphology and prior immunohistochemistry staining. Immunohistochemistry for p16 (INK4a) expression was performed in all cases. DNA was extracted from formalin-fixed, paraffin-embedded specimens and run on the Roche Linear Array HPV Genotyping test and a real-time polymerase chain reaction HPV assay. Pathologic tumor stage was collected from surgical pathology reports. High-risk HPV genotypes were not detected in any of the 20 SCLC specimens, whereas p16 was up-regulated in 14 (70%) of 20. p16 up-regulation can be used as an indicator of disruption of the Rb pathway either by integration of the HPV E7 oncoprotein or other mechanisms. In conclusion, our findings indicate that, unlike some other small cell neuroendocrine carcinomas, the pathogenesis of SCLC does not appear to be associated with high-risk HPV infection, a potentially very useful characteristic when determining primary from metastatic tumors.

Breast MRI-detected cystic apocrine metaplasia: imaging features with microvessel analysis and histologic correlation.

OBJECTIVE: The purpose of this article is to characterize the histologic vascular features and distinguishing MRI features of cystic apocrine metaplasia to better understand imaging-pathology concordance. MATERIALS AND METHODS: Retrospective review of 261 consecutive MRI-guided biopsy cases was performed. Pathology results were reviewed for all biopsies; cystic apocrine metaplasia was identified as the predominant finding in 19 cases (7%). CD31 immunohistochemistry was subsequently performed on the most representative block of cystic apocrine metaplasia, and microvasculature was evaluated using computer-assisted image analysis. The contrast-enhanced MRI examinations correlating with the cystic apocrine metaplasia cases were independently reviewed by two radiologists specializing in breast imaging; lesions were analyzed for morphologic, kinetic, and T2 characteristics. RESULTS: On MRI review, 17 of 19 (89%) lesions were 10 mm or smaller. Washout kinetics were present in 11 of 19 (58%) lesions, and 14 of 19 (74%) lesions were at least partially hyperintense on T2-weighted sequences relative to adjacent glandular tissue. Cystic apocrine metaplasia had a higher percentage area (mean, 4.1%) of CD31-immunostained microvessels compared with background fibroglandular tissue (mean, 1.2%). CONCLUSION: Cystic apocrine metaplasia should be considered in the differential diagnosis of a T2-hyperintense enhancing focus or subcentimeter smoothly marginated mass, even if associated with washout kinetics. Cystic apocrine metaplasia contains a statistically significant increase in microvessel area compared with background fibroglandular tissue and fat and, therefore, may be considered a concordant result for this set of imaging findings.

Failure of Primary Total Hip Arthroplasty Secondary to New Onset of Paget Disease: A Case Report.

CASE: Paget disease of bone occurs in 3% to 4% of the population over fifty years of age, more often in men than in women. There is no known genetic basis for the disease, which is often diagnosed via incidental radiographic findings. We present a seventy-seven-year-old man with new-onset Paget disease after total hip arthroplasty, leading to loosening of the femoral stem and necessitating revision. CONCLUSION: This case emphasizes the need for thoughtful evaluation of patients with failed arthroplasty and proposes steps for an appropriate workup prior to revision surgery.

All-trans-retinoic acid antagonizes the Hedgehog pathway by inducing patched.

Male germ cell tumors (GCTs) are a model for a curable solid tumor. GCTs can differentiate into mature teratomas. Embryonal carcinomas (ECs) represent the stem cell compartment of GCTs and are the malignant counterpart to embryonic stem (ES) cells. GCTs and EC cells are useful to investigate differentiation therapy and chemotherapy response. This study explored mechanistic interactions between all-trans-retinoic acid (RA), which induces differentiation of EC and ES cells, and the Hedgehog (Hh) pathway, a regulator of self-renewal and proliferation. RA was found to induce mRNA and protein expression of Patched 1 (Ptch1), the Hh ligand receptor and negative regulator of this pathway. PTCH1 is also a target gene of Hh signaling through Smoothened (Smo) activation. Yet, this observed RA-mediated Ptch1 induction was independent of Smo. It occurred despite co-treatment with RA and Smo inhibitors. Retinoid induction of Ptch1 also occurred in other RA-responsive cancer cell lines and in normal ES cells. Notably, this enhanced Ptch1 expression was preceded by induction of the homeobox transcription factor Meis1, a direct RA target. Direct interaction between Meis1 and Ptch1 was confirmed using chromatin immunoprecipitation assays. To establish the translational relevance of this work, Ptch1 expression was shown to be deregulated in human ECs relative to mature teratoma and the normal seminiferous tubule. Taken together, these findings reveal a previously unrecognized mechanism through which RA can inhibit the Hh pathway via Ptch1 induction. Engaging this pathway is a new way to repress the Hh pathway that can be translated into the cancer clinic.

Comparing histone deacetylase inhibitor responses in genetically engineered mouse lung cancer models and a window of opportunity trial in patients with lung cancer.

Histone deacetylase inhibitor (HDACi; vorinostat) responses were studied in murine and human lung cancer cell lines and genetically engineered mouse lung cancer models. Findings were compared with a window of opportunity trial in aerodigestive tract cancers. In human (HOP62, H522, and H23) and murine transgenic (ED-1, ED-2, LKR-13, and 393P, driven, respectively, by cyclin E, degradation-resistant cyclin E, KRAS, or KRAS/p53) lung cancer cell lines, vorinostat reduced growth, cyclin D1, and cyclin E levels, but induced p27, histone acetylation, and apoptosis. Other biomarkers also changed. Findings from transgenic murine lung cancer models were integrated with those from a window of opportunity trial that measured vorinostat pharmacodynamic responses in pre- versus posttreatment tumor biopsies. Vorinostat repressed cyclin D1 and cyclin E expression in murine transgenic lung cancers and significantly reduced lung cancers in syngeneic mice. Vorinostat also reduced cyclin D1 and cyclin E expression, but increased p27 levels in post- versus pretreatment human lung cancer biopsies. Notably, necrotic and inflammatory responses appeared in posttreatment biopsies. These depended on intratumoral HDACi levels. Therefore, HDACi treatments of murine genetically engineered lung cancer models exert similar responses (growth inhibition and changes in gene expression) as observed in lung cancer cell lines. Moreover, enhanced pharmacodynamic responses occurred in the window of opportunity trial, providing additional markers of response that can be evaluated in subsequent HDACi trials. Thus, combining murine and human HDACi trials is a strategy to translate preclinical HDACi treatment outcomes into the clinic. This study uncovered clinically tractable mechanisms to engage in future HDACi trials.

Cytomorphologic features of advanced lung adenocarcinomas tested for EGFR and KRAS mutations: a retrospective review of 50 cases.

Associations between bronchioloalveolar carcinoma (BAC), mucinous differentiation, and epidermal growth factor receptor (EGFR) and KRAS mutations have been previously reported in studies of surgical specimens. We present the cytomorphology of lung adenocarcinomas, including metastases that were diagnosed by cytologic methods and the relationship to both EGFR and KRAS mutational status. We retrospectively reviewed the clinical and cytomorphologic features of 50 lung adenocarcinomas that were tested for both EGFR and KRAS mutations. Cytomorphologic features evaluated included cell size, architectural pattern, nucleoli, intranuclear cytoplasmic inclusions (INCI), mucin, necrosis, squamoid features, lymphocytic response, and histologic features of BAC differentiation. DNA was extracted from a paraffin-embedded cell block or frozen needle core fragments. Exon 19 deletions and the L858R mutation in exon 21 of EGFR were detected using PCR followed by capillary electrophoresis for fragment sizing. KRAS mutational analysis was performed by real-time PCR using a set of seven different Taqman(r) allelic discrimination assays to detect six mutations in codon 12 and one mutation in codon 13. Six cases (12%) showed EGFR mutations, 12 (24%) showed KRAS mutations, and 38 (62%) contained neither EGFR nor KRAS mutations. The majority of patients had stage IV disease (78%); 20 samples (40%) were from metastatic sites. The presence of prominent INCI (P = 0.036), papillary fragments (P = 0.041), and histologic features of BAC on paraffin block (P = 0.039) correlated with the presence of EGFR mutations. The presence of necrosis (P = 0.030), squamoid features (P = 0.048), and poorly differentiated tumors (P = 0.025) were more likely to be identified in the KRAS positive group.

Response to inhibition of smoothened in diverse epithelial cancer cells that lack smoothened or patched 1 mutations.

Hedgehog (HH) pathway Smoothened (Smo) inhibitors are active against Gorlin syndrome-associated basal cell carcinoma (BCC) and medulloblastoma where Patched (Ptch) mutations occur. We interrogated 705 epithelial cancer cell lines for growth response to the Smo inhibitor cyclopamine and for expressed HH pathway-regulated species in a linked genetic database. Ptch and Smo mutations that respectively conferred Smo inhibitor response or resistance were undetected. Previous studies revealed HH pathway activation in lung cancers. Therefore, findings were validated using lung cancer cell lines, transgenic and transplantable murine lung cancer models, and human normal-malignant lung tissue arrays in addition to testing other Smo inhibitors. Cyclopamine sensitivity most significantly correlated with high cyclin E (P=0.000009) and low insulin-like growth factor binding protein 6 (IGFBP6) (P=0.000004) levels. Gli family members were associated with response. Cyclopamine resistance occurred with high GILZ (P=0.002) expression. Newer Smo inhibitors exhibited a pattern of sensitivity similar to cyclopamine. Gain of cyclin E or loss of IGFBP6 in lung cancer cells significantly increased Smo inhibitor response. Cyclin E-driven transgenic lung cancers expressed a gene profile implicating HH pathway activation. Cyclopamine treatment significantly reduced proliferation of murine and human lung cancers. Smo inhibition reduced lung cancer formation in a syngeneic mouse model. In human normal-malignant lung tissue arrays cyclin E, IGFBP6, Gli1 and GILZ were each differentially expressed. Together, these findings indicate that Smo inhibitors should be considered in cancers beyond those with activating HH pathway mutations. This includes tumors that express genes indicating basal HH pathway activation.

Evidence for the ubiquitin protease UBP43 as an antineoplastic target.

New pharmacologic targets are needed for lung cancer. One candidate pathway to target is composed of the E1-like ubiquitin-activating enzyme (UBE1L) that associates with interferon-stimulated gene 15 (ISG15), which complexes with and destabilizes cyclin D1. Ubiquitin protease 43 (UBP43/USP18) removes ISG15 from conjugated proteins. This study reports that gain of UBP43 stabilized cyclin D1, but not other D-type cyclins or cyclin E. This depended on UBP43 enzymatic activity; an enzymatically inactive UBP43 did not affect cyclin D1 stability. As expected, small interfering RNAs that reduced UBP43 expression also decreased cyclin D1 levels and increased apoptosis in a panel of lung cancer cell lines. Forced cyclin D1 expression rescued UBP43 apoptotic effects, which highlighted the importance of cyclin D1 in conferring this. Short hairpin RNA-mediated reduction of UBP43 significantly increased apoptosis and reduced murine lung cancer growth in vitro and in vivo after transplantation of these cells into syngeneic mice. These cells also exhibited increased response to all-trans-retinoic acid, interferon, or cisplatin treatments. Notably, gain of UBP43 expression antagonized these effects. Normal-malignant human lung tissue arrays were examined independently for UBP43, cyclin D1, and cyclin E immunohistochemical expression. UBP43 was significantly (P < 0.01) increased in the malignant versus normal lung. A direct relationship was found between UBP43 and cyclin D1 (but not cyclin E) expression. Differential UBP43 expression was independently detected in a normal-malignant tissue array with diverse human cancers. Taken together, these findings uncovered UBP43 as a previously unrecognized antineoplastic target.