Accuracy of endobronchial ultrasound-guided transbronchial needle aspiration for staging of non-small cell lung cancer.

BACKGROUND: Endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) is routinely performed to confirm a lung cancer diagnosis and/or to clinically stage disease. EBUS-TBNA findings may be used to determine whether patients can be offered potentially curative surgery. In this study, we evaluated the reporting in our service of EBUS-TBNA cytology for early-stage (operable) non-small cell lung cancer (NSCLC), focusing on diagnostic accuracy and analyzing cases with discordant cytologic and post-surgical histopathologic conclusions. METHODS: Cytology slides and cytopathology reports of 120 NSCLC patients who had undergone EBUS-TBNA and lobectomy in our hospital system between 2015 and 2021 were retrospectively reviewed. RESULTS: Of 290 lymph nodes (110 cases) able to be reviewed, interpretation of 48 lymph nodes was discordant with the original cytopathology report. This included 31 lymph nodes originally reported as adequate, which were found to be non-diagnostic on review. The diagnostic accuracy (benign/malignant) of lymph nodes that were sampled at EBUS-TBNA and excised at surgery was 89%. Specific examination of cases where EBUS-TBNA cytology did not reflect post-surgical findings illustrated important features and limitations of the procedure. These included potential misclassification of lymph node stations, the presence of multiple, variably involved nodes at lymph node stations, and the failure to detect small volume disease. CONCLUSIONS: Continuous evaluation of EBUS-TBNA performance identifies technical limitations and areas of improvement for cytopathology reporting. This is increasingly important in an era where lung cancer screening is expected to increase diagnosis of early-stage disease and with the advent of novel treatments, including non-surgical management options.

Detection of metastases using circulating tumour DNA in uveal melanoma.

BACKGROUND: Approximately 50% of uveal melanoma (UM) patients will develop metastatic disease depending on the genetic features of the primary tumour. Patients need 3-12 monthly scans, depending on their prognosis, which is costly and often non-specific. Circulating tumour DNA (ctDNA) quantification could serve as a test to detect and monitor patients for early signs of metastasis and therapeutic response. METHODS: We assessed ctDNA as a biomarker in three distinct UM cohorts using droplet-digital PCR: (A) a retrospective analysis of primary UM patients to predict metastases; (B) a prospective analysis of UM patients after resolution of their primary tumour for early detection of metastases; and (C) monitoring treatment response in metastatic UM patients. RESULTS: Cohort A: ctDNA levels were not associated with the development of metastases. Cohort B: ctDNA was detected in 17/25 (68%) with radiological diagnosis of metastases. ctDNA was the strongest predictor of overall survival in a multivariate analysis (HR = 15.8, 95% CI 1.7-151.2, p = 0.017). Cohort C: ctDNA monitoring of patients undergoing immunotherapy revealed a reduction in the levels of ctDNA in patients with combination immunotherapy. CONCLUSIONS: Our proof-of-concept study shows the biomarker feasibility potential of ctDNA monitoring in for the clinical management of uveal melanoma patients.

Discordance between clinical and pathologic staging and the timeliness of care of non-small cell lung cancer patients diagnosed with operable tumors.

AIM: This study was performed to evaluate concordance between clinical and pathologic staging of non-small cell lung cancer (NSCLC) in our hospital network. METHODS: We retrospectively reviewed records of 417 patients with NSCLC who received curative surgery and whose pathology was evaluated in our hospital between 2016 and 2021. Cytology, tissue pathology, and associated clinical, surgical, and imaging information were retrieved from hospital digital records. RESULTS: The cohort included 214 female and 203 male patients aged 20.6-85.8 years. Median times among staging computed tomography and surgery (105 days [interquartile range (IQR) 77.0-143.0]), positron emission tomography and surgery (78.5 days [IQR 56.0-109.0]), and endobronchial ultrasound-guided transbronchial needle aspiration and surgery (59 days [IQR 42-94]) indicated that Australian guidelines of <42 days between original referral and commencement of treatment were not being met in the majority of cases. Discordance between clinical TNM (cTNM) and pathologic TNM staging was 25.9%, including 18.4% cases that were clinically understaged and two patients with undetected stage IVA disease. cTNM understaging was significantly associated with time between the final staging investigation and surgery (p = .023), pleural (p < .05) and vessel (p < .05) invasion, and diagnosis of high-grade adenocarcinoma (p = .001). CONCLUSION: Discordance between clinical and pathologic staging of NSCLC is associated with tumor histopathologic characteristics and treatment delays. Although tumor factors that lead to discordant staging cannot be controlled, reduced time to surgery may have resulted in better outcomes for some patients in this potentially curable lung cancer cohort.

Application of multiplex ligation-dependent probe amplification (MLPA) and low pass whole genome sequencing (LP-WGS) to the classification / characterisation of low grade glioneuronal tumours.

AIMS: Glioneuronal tumours, although rare, are an important cause of treatment-resistant epilepsy. Differential diagnosis of glioneuronal tumour subtypes is complicated by their variable histological appearance and the lack of pathognomonic histological or molecular biomarkers. In this study we have applied techniques available in a diagnostic laboratory setting to characterise molecular and cytogenetic abnormalities in a single institution cohort of glioneuronal tumours. METHODS: A cohort of 29 glioneuronal tumours that included 21 gangliogliomas and 5 dysembryoplastic neuroepithelial tumours (DNETs) was analysed using low pass whole genome sequencing (WGS) and 2 multiplex ligation-dependent probe amplification (MLPA) central nervous system (CNS) tumour probesets. RESULTS: Low pass WGS identified chromosomal or subchromosomal alterations in 15 specimens. The most common chromosomal alterations were gains of chromosome 7 (n = 8) and chromosome 16 (n = 3). The BRAFV600E mutation was detected by MLPA in 9/21 (42.9%) gangliogliomas and 2/2 pleomorphic xanthoastrocytomas (PXAs). Chromosome 7 gains detected by WGS were reflected in MLPAs by overall gains of chromosome 7 gene probes, including those for BRAF, KIAA1549 and EGFR, while an internal BRAF/MKRN1 duplication was detected in a single ganglioglioma. Homozygous CDKN2A/B loss was detected by MLPA in 3 gangliogliomas, with p16 immunohistochemistry supporting these results. CONCLUSIONS: The combination of low pass WGS and MLPA identifies multiple, diverse genetic and chromosomal alterations in glioneuronal tumours, irrespective of histological tumour grade.

Analysis of Circulating Tumour Cells in Early-Stage Uveal Melanoma: Evaluation of Tumour Marker Expression to Increase Capture.

(1) Background: The stratification of uveal melanoma (UM) patients into prognostic groups is critical for patient management and for directing patients towards clinical trials. Current classification is based on clinicopathological and molecular features of the tumour. Analysis of circulating tumour cells (CTCs) has been proposed as a tool to avoid invasive biopsy of the primary tumour. However, the clinical utility of such liquid biopsy depends on the detection rate of CTCs. (2) Methods: The expression of melanoma, melanocyte, and stem cell markers was tested in a primary tissue microarray (TMA) and UM cell lines. Markers found to be highly expressed in primary UM were used to either immunomagnetically isolate or immunostain UM CTCs prior to treatment of the primary lesion. (3) Results: TMA and cell lines had heterogeneous expression of common melanoma, melanocyte, and stem cell markers. A multi-marker panel of immunomagnetic beads enabled isolation of CTCs in 37/43 (86%) patients with UM. Detection of three or more CTCs using the multi-marker panel, but not MCSP alone, was a significant predictor of shorter progression free (p = 0.040) and overall (p = 0.022) survival. (4) Conclusions: The multi-marker immunomagnetic isolation protocol enabled the detection of CTCs in most primary UM patients. Overall, our results suggest that a multi-marker approach could be a powerful tool for CTC separation for non-invasive prognostication of UM.

Lack of correlation between MSH3 immunohistochemistry and microsatellite analysis for the detection of elevated microsatellite alterations at selected tetranucleotide repeats (EMAST) in colorectal cancers.

Immunohistochemical evaluation of mismatch repair protein (MMR) expression is an important screening tool in diagnostic pathology, where it is routinely used to identify subsets of colorectal cancers (CRCs) with either inherited or sporadic forms of microsatellite instability (MSI). MSH3 is not included in current MMR panels, although aberrant MSH3 expression is reported to occur in 40-60% of CRCs and is associated with elevated microsatellite alterations at selected tetranucleotide repeats (EMAST) and a worse prognosis. In this study, we applied MSH3 immunohistochemistry and tetranucleotide MSI analysis to a cohort of 250 unselected CRCs to evaluate the potential use of the methods in routine practice. Partial, complete, and focal loss of nuclear MSH3 and its cytoplasmic mislocalization were evident in 67% of tumors, whereas MSI was evident in two to six of a panel of six tetranucleotide repeats in 46% of cases. However, concordance between MSH3 immunohistochemistry and tetranucleotide MSI results was only 61%, indicating the unsuitability of this combination of tests in routine pathology practice. MSH3 immunostaining was compromised in areas of tissue crush and autolysis, which are common in biopsy and surgical samples, potentially mitigating against its routine use. Although tetranucleotide MSI is clearly evident in a subset of CRCs, further development of validated sets of tetranucleotide repeats and either MSH3 or other immunohistochemical markers will be required to include EMAST testing in the routine evaluation of CRCs in clinical practice.

Low-Pass Whole-Genome Sequencing as a Method of Determining Copy Number Variations in Uveal Melanoma Tissue Samples.

Analysis of specific somatic copy number alterations (SCNAs) using multiplex ligation-dependent probe amplification (MLPA) is used routinely as a prognostic test for uveal melanoma (UM). This technique requires relatively large amounts of input DNA, unattainable from many small fine-needle aspirate biopsy specimens. Herein, we compared the use of MLPA with whole-genome amplification (WGA) combined with low-pass whole-genome sequencing (LP-WGS) for detection of SCNA profiles in UM biopsy specimens. DNA was extracted from 21 formalin-fixed, paraffin-embedded UM samples and SCNAs were assessed using MLPA and WGA followed by LP-WGS. Cohen's κ was used to assess the concordance of copy number calls of each individual chromosome arm for each patient. MLPA and WGA/LP-WGS detection of SCNAs in chromosomes 1p, 3, 6, and 8 were compared and found to be highly concordant with a Cohen's κ of 0.856 (bias-corrected and accelerated 95% CI, 0.770-0.934). Only 13 of 147 (8.8%) chromosomal arms investigated resulted in discordant calls, predominantly SCNAs detected by WGA/LP-WGS but not MLPA. These results indicate that LP-WGS might be a suitable alternative or adjunct to MLPA for the detection of SCNAs associated with prognosis of UM, for cases with limiting tissue or DNA yields.

ETS1 induces transforming growth factor ß signaling and promotes epithelial-to-mesenchymal transition in prostate cancer cells.

Expression of the transcriptional regulator, E26 transformation-specific 1 (ETS1), is elevated in human prostate cancers, and this is associated with more aggressive tumor behavior and a rapid progression to castrate-resistant disease. Multiple ETS1 isoforms with distinct biological activities have been characterized and in 44 matched nonmalignant and malignant human prostate specimens, messenger RNAs for two ETS1 isoforms, ETS1p51 and ETS1p42, were detected, with ETS1p51 levels significantly lower in prostate tumor compared to matched nonmalignant prostate tissues. In contrast, ETS1p51 protein, the only ETS1 isoform detected, was expressed at significantly higher levels in malignant prostate. Analysis of epithelial-to-mesenchymal transition (EMT)-associated genes regulated following overexpression of ETS1p51 in the LNCaP prostate cancer cell line predicted promotion of transforming growth factor ß (TGFß) signaling and of EMT. ETS1p51 overexpression upregulated cellular levels of the EMT transcriptional regulators, ZEB1 and SNAIL1, resulted in reduced expression of the mesenchymal marker vimentin with concomitantly elevated levels of claudin 1, an epithelial tight junction protein, and increased prostate cancer cell migration and invasion. ETS1p51-induced activation of the pro-EMT TGFß signaling pathway that was predicted in polymerase chain reaction arrays was verified by demonstration of elevated SMAD2 phosphorylation following ETS1p51 overexpression. Attenuation of ETS1p51 effects on prostate cancer cell migration and invasion by inhibition of TGFß pathway signaling indicated that ETS1p51 effects were in part mediated by induction of TGFß signaling. Thus, overexpression of ETS1p51, the predominant ETS1 isoform expressed in prostate tumors, promotes an EMT program in prostate cancer cells in part via activation of TGFß signaling, potentially accounting for the poor prognosis of ETS1-overexpressing prostate tumors.

The case for DNA methylation based molecular profiling to improve diagnostic accuracy for central nervous system embryonal tumors (not otherwise specified) in adults.

Central nervous system primitive neuro-ectodermal tumors (CNS-PNETs), have recently been re-classified in the most recent 2016 WHO Classification into a standby catch all category, "CNS Embryonal Tumor, not otherwise specified" (CNS embryonal tumor, NOS) based on epigenetic, biologic and histopathologic criteria. CNS embryonal tumors (NOS) are a rare, histologically and molecularly heterogeneous group of tumors that predominantly affect children, and occasionally adults. Diagnosis of this entity continues to be challenging and the ramifications of misdiagnosis of this aggressive class of brain tumors are significant. We report the case of a 45-year-old woman who was diagnosed with a central nervous system embryonal tumor (NOS) based on immunohistochemical analysis of the patient's tumor at diagnosis. However, later genome-wide methylation profiling of the diagnostic tumor undertaken to guide treatment, revealed characteristics most consistent with IDH-mutant astrocytoma. DNA sequencing and immunohistochemistry confirmed the presence of IDH1 and ATRX mutations resulting in a revised diagnosis of high-grade small cell astrocytoma, and the implementation of a less aggressive treatment regime tailored more appropriately to the patient's tumor type. This case highlights the inadequacy of histology alone for the diagnosis of brain tumours and the utility of methylation profiling and integrated genomic analysis for the diagnostic verification of adults with suspected CNS embryonal tumor (NOS), and is consistent with the increasing realization in the field that a combined diagnostic approach based on clinical, histopathological and molecular data is required to more accurately distinguish brain tumor subtypes and inform more effective therapy.

miR-331-3p and Aurora Kinase inhibitor II co-treatment suppresses prostate cancer tumorigenesis and progression.

RNA-based therapeutics could represent a new avenue of cancer treatment. miRNA 331-3p (miR-331-3p) is implicated in prostate cancer (PCa) as a putative tumor suppressor, but its functional activity and synergy with other anti-tumor agents is largely unknown. We found miR-331-3p expression in PCa tumors was significantly decreased compared to non-malignant matched tissue. Analysis of publicly available PCa gene expression data sets showed miR-331-3p expression negatively correlated with Gleason Score, tumor stage, lymph node involvement and PSA value, and was significantly down regulated in tumor tissue relative to normal prostate tissue. Overexpression of miR-331-3p reduced PCa cell growth, migration and colony formation, as well as xenograft tumor initiation, proliferation and survival of mice. Microarray analysis identified seven novel targets of miR-331-3p in PCa. The 3'-untranslated regions of PLCγ1 and RALA were confirmed as targets of miR-331-3p, with mutation analyses confirming RALA as a direct target. Expression of miR-331-3p or RALA siRNA in PCa cells reduced RALA expression, proliferation, migration and colony formation in vitro. RALA expression positively correlated with Gleason grade in two separate studies, as well as in a PCa tissue microarray. Co-treatment using siRALA with an Aurora Kinase inhibitor (AKi-II) decreased colony formation of PCa cells while the combination of AKi-II with miR-331-3p resulted in significant reduction of PCa cell proliferation in vitro and PCa xenograft growth in vivo. Thus, miR-331-3p directly targets the RALA pathway and the addition of the AKi-II has a synergistic effect on tumor growth inhibition, suggesting a potential role as combination therapy in PCa.

Erdheim-Chester disease associated with a novel, complex BRAF p.Thr599_Val600delinsArgGlu mutation.

BRAF mutation testing to determine eligibility for treatment with vemurafenib was performed on archival skin lesions of a 54-year-old patient diagnosed with Erdheim-Chester disease (ECD) in 1999. Sanger sequencing of DNA extracted from a 2008 skin lesion identified two non-contiguous base substitutions in BRAF, which were shown by next-generation sequencing (NGS) to be located in the same allele. Due to its long-standing duration, molecular evolution of disease was possible; however, both Sanger and NGS of a 2000 skin lesion were unsuccessful due to the poor quality of DNA. Finally, droplet digital PCR using a probe specific for this novel mutation detected the complex BRAF mutation in both the 2000 and 2008 lesions, indicating this case to be ECD with a novel underlying BRAF p.Thr599_Val600delinsArgGlu mutation. Although well at present, molecular modelling of the mutant BRAF suggests suboptimal binding of vemurafenib and hence reduced therapeutic effectiveness.

Activity of ABCG2 Is Regulated by Its Expression and Localization in DHT and Cyclopamine-Treated Breast Cancer Cells.

Elevated expression of the efflux transporter, ATP-binding cassette subfamily G isoform 2 (ABCG2) on the plasma membrane of cancer cells contributes to the development of drug resistance and is a key characteristic of cancer stem cells. In this study, gene expression analysis identified that treatment of the MCF-7 and T-47D breast cancer cell lines with the androgen, 5α-dihydrotestosterone (DHT), and the Hedgehog signaling inhibitor, cyclopamine downregulated ABCG2 mRNA levels. In MCF-7 cells, and in Hoechst 33342(lo) /CD44(hi) /CD24(lo) breast cancer stem-like cells isolated from MCF-7 cultures, ABCG2 was accumulated in cell-to-cell junction complexes and in large cytoplasmic aggresome-like vesicles. DHT treatments, which decreased cellular ABCG2 protein levels, led to diminished ABCG2 localization in both cell-to-cell junction complexes and in cytoplasmic vesicles. In contrast, cyclopamine, which did not alter ABCG2 protein levels, induced accumulation of ABCG2 in cytoplasmic vesicles, reducing its localization in cell-to-cell junction complexes. The reduced localization of ABCG2 at the plasma membrane of MCF-7 cells was associated with decreased efflux of the ABCG2 substrate, mitoxantrone, and increased sensitivity of cyclopamine-treated cultures to the cytotoxic effects of mitoxantrone. Together, these findings indicate that DHT and cyclopamine reduce ABCG2 activity in breast cancer cells by distinct mechanisms, providing evidence to advocate the adjunct use of analogous pharmaceutics to increase or prolong the efficacy of breast cancer treatments. J. Cell. Biochem. 117: 2249-2259, 2016. © 2016 Wiley Periodicals, Inc.

Loss of the nuclear receptor corepressor SLIRP compromises male fertility.

Nuclear receptors (NRs) and their coregulators play fundamental roles in initiating and directing gene expression influencing mammalian reproduction, development and metabolism. SRA stem Loop Interacting RNA-binding Protein (SLIRP) is a Steroid receptor RNA Activator (SRA) RNA-binding protein that is a potent repressor of NR activity. SLIRP is present in complexes associated with NR target genes in the nucleus; however, it is also abundant in mitochondria where it affects mitochondrial mRNA transcription and energy turnover. In further characterisation studies, we observed SLIRP protein in the testis where its localization pattern changes from mitochondrial in diploid cells to peri-acrosomal and the tail in mature sperm. To investigate the in vivo effects of SLIRP, we generated a SLIRP knockout (KO) mouse. This animal is viable, but sub-fertile. Specifically, when homozygous KO males are crossed with wild type (WT) females the resultant average litter size is reduced by approximately one third compared with those produced by WT males and females. Further, SLIRP KO mice produced significantly fewer progressively motile sperm than WT animals. Electron microscopy identified disruption of the mid-piece/annulus junction in homozygous KO sperm and altered mitochondrial morphology. In sum, our data implicates SLIRP in regulating male fertility, wherein its loss results in asthenozoospermia associated with compromised sperm structure and mitochondrial morphology.

ETS1 regulates NKX3.1 5' promoter activity and expression in prostate cancer cells.

BACKGROUND: NKX3.1 controls the differentiation and proliferation of prostatic epithelial cells both during development and in the adult, while its expression is frequently downregulated in prostate cancers. Transcriptional control of NKX3.1 expression and in particular, factors that function via the NKX3.1 5' proximal promoter are poorly characterized. METHODS: Deletion reporter analyses, bioinformatics, electromobility shift assays (EMSA), chromatin immunoprecipitation (ChIP) and Western blotting were performed to identify and functionally characterize sites of transcription factor binding within the initial 2,062 bp of the NKX3.1 5' promoter. RESULTS: Deletion reporter studies of the 2,062 bp NKX3.1 5' promoter sequence localized positive transcriptional activity between -1069 and -993. Bioinformatic analyses identified the presence of two overlapping ETS1 binding sites within this region, designated EBS1 and EBS2, which exhibited 82% and 74% homology, respectively, to the ETS consensus binding sequence. EMSA and supershift assays indicated binding of both endogenous ETS1 and a recombinant GST-ETS1 protein solely to EBS1, a result that was confirmed in vivo by ChIP analysis. ETS1 overexpression transactivated NKX3.1 promoter reporter activity and upregulated endogenous NKX3.1 mRNA and protein levels in the LNCaP prostate cancer cell line, demonstrating a functional role for ETS1 in the regulation of NKX3.1 expression. CONCLUSIONS: ETS1 upregulation of NKX3.1 expression in LNCaP cells is mediated in part via its interaction with an EBS located in the NKX3.1 5' proximal promoter. ETS1 may regulate NKX3.1 during prostate development, with the aberrant ETS1 expression and cellular localization frequently observed in human prostate tumors potentially contributing to the abnormal expression of NKX3.1.

Androgen regulation of the prostatic tumour suppressor NKX3.1 is mediated by its 3' untranslated region.

The homeodomain transcription factor NKX3.1 is a prostate-specific tumour suppressor, expression of which is reduced or undetectable in the majority of metastatic prostate tumours. In the normal prostate and in prostate cancer cells, NKX3.1 expression is under tight androgenic control that we have shown to be mediated by its ~2.5 kb 3'UTR (3' untranslated region). Reporter deletion analysis of the NKX3.1 3'UTR identified three regions that were transactivated by DHT (5alpha-dihydrotestosterone) in the AR (androgen receptor)-expressing prostate cancer cell line LNCaP. Reversal of DHT effects by the anti-androgen bicalutamide supported an AR-mediated mechanism, and bioinformatic analysis of the NKX3.1 3'UTR identified canonical AREs (androgen-response elements) in each of the androgen-responsive regions. EMSAs (electrophoretic mobility-shift assays) indicated binding of the AR DNA-binding domain to two of the AREs, a proximal ARE at +2378-2392 from the transcription start site, and a more distal ARE at +3098-3112. ChIP (chromatin immunoprecipitation) analysis provided further evidence of ligand-dependent recruitment of endogenous AR to sequence encompassing each of the two elements, and site-directed mutagenesis and deletion analysis confirmed the contribution of each of the AREs in reporter assays. The present studies have therefore demonstrated that the NKX3.1 3'UTR functions as an androgen-responsive enhancer, with the proximal ARE contributing the majority and the distal ARE providing a smaller, but significant, proportion of the androgen responsiveness of the NKX3.1 3'UTR. Characterization of androgen-responsive regions of the NKX3.1 gene will assist in the identification of transcriptional regulatory mechanisms that lead to the deregulation of NKX3.1 expression in advanced prostate cancers.

ERK/MAPK regulation of the androgen responsiveness of breast cancer cells.

The androgen receptor (AR) is the most widely expressed steroid hormone receptor in human breast cancers and androgens including 5alpha-dihydrotestosterone are potent inhibitors of breast cancer cell proliferation. The extracellular signal-regulated mitogen activated protein kinase (ERK/MAPK) pathway is hyperactivated in a proportion of breast tumors and can interact with steroid hormone receptor signaling by altering receptor phosphorylation, turnover, ligand, and cofactor interactions. To examine the effects of ERK/ MAPK hyperactivity on AR levels, MCF-7 cells were stably transfected with a plasmid encoding a constitutively active MEK1 protein to create MCF-7-DeltaMEK1 cells. Treatment of MCF-7-DeltaMEK1 with androgens caused a transient increase in AR protein levels, similar to that observed in untransfected MCF-7 cells treated with androgens. Androgens also inhibited the proliferation of MCF-7-DeltaMEK1 cells by 50-60% following 8 days of treatment in association with increased accumulation of cells in the G1 phase of the cell cycle. These results indicate that although ERK/MAPK hyperactivation in breast cancer cells is associated with reduced estrogen receptor (ERalpha) levels and antiestrogen resistance, AR levels are maintained and breast cancer cells remain susceptible to the growth inhibitory effects of androgens.

The intracellular and nuclear-targeted delivery of an antiandrogen drug by carrier peptides.

Cell permeable carrier peptides are currently of interest for their potential to improve the delivery of bioactive molecules into cells and to specific cellular compartments. We have investigated the activity of a derivative of the antiandrogen drug, bicalutamide, attached to the cell-permeable carrier peptide penetratin(R). We have used both disulfide (labile) and thioether (nonlabile) linkages to attach the bicalutamide derivative to the peptide in order to assess whether one type of chemistry has advantages over the other. In addition we have added a nuclear localization sequence (NLS) to the carrier peptide to investigate whether localization of the drug to the nucleus of the cell affects the activity of the drug. Biotin-labeled peptides were used to demonstrate that the carrier peptide is rapidly accumulated inside cultured cells, and that the incorporation of an NLS in the sequence results in its nuclear targeting. The bicalutamide derivative linked to carrier peptides via a disulfide-linkage exerted no greater antiproliferative effect in LNCaP cells, than the bicalutamide derivative alone. The bicalutamide derivative linked to the carrier peptide by a non-labile thioether linkage showed a similar activity profile. When the construct includes a nuclear targeting sequence, however, a markedly increased antiproliferative effect was observed. This study has thus shown that the activity of bicalutamide may be enhanced by the nonlabile attachment of a cell-permeable and nuclear-targeted peptide, which has implications for the development of novel antiandrogens for the treatment of prostate cancer.

Characterization of columnar cell lesions of the breast: immunophenotypic analysis of columnar alteration of lobules with prominent apical snouts and secretions.

Columnar cell lesions of the breast are detected with increasing frequency in routine pathology practice, in part as a result of the widespread biopsy of nonpalpable breast abnormalities detected by screening mammography. Immunohistochemical investigation of the lesions in relation to the normal breast or to other breast pathologies is not well characterized, and the malignant potential of this spectrum of lesions has not been examined clinically. In this study, a cohort of 45 breast specimens containing columnar cell lesions, in particular, columnar alteration of lobules with prominent apical snouts and secretions (CAPSS), was investigated for expression of a series of breast tumor biomarkers. Using a semiquantitative immunohistochemical scoring system, up-regulation of estrogen, progesterone, and androgen receptors in CAPSS lesions to levels not significantly different from that in in situ or invasive breast tumors was identified. In four cases where CAPSS within a specimen lacked expression of a steroid hormone receptor, the coexisting in situ or invasive carcinoma also lacked expression of that receptor. In 81% of CAPSS lesions, E-cadherin immunostaining was reduced in isolated foci of cells or was decreased in intensity in all cells within the lesion. Quantitation of Ki-67 immunostaining demonstrated that proliferation of cells within CAPSS lesions was increased, compared with normal breast epithelium, but was lower than that detected in in situ or invasive cancers within the same specimens. Results of these analyses indicate that CAPSS shares immunophenotypic alterations with other premalignant lesions, the clinical implications of which may be investigated using established breast tumor biomarkers.

Transcriptional regulation of the homeobox gene NKX3.1 by all-trans retinoic acid in prostate cancer cells.

NKX3.1 is a homeobox gene, expression of which is largely restricted to the adult prostatic epithelium. Loss of NKX3.1 expression has been linked to prostate carcinogenesis and disease progression and occurs in the absence of mutations in the coding region of the NKX3.1 gene. In this study, we have characterized regulation of NKX3.1 expression by all-trans retinoic acid (tRA), a naturally occurring vitamin A metabolite that is accumulated at high levels in the prostate. Using the prostate cancer cell line LNCaP, Western blot analysis revealed a approximately twofold induction of NKX3.1 protein levels following tRA exposure, with sequential analysis of NKX3.1 protein levels in cycloheximide co-treated cells indicating that tRA does not alter NKX3.1 protein turnover. The approximately 1.6-fold increase in NKX3.1 mRNA levels detected in tRA-treated LNCaP cells also occurred independently of new protein synthesis and was not mediated by changes in NKX3.1 mRNA stability. In contrast, nuclear run-on assays indicated that tRA treatment increased NKX3.1 transcription. To identify retinoid responsive regions of the NKX3.1 gene, DNA sequences encompassing approximately 2 kb of the NKX3.1 promoter or the entire 3'untranslated region (UTR) were cloned into luciferase reporter plasmids. Analysis of induced luciferase activity following transfection of these constructs into prostate cancer cells did not identify tRA responsiveness, however the 3'UTR was found to be strongly androgen responsive. These studies demonstrate that the NKX3.1 gene is a direct target of retinoid receptors and suggest that androgen regulation of NKX3.1 expression is mediated in part by the 3'UTR.

Differential modulation of cell cycle, apoptosis and PPARgamma2 gene expression by PPARgamma agonists ciglitazone and 9-hydroxyoctadecadienoic acid in monocytic cells.

We sought to compare the effects of the thiazolidinedione ciglitazone with the endogenous fatty acid PPARgamma agonists 9- and 13-hydroxyoctadecadienoic acid (9- and 13-HODE), in U937 monocytic cells. Ciglitazone and 9-HODE inhibited cell proliferation and all three agonists increased cellular content of C18:0 fatty acids. Ciglitazone and 13-HODE resulted in an increased percentage of cells in S phase and ciglitazone reduced the percentage of cells in G2/M phase of cell cycle, whilst 9-HODE increased the percentage of cells in G0/1 and reduced the fraction in S and G2/M phases. 9-HODE selectively induced apoptosis in U937 cells, and increased PPARgamma2 gene expression. Induction of apoptosis by 9-HODE was not abrogated by the presence of the PPARgamma antagonist GW9662. Synthetic (TZD) and endogenous fatty acid ligands for PPARgamma, ciglitazone and 9- and 13-HODE, possess differential, ligand specific actions in monocytic cells to regulate cell cycle progression, apoptosis and PPARgamma2 gene expression.