A Multimodal Approach to Discover Biomarkers for Taxane-Induced Peripheral Neuropathy (TIPN): A Study Protocol.

Introduction: Taxanes are a class of chemotherapeutics commonly used to treat various solid tumors, including breast and ovarian cancers. Taxane-induced peripheral neuropathy (TIPN) occurs in up to 70% of patients, impacting quality of life both during and after treatment. TIPN typically manifests as tingling and numbness in the hands and feet and can cause irreversible loss of function of peripheral nerves. TIPN can be dose-limiting, potentially impacting clinical outcomes. The mechanisms underlying TIPN are poorly understood. As such, there are limited treatment options and no tools to provide early detection of those who will develop TIPN. Although some patients may have a genetic predisposition, genetic biomarkers have been inconsistent in predicting chemotherapy-induced peripheral neuropathy (CIPN). Moreover, other molecular markers (eg, metabolites, mRNA, miRNA, proteins) may be informative for predicting CIPN, but remain largely unexplored. We anticipate that combinations of multiple biomarkers will be required to consistently predict those who will develop TIPN. Methods: To address this clinical gap of identifying patients at risk of TIPN, we initiated the Genetics and Inflammatory Markers for CIPN (GENIE) study. This longitudinal multicenter observational study uses a novel, multimodal approach to evaluate genomic variation, metabolites, DNA methylation, gene expression, and circulating cytokines/chemokines prior to, during, and after taxane treatment in 400 patients with breast cancer. Molecular and patient reported data will be collected prior to, during, and after taxane therapy. Multi-modal data will be used to develop a set of comprehensive predictive biomarker signatures of TIPN. Conclusion: The goal of this study is to enable early detection of patients at risk of developing TIPN, provide a tool to modify taxane treatment to minimize morbidity from TIPN, and improved patient quality of life. Here we provide a brief review of the current state of research into CIPN and TIPN and introduce the GENIE study design.

MCL1 nuclear translocation induces chemoresistance in colorectal carcinoma.

Colorectal cancer (CRC) is one of the most common and deadliest forms of cancer. Myeloid Cell Leukemia 1 (MCL1), a pro-survival member of the Bcl-2 protein family is associated with chemo-resistance in CRC. The ability of MCL1 to inhibit apoptosis by binding to the BH3 domains of pro-apoptotic Bcl-2 family members is a well-studied means by which this protein confers resistance to multiple anti-cancer therapies. We found that specific DNA damaging chemotherapies promote nuclear MCL1 translocation in CRC models. In p53null CRC, this process is associated with resistance to chemotherapeutic agents, the mechanism of which is distinct from the classical mitochondrial protection. We previously reported that MCL1 has a noncanonical chemoresistance capability, which requires a novel loop domain that is distinct from the BH3-binding domain associated with anti-apoptotic function. Herein we disclose that upon treatment with specific DNA-damaging chemotherapy, this loop domain binds directly to alpha-enolase which in turn binds to calmodulin; we further show these protein-protein interactions are critical in MCL1's nuclear import and chemoresistance. We additionally observed that in chemotherapy-treated p53-/- CRC models, MCL1 nuclear translocation confers sensitivity to Bcl-xL inhibitors, which has significant translational relevance given the co-expression of these proteins in CRC patient samples. Together these findings indicate that chemotherapy-induced MCL1 translocation represents a novel resistance mechanism in CRC, while also exposing an inherent and targetable Bcl-xL co-dependency in these cancers. The combination of chemotherapy and Bcl-xL inhibitors may thus represent a rational means of treating p53-/- CRC via exploitation of this unique MCL1-based chemoresistance mechanism.

Evaluation of Urinalysis-Based Screening for Urothelial Carcinoma in Patients With Lynch Syndrome.

BACKGROUND: Approximately 5% to 10% of patients with Lynch syndrome develop urothelial carcinoma. Current screening recommendations vary and are based on expert opinion. Practices need to be evaluated for clinical effectiveness. Our program utilizes urinalysis as a screening test, followed by additional evaluation of microscopic hematuria. OBJECTIVE: This study aimed to determine the clinical utility of a urinalysis-based screening approach for urothelial cancers in patients with Lynch syndrome. DESIGN: This is a retrospective review of a prospectively maintained cohort. SETTING: Patients with Lynch syndrome were managed at a tertiary referral center. PATIENTS: All patients with a Lynch syndrome diagnosis who had a screening urinalysis done as part of our institutional screening protocol (N = 204) were included. MAIN OUTCOME MEASURES: A single-institution hereditary colorectal cancer syndrome registry was queried for patients with Lynch syndrome who had been screened for urothelial carcinomas by urinalysis. Demographics, genotype, family history of urothelial carcinoma, urinalysis results, and subsequent screenings and final diagnosis were gathered for patients between 2008 and 2017. RESULTS: Two hundred four asymptomatic patients underwent screening by urinalysis. Nineteen patients (9.3%) had microscopic hematuria and were further evaluated with urine cytology, imaging, cystoscopy, and/or Urology consultation. None of the 19 patients with microscopic hematuria had urothelial carcinoma. During the same study period, 5 of 204 (2.4%) patients with Lynch syndrome were diagnosed with urothelial cancer, and all presented with symptoms between screening intervals. LIMITATIONS: This is a retrospective study, and not all patients underwent the same secondary evaluation. CONCLUSIONS: No urothelial carcinomas were detected by screening urinalysis in our cohort of asymptomatic patients with Lynch syndrome. False-positive testing led to extensive, mostly uninformative, workups. If urothelial cancer screening is to continue, more effective screening approaches need to be identified. See Video Abstract at http://links.lww.com/DCR/B702. EVALUACIN DEL CRIBADO BASADO EN ANLISIS DE ORINA PARA CARCINOMA UROTELIAL EN PACIENTES CON SNDROME DE LYNCH: ANTECEDENTES:Aproximadamente el 5-10% de los pacientes con síndrome de Lynch desarrollan carcinoma urotelial. Las recomendaciones actuales de detección varían y se basan en la opinión de expertos. Las prácticas deben evaluarse para determinar su eficacia clínica. Nuestro programa utiliza el análisis de orina como prueba de detección, seguido de una evaluación adicional con hematuria microscópica.OBJETIVO:Determinar la utilidad clínica desde un enfoque de cribado basado en análisis de orina, para cánceres uroteliales en pacientes con síndrome de Lynch.DISEÑO:Revisión retrospectiva de una cohorte mantenida prospectivamente.ENTORNO CLINICO:Pacientes con síndrome de Lynch atendidos en un centro de referencia terciario.PACIENTES:Criterios de inclusión fueron todos los pacientes con diagnóstico de síndrome de Lynch realizándoles un análisis de orina de detección como parte de nuestro protocolo de detección institucional (N = 204).PRINCIPALES MEDIDAS DE VALORACION:Solicitando un registro de síndrome de cáncer colorrectal hereditario de una sola institución para pacientes con síndrome de Lynch previamente evaluados para carcinomas uroteliales mediante análisis de orina. Se recopilaron para los pacientes entre 2008 y 2017, datos demográficos, genotipo, antecedentes familiares de carcinoma urotelial, resultados del análisis de orina, posteriores exámenes de detección posteriores y diagnóstico final.RESULTADOS:Doscientos cuatro pacientes asintomáticos fueron sometidos a cribado mediante análisis de orina. Diecinueve pacientes (9,3%) tenían hematuria microscópica y fueron investigados más a fondo con citología de orina, imágenes, cistoscopia y / o consulta de urología. Ninguno de los 19 pacientes con hematuria microscópica tenían carcinoma urotelial. Durante el mismo período de estudio, 5 de 204 (2,4%) pacientes con síndrome de Lynch fueron diagnosticados con cáncer urotelial y todos presentaron presentando síntomas entre los intervalos de detección.LIMITACIONES:Estudio retrospectivo y no todos los pacientes sometidos a la misma evaluación secundaria.CONCLUSIONES:No se detectaron carcinomas uroteliales mediante análisis de orina de detección en nuestra cohorte de pacientes asintomáticos con síndrome de Lynch. Las pruebas de falsos positivos. Condujeron a estudios exhaustivos y en su mayoría poco informativos. Si se desea continuar con la detección del cáncer de urotelio, es necesario identificar enfoques de detección más efectivos. Consulte Video Resumen en http://links.lww.com/DCR/B702.

Prediction of Poor Response to Neoadjuvant Chemoradiation in Patients With Rectal Cancer Using a DNA Repair Deregulation Score: Picking the Losers Instead of the Winners.

BACKGROUND: Patients with rectal cancer may undergo neoadjuvant chemoradiation even in early stages in an attempt to achieve complete clinical response and undergo organ preservation. However, prediction of tumor response is unavailable. In this setting, accurate identification of poor responders could spare patients with early stage disease from potentially unnecessary chemoradiation. OBJECTIVE: This study focused on development/test of a score based on DNA repair gene expression to predict response to neoadjuvant chemoradiation in patients with rectal cancer. DESIGN: Pretreatment biopsy samples from patients with rectal cancer undergoing neoadjuvant chemoradiation were collected and underwent gene expression analysis using RNA-Seq (test cohort). A score was constructed using 8 differentially expressed DNA repair genes between good (complete clinical) and poor responders (incomplete clinical) to treatment. The score was validated in 2 independent cohorts of patients undergoing similar treatment strategies and using quantitative polymerase chain reaction and microarray gene expression data. SETTINGS: This was a retrospective analysis of gene expression data from 3 independent institutions. PATIENTS: Patients with rectal cancer undergoing neoadjuvant chemoradiation (50.4-54.0 Gy and 5-fluorouracil-based chemotherapy) were eligible. Patients with complete clinical response, complete pathological response, or ≤10% residual cancer cells were considered good responders. Patients with >10% residual cancer cells were considered poor responders. The test cohort included 25 patients (16 poor responders). Validation cohort 1 included 28 patients (18 poor responders), and validation cohort 2 included 46 patients (22 poor responders). MAIN OUTCOMES MEASURES: Response was correlated with the DNA repair score calculated using the expression levels of 8 DNA repair genes. DNA repair score sensitivity, specificity, and positive and negative predictive values were determined in test and validation cohorts. RESULTS: Poor responders had significantly lower DNA repair scores when compared with good responders across all 3 cohorts, regardless of the gene expression platform used. A low score correctly predicted poor response in 93%, 90%, and 71% in test, validation 1, and validation 2 cohorts. LIMITATIONS: This study was limited by its small sample size, different gene expression platforms, and treatment regimens across different cohorts used. CONCLUSIONS: A DNA repair gene score may predict patients likely to have poor response to chemoradiation. This score may be a relevant tool to be investigated in future studies focused on chemoradiation used in the context of organ preservation. See Video Abstract at http://links.lww.com/DCR/B104. PREDICCIÓN DE RESPUESTA DEFICIENTE A LA RADIO-QUIMIOTERAPIA NEOADYUVANTE EN PACIENTES CON CÁNCER RECTAL UTILIZANDO UNA PUNTUACIÓN DE DESREGULACIÓN DE REPARACIÓN DE ADN: ESCOGER LOS PERDEDORES EN LUGAR DE LOS GANADORES: Los pacientes con cáncer rectal pueden someterse a radio-quimioterapia neoadyuvante incluso en estadios tempranos en el intento por lograr una respuesta clínica completa y permitir una preservación de órgano. Sin embargo, aun no existen herramientas disponible para la prediccion de la respuesta tumoral al tratamiento. En este contexto, la identificación precisa de los tumores con mala respuesta al tratamiento podría evitar que los pacientes con enfermedad en estadio temprano sean sometidos a radio-quimioterapia potencialmente innecesaria.Desarrollo / testeo de una puntuación basada en la expresión genes reparadores del ADN para predecir la respuesta a la nCRT en pacientes con cáncer rectal.Se recogieron muestras de biopsia de pre-tratamiento de pacientes con cáncer rectal sometidos a radio-quimioterapia neoadyuvante y se les realizó un análisis de expresión génica utilizando RNAseq (cohorte de prueba). Se construyó una puntuación utilizando 8 genes de reparación de ADN expresados diferencialmente entre buenos (respuesta clinica completa) y pobres respondedores (respuesta clinica incompleta) al tratamiento. La puntuación se validó en 2 cohortes independientes de pacientes sometidos a estrategias de tratamiento similares y utilizando qPCR y datos de expresión de genes en chips ADN (biotecnología -microarrays).Análisis retrospectivo de los datos de expresión génica de 3 instituciones independientes.Fueron incluidos aquellos pacientes con cáncer rectal sometidos a radio-quimioterapia neoadyuvante (50,4-54 Gy y quimioterapia basada en 5FU). Los pacientes con respuesta clínica completa, respuesta patológica completa o ≤10% de células cancerosas residuales se consideraron buenos respondedores. Los pacientes con> 10% de células cancerosas residuales se consideraron de respuesta deficiente. La cohorte de prueba incluyó a 25 pacientes (16 respondedores pobres). La cohorte de validación n. ° 1 incluyó a 28 pacientes (18 respondedores pobres) y la cohorte de validación n. ° 2 incluyó a 46 pacientes (22 respondedores pobres).La respuesta se correlacionó con la puntuación de reparación de ADN calculada utilizando los niveles de expresión de 8 genes de reparación de ADN. La sensibilidad del puntaje de reparación del ADN, la especificidad, los valores predictivos positivos y negativos se determinaron en las cohortes de prueba y validación.Los malos respondedores tuvieron puntuaciones de reparación de ADN significativamente más bajas en comparación con los buenos respondedores en las 3 cohortes, independientemente de la plataforma de expresión génica utilizada. Una puntuación baja predijo correctamente una respuesta pobre en el 93%, 90% y 71% en las cohortes de prueba, validación n. ° 1 y validación n. ° 2, respectivamente.Pequeño tamaño de la muestra, diferentes plataformas de expresión génica y regímenes de tratamiento en diferentes cohortes utilizadas.La puntuacion basada en genes de reparación del ADN puede predecir los pacientes con respuesta pobre a la radio-quimioterapia. Esta puntuación puede ser una herramienta relevante para investigar en futuros estudios centrados en la radio-quimioterapia utilizada en el contexto de la preservación de órganos. Consulte Video Resumen en http://links.lww.com/DCR/B104. (Traducción-Dr. Xavier Delgadillo and Dr. Laura Melina Fernandez).

CoA Synthase (COASY) Mediates Radiation Resistance via PI3K Signaling in Rectal Cancer.

Neoadjuvant radiation is standard of care for locally advanced rectal cancer. Response to radiation is highly variable and directly linked with survival. However, there currently are no validated biomarkers or molecular targets to predict or improve radiation response, which would help develop personalized treatment and ideally targeted therapies. Here, we identified a novel biomarker, coenzyme A synthase (COASY), whose mRNA expression was consistently elevated in radioresistant human rectal cancers. This observation was validated in independent patient cohorts and further confirmed in colorectal cancer cell lines. Importantly, genetic overexpression and knockdown yielded radioresistant and sensitive phenotypes, respectively, in vitro and in vivo. COASY-knockdown xenografts were more vulnerable to radiation, showing delayed tumor growth, decreased proliferation, and increased apoptosis. Mechanistically, COASY protein directly interacted with the PI3K regulatory subunit PI3K-P85α, which increased AKT and mTOR phosphorylation, enhancing cell survival. Furthermore, shRNA COASY knockdown disrupted downstream PI3K pathway activation and also hindered DNA double-strand break repair, which both led to improved radiosensitivity. Collectively, this work reveals for the first time the biological relevance of COASY as a predictive rectal cancer biomarker for radiation response and offers mechanistic evidence to support COASY as a potential therapeutic target. SIGNIFICANCE: COASY is a novel radiotherapy response modulator in rectal cancer that regulates PI3K activation and DNA repair. Furthermore, COASY levels directly correlate with radiation response and serve as a predictive biomarker.

Methylated SEPTIN9 plasma test for colorectal cancer detection may be applicable to Lynch syndrome.

OBJECTIVE: The plasma-based methylated SEPTIN9 (mSEPT9) is a colorectal cancer (CRC) screening test for adults aged 50-75 years who are at average risk for CRC and have refused colonoscopy or faecal-based screening tests. The applicability of mSEPT9 for high-risk persons with Lynch syndrome (LS), the most common hereditary CRC condition, has not been assessed. This study sought preliminary evidence for the utility of mSEPT9 for CRC detection in LS. DESIGN: Firstly, SEPT9 methylation was measured in LS-associated CRC, advanced adenoma, and subject-matched normal colorectal mucosa tissues by pyrosequencing. Secondly, to detect mSEPT9 as circulating tumor DNA, the plasma-based mSEPT9 test was retrospectively evaluated in LS subjects using the Epi proColon 2.0 CE assay adapted for 1mL plasma using the "1/1 algorithm". LS case groups included 20 peri-surgical cases with acolonoscopy-based diagnosis of CRC (stages I-IV), 13 post-surgical metastatic CRC, and 17 pre-diagnosis cases. The control group comprised 31 cancer-free LS subjects. RESULTS: Differential hypermethylation was found in 97.3% (36/37) of primary CRC and 90.0% (18/20) of advanced adenomas, showing LS-associated neoplasia frequently produce the mSEPT9 biomarker. Sensitivity of plasma mSEPT9 to detect CRC was 70.0% (95% CI, 48%-88%)in cases with a colonoscopy-based CRC diagnosis and 92.3% (95% CI, 64%-100%) inpost-surgical metastatic cases. In pre-diagnosis cases, plasma mSEPT9 was detected within two months prior to colonoscopy-based CRC diagnosis in 3/5 cases. Specificity in controls was 100% (95% CI 89%-100%). CONCLUSION: These preliminary findings suggest mSEPT9 may demonstrate similar diagnostic performance characteristics in LS as in the average-risk population, warranting a well-powered prospective case-control study.

A Changing Spectrum of Colorectal Cancer Biology With Age: Implications for the Young Patient.

BACKGROUND: The methylator pathway of colorectal carcinogenesis, characterized by CpG island hypermethylation and BRAF mutations, accounts for ≈25% of colorectal cancers. Because these cancers tend to be right sided and because DNA methylation in the right colon increases with age, we expect an increasing proportion of right-sided cancer over time. Conversely, we expect young patients (age <50 y) to have less methylated and fewer right-sided cancers OBJECTIVE:: The purpose of this study was to analyze the distribution and genetic traits of colorectal cancer from different age groups. DESIGN: This was a retrospective cohort study. SETTING: The study was conducted at a high-volume tertiary referral center. PATIENTS: Patient samples included those from our colorectal cancer biobank of resected colorectal cancer specimens. MAIN OUTCOME MEASURES: Tumor CpG island hypermethylation, microsatellite instability, and mutations in KRAS and BRAF oncogenes were analyzed in resected specimens and stratified by age and tumor location. Comparisons included age >50 or <50 years and decade of diagnosis (≤50, 51-60, 61-70, 71-80, and >81 y). Patients with IBD or hereditary syndromes were excluded. RESULTS: A total of 497 colorectal cancers were analyzed (266 men and 231 women); 57 patients (11.5%) were ≤50 years of age. No young cancers (0/57) were hypermethylated compared with 97 (22%) of 440 cancers of patients aged >50 years (p < 0.001). An increasing percentage of tumors were CpG island phenotype high with each decade of age at diagnosis. No cancers in patients <50 years of age were microsatellite unstable compared with 91 (23.6%) of 346 for those >50 years of age. No young cancers contained a BRAF mutation compared with 46 (10.6%) of 434 in older cancers (p < 0.001). KRAS mutations were less common in young cancers compared with older cancers (13/57 (22.8%) vs 126/410 (30.7%); p < 0.01). Eleven (19.3%) of 57 young cancers were proximal compared with 228 (51.8%) of 440 (p < 0.001) older cancers. LIMITATIONS: This study was limited by its retrospective design. CONCLUSIONS: The lack of CpG island methylator phenotype tumors in young patients is consistent with the dominant left-sided cancer distribution seen in the young and focuses efforts to understand and prevent cancer in this age group on causes of chromosomal instability. See Video Abstract at http://links.lww.com/DCR/A709.

Poly(ADP-Ribose) Polymerase Inhibition Sensitizes Colorectal Cancer-Initiating Cells to Chemotherapy.

Colorectal cancer (CRC) remains a leading killer in the U.S. with resistance to treatment as the largest hurdle to cure. Colorectal cancer-initiating cells (CICs) are a self-renewing tumor population that contribute to tumor relapse. Here, we report that patient-derived CICs display relative chemoresistance compared with differentiated progeny. In contrast, conventional cell lines failed model therapeutic resistance. CICs preferentially repaired chemotherapy-induced DNA breaks, prompting us to interrogate DNA damage pathways against which pharmacologic inhibitors have been developed. We found that CICs critically depended on the key single-strand break repair mediator, poly(ADP-ribose) polymerase (PARP), to survive treatment with standard-of-care chemotherapy. Small molecule PARP inhibitors (PARPi) sensitized CICs to chemotherapy and reduced chemotherapy-treated CIC viability, self-renewal, and DNA damage repair. Although PARPi monotherapy failed to kill CICs, combined PARPi therapy with chemotherapy attenuated tumor growth in vivo. Clinical significance of PARPi for CRC patients was supported by elevated PARP levels in colorectal tumors compared with normal colon, with further increases in metastases. Collectively, our results suggest that PARP inhibition serves as a point of fragility for CICs by augmenting therapeutic efficacy of chemotherapy. Stem Cells 2019;37:42-53.

Epigenetically regulated miR-1247 functions as a novel tumour suppressor via MYCBP2 in methylator colon cancers.

BACKGROUND: Colorectal cancer (CRC) is a heterogeneous disease with distinct clinical subsets based on underlying genetic and epigenetic changes. DNA hypermethylation yields a unique CRC subset with a distinct phenotype and clinical behaviour, but this oncogenic pathway is not fully characterised. This study identifies and characterises miR-1247 as a novel tumour suppressor microRNA in methylated human colon cancers. METHOD: Tumour samples from patients with hypermethylated and non-methylated colon cancer and cell lines were evaluated for miR-1247 expression and function. A murine subcutaneous xenograft model was used for in vivo functional studies. RESULTS: miR-1247 was methylated and underexpressed in methylator colon cancers. Overexpression of miR-1247 significantly inhibited cell proliferation, decreased tumour cell motility, induced apoptosis, and mitigated tumour formation capacity both in vivo and in vitro. Pharmacologic demethylation increased miR-1247 expression and produced similar anti-tumour activities. Mechanistic investigations revealed that MYCBP2, a member of the c-myc oncogene family, is a direct functional target of miR-1247. Furthermore, in CRC patients, MYCBP2 protein levels are associated with miR-1247 levels and survival. CONCLUSIONS: miR-1247 acts as a tumour suppressor by inhibiting MYCBP2 in methylator colon cancer. The MYCBP2/c-myc axis may underlie the anti-tumour activities of miR-1247 and is a potential therapeutic target via demethylation agents.

Simvastatin enhances radiation sensitivity of colorectal cancer cells.

INTRODUCTION: Neoadjuvant chemoradiation (CRT) for rectal cancer induces variable responses, and better response has been associated with improved oncologic outcomes. Our group has previously shown that the administration of HMG-CoA reductase inhibitors, commonly known as statins, is associated with improved response to neoadjuvant CRT in rectal cancer patients. The purpose of this study was to study the effects of simvastatin on colorectal cancer cells and explore its potential as a radiation-sensitizer in vitro. METHODS: Four colorectal cancer cell lines (SW480, DLD1, SW837, and HRT18) were used to test the effects of simvastatin alone, radiation alone, and combination therapy. Outcome measures included ATP-based cell viability, colony formation, and protein (immunoblot) assays. RESULTS: The combination of radiation and simvastatin inhibited colony formation and cell viability of all four CRC lines, to a greater degree than either treatment alone (p < 0.01). In addition, the effects of simvastatin in this combination therapy were dose dependent, with increased concentrations resulting in more potentiated inhibitory effects. The radiosensitizing effects of simvastatin on cell viability were negated by the presence of exogenous GGPP in the media. On protein analyses of irradiated cells, simvastatin treatment inhibited phosphorylation of ERK1/2, in a dose-dependent manner, while the total levels of ERK1/2 remained stable. In addition, the combined treatment resulted in increased levels of cleaved caspase 3, indicating greater apoptotic activity in the cells treated with radiation and simvastatin together. CONCLUSIONS: Treatment with simvastatin hindered CRC cell viability and enhanced radiation sensitivity in vitro. These effects were tied to the depletion of GGPP and the decreased phosphorylation of ERK1/2, suggesting a prominent role for the EGFR-RAS-ERK1/2 pathway, through which statin enhances radiation sensitivity.

RBP4-STRA6 Pathway Drives Cancer Stem Cell Maintenance and Mediates High-Fat Diet-Induced Colon Carcinogenesis.

The transmembrane protein, STRA6, functions as a vitamin A transporter and a cytokine receptor when activated by vitamin A-bound serum retinol binding protein 4 (RBP4). STRA6 activation transduces a JAK2-STAT3 signaling cascade and promotes tumorigenesis in a xenograft mouse model of colon cancer. We show here that RBP4 and STRA6 expression is associated with poor oncologic prognosis. Downregulating STRA6 or RBP4 in colon cancer cells decreased the fraction of cancer stem cells and their sphere and tumor initiation frequency. Furthermore, we show that high-fat diet (HFD) increases LGR5 expression and promotes tumor growth in a xenograft model independent of obesity. HFD increased STRA6 levels, and downregulation of STRA6 delays and impairs tumor initiation, tumor growth, and expression of stemness markers. Together, these data demonstrate a key role of STRA6 and RBP4 in the maintenance of colon cancer self-renewal and that this pathway is an important link through which consumption of HFD contributes to colon carcinogenesis.

NPTX2 is associated with neoadjuvant therapy response in rectal cancer.

BACKGROUND: Neoadjuvant chemoradiation (CRT) is recommended for locally advanced rectal cancer. Tumor response varies from pathologic complete response (pCR) to no tumor regression. The mechanisms behind CRT resistance remain undefined. In our previously generated complementary DNA microarrays of pretreatment biopsies from rectal cancer patients, neuronal pentraxin 2 (NPTX2) expression discriminated patients with pCR from those with residual tumor. As tumor response is prognostic for survival, we sought to evaluate the clinical relevance of NPTX2 in rectal cancer. MATERIALS AND METHODS: Real-time quantitative polymerase chain reaction was used to evaluate NPTX2 messenger RNA expression in individual rectal cancers before CRT. Tumors with NPTX2 expression <50% of normal rectum were defined as NPTX2-low and those with >50% were defined as NPTX2-high. NPTX2 levels were compared to response to therapy and oncologic outcomes using Mann-Whitney, Kruskal-Wallis, chi-square, and Mantel-Cox (log-rank) tests, as appropriate. RESULTS: Rectal cancers from 40 patients were included. The mean patient age was 56.8 years, and 30% were female. pCR was achieved in eight of 40 patients (20%). In these patients, messenger RNA NPTX2 levels were significantly decreased compared to those with residual cancer (fold change 30.4, P = 0.017). Patients with NPTX2-low tumors (n = 13) achieved improved response to treatment (P = 0.012 versus NPXT2-high tumors), with 38.5% and 46.1% of patients achieving complete or moderate response, respectively. Of patients with NPTX2-high tumors (n = 27), 11.1% and 18.5% achieved complete or moderate response, respectively. No recurrence or death was recorded in patients with NPTX2-low tumors, reflecting more favorable disease-free survival (P = 0.045). CONCLUSIONS: Decreased NPTX2 expression in rectal adenocarcinomas is associated with improved response to CRT and improved prognosis. Further studies to validate these results and elucidate the biological role of NPTX2 in rectal cancer are needed.

Structure-Function Analysis of the Mcl-1 Protein Identifies a Novel Senescence-regulating Domain.

Unlike other antiapoptotic Bcl-2 family members, Mcl-1 also mediates resistance to cancer therapy by uniquely inhibiting chemotherapy-induced senescence (CIS). In general, Bcl-2 family members regulate apoptosis at the level of the mitochondria through a common prosurvival binding groove. Through mutagenesis, we determined that Mcl-1 can inhibit CIS even in the absence of its apoptotically important mitochondrion-localizing domains. This finding prompted us to generate a series of Mcl-1 deletion mutants from both the N and C termini of the protein, including one that contained a deletion of all of the Bcl-2 homology domains, none of which impacted anti-CIS capabilities. Through subsequent structure-function analyses of Mcl-1, we identified a previously uncharacterized loop domain responsible for the anti-CIS activity of Mcl-1. The importance of the loop domain was confirmed in multiple tumor types, two in vivo models of senescence, and by demonstrating that a peptide mimetic of the loop domain can effectively inhibit the anti-CIS function of Mcl-1. The results from our studies appear to be highly translatable because we discerned an inverse relationship between the expression of Mcl-1 and of various senescence markers in cancerous human tissues. In summary, our findings regarding the unique structural properties of Mcl-1 provide new approaches for targeted cancer therapy.

Glioma cancer stem cells secrete Gremlin1 to promote their maintenance within the tumor hierarchy.

Glioblastomas are the most prevalent and lethal primary brain tumor and are comprised of hierarchies with self-renewing cancer stem cells (CSCs) at the apex. Like neural stem cells (NSCs), CSCs reside in functional niches that provide essential cues to maintain the cellular hierarchy. Bone morphogenetic proteins (BMPs) instruct NSCs to adopt an astrocyte fate and are proposed as anti-CSC therapies to induce differentiation, but, paradoxically, tumors express high levels of BMPs. Here we demonstrate that the BMP antagonist Gremlin1 is specifically expressed by CSCs as protection from endogenous BMPs. Gremlin1 colocalizes with CSCs in vitro and in vivo. Furthermore, Gremlin1 blocks prodifferentiation effects of BMPs, and overexpression of Gremlin1 in non-CSCs decreases their endogenous BMP signaling to promote stem-like features. Consequently, Gremlin1-overexpressing cells display increased growth and tumor formation abilities. Targeting Gremlin1 in CSCs results in impaired growth and self-renewal. Transcriptional profiling demonstrated that Gremlin1 effects were associated with inhibition of p21(WAF1/CIP1), a key CSC signaling node. This study establishes CSC-derived Gremlin1 as a driving force in maintaining glioblastoma tumor proliferation and glioblastoma hierarchies through the modulation of endogenous prodifferentiation signals.

Chemotherapy activates cancer-associated fibroblasts to maintain colorectal cancer-initiating cells by IL-17A.

Many solid cancers display cellular hierarchies with self-renewing, tumorigenic stemlike cells, or cancer-initiating cells (CICs) at the apex. Whereas CICs often exhibit relative resistance to conventional cancer therapies, they also receive critical maintenance cues from supportive stromal elements that also respond to cytotoxic therapies. To interrogate the interplay between chemotherapy and CICs, we investigated cellular heterogeneity in human colorectal cancers. Colorectal CICs were resistant to conventional chemotherapy in cell-autonomous assays, but CIC chemoresistance was also increased by cancer-associated fibroblasts (CAFs). Comparative analysis of matched colorectal cancer specimens from patients before and after cytotoxic treatment revealed a significant increase in CAFs. Chemotherapy-treated human CAFs promoted CIC self-renewal and in vivo tumor growth associated with increased secretion of specific cytokines and chemokines, including interleukin-17A (IL-17A). Exogenous IL-17A increased CIC self-renewal and invasion, and targeting IL-17A signaling impaired CIC growth. Notably, IL-17A was overexpressed by colorectal CAFs in response to chemotherapy with expression validated directly in patient-derived specimens without culture. These data suggest that chemotherapy induces remodeling of the tumor microenvironment to support the tumor cellular hierarchy through secreted factors. Incorporating simultaneous disruption of CIC mechanisms and interplay with the tumor microenvironment could optimize therapeutic targeting of cancer.

Hedgehog signaling regulates telomerase reverse transcriptase in human cancer cells.

The Hedgehog (HH) signaling pathway is critical for normal embryonic development, tissue patterning and cell differentiation. Aberrant HH signaling is involved in multiple human cancers. HH signaling involves a multi-protein cascade activating the GLI proteins that transcriptionally regulate HH target genes. We have previously reported that HH signaling is essential for human colon cancer cell survival and inhibition of this signal induces DNA damage and extensive cell death. Here we report that the HH/GLI axis regulates human telomerase reverse transcriptase (hTERT), which determines the replication potential of cancer cells. Suppression of GLI1/GLI2 functions by a C-terminus truncated GLI3 repressor mutant (GLI3R), or by GANT61, a pharmacological inhibitor of GLI1/GLI2, reduced hTERT protein expression in human colon cancer, prostate cancer and Glioblastoma multiforme (GBM) cell lines. Expression of an N-terminus deleted constitutively active mutant of GLI2 (GLI2ΔN) increased hTERT mRNA and protein expression and hTERT promoter driven luciferase activity in human colon cancer cells while GANT61 inhibited hTERT mRNA expression and hTERT promoter driven luciferase activity. Chromatin immunoprecipitation with GLI1 or GLI2 antibodies precipitated fragments of the hTERT promoter in human colon cancer cells, which was reduced upon exposure to GANT61. In contrast, expression of GLI1 or GLI2ΔN in non-malignant 293T cells failed to alter the levels of hTERT mRNA and protein, or hTERT promoter driven luciferase activity. Further, expression of GLI2ΔN increased the telomerase enzyme activity, which was reduced by GANT61 administration in human colon cancer, prostate cancer, and GBM cells. These results identify hTERT as a direct target of the HH signaling pathway, and reveal a previously unknown role of the HH/GLI axis in regulating the replication potential of cancer cells. These findings are of significance in understanding the important regulatory mechanisms that determine the functions of HH/GLI signaling in cancer cells.

The GLI genes as the molecular switch in disrupting Hedgehog signaling in colon cancer.

The Hedgehog (HH) signaling pathway leads to activation of GLI, which transcriptionally regulate target genes. Regulated HH signaling activity is critical during embryogenesis while aberrantly activated HH signaling is evident in a variety of human cancers. Canonical HH signaling engages the transmembrane receptor Patched (PTCH) and the signaling intermediate Smoothened (SMO) to activate GLI1 and GLI2. In addition GLI1 and GLI2 are activated by non-canonical oncogenic signaling pathways to further drive HH-dependent survival. We have demonstrated in human colon carcinoma cells that inhibition of the RAS/RAF pathway by U0126 decreases p-ERK protein expression and also inhibits GLI-luciferase activity and GLI1 mRNA and protein levels. Of importance is the demonstration that targeting of SMO (using cyclopamine) has minimal effect on cell survival in comparison to the inhibition of GLI (using GANT61), which induced extensive cell death in 7/7 human colon carcinoma cell lines. Genetic inhibition of the function of GLI1 and GLI2 by transient transfection of the C-terminus deleted repressor GLI3R, reduced proliferation and induced cleavage of caspase-3 and cell death in HT29 cells, similar to the effects of GANT61. Mechanistically, downstream of GLI1 and GLI2 inhibition, γH2AX (a marker of DNA double strand breaks) expression was upregulated, and γH2AX nuclear foci were demonstrated in cells that expressed GLI3R. Activation of the ATM/Chk2 axis with co-localization of γH2AX and p-Chk2 nuclear foci were demonstrated following GLI1/GLI2 inhibition. GANT61 induced cellular accumulation at G1/S and early S with no further progression before cells became subG1, while cDNA microarray gene profiling demonstrated downregulation of genes involved in DNA replication, the DNA damage response, and DNA repair, mechanisms that are currently being pursued. These studies highlight the importance of targeting the GLI genes downstream of SMO for terminating HH-dependent survival, suggesting that GLI may constitute a molecular switch that determines the balance between cell survival and cell death in human colon carcinoma.

An antiapoptotic BCL-2 family expression index predicts the response of chronic lymphocytic leukemia to ABT-737.

The antiapoptotic BCL-2 proteins regulate lymphocyte survival and are over-expressed in lymphoid malignancies, including chronic lymphocytic leukemia. The small molecule inhibitor ABT-737 binds with high affinity to BCL-2, BCL-XL, and BCL-W but with low affinity to MCL-1, BFL-1, and BCL-B. The active analog of ABT-737, navitoclax, has shown a high therapeutic index in lymphoid malignancies; developing a predictive marker for it would be clinically valuable for patient selection or choice of drug combinations. Here we used RT-PCR as a highly sensitive and quantitative assay to compare expression of antiapoptotic BCL-2 genes that are known to be targeted by ABT-737. Our findings reveal that the relative ratio of MCL-1 and BFL-1 to BCL-2 expression provides a highly significant linear correlation with ABT-737 sensitivity (r = 0.6, P < .001). In contrast, antiapoptotic transcript levels, used individually or in combination for high or low affinity ABT-737-binding proteins, could not predict ABT-737 sensitivity. The (MCL-1 + BFL-1)/BCL-2 ratio was validated in a panel of leukemic cell lines subjected to genetic and pharmacologic manipulations. Changes after ABT-737 treatment included increased expression of BFL-1 and BCL-B that may contribute to treatment resistance. This study defines a highly significant BCL-2 expression index for predicting the response of CLL to ABT-737.

Blocking Hedgehog survival signaling at the level of the GLI genes induces DNA damage and extensive cell death in human colon carcinoma cells.

Canonical Hedgehog (HH) signaling is characterized by Smoothened (Smo)-dependent activation of the transcription factors Gli1 and Gli2, which regulate HH target genes. In human colon carcinoma cells, treatment with the Gli small-molecule inhibitor GANT61 induces extensive cell death in contrast to the Smo inhibitor cyclopamine. Here we elucidate cellular events upstream of cell death elicited by GANT61, which reveal the basis for its unique cytotoxic activity in colon carcinoma cells. Unlike cyclopamine, GANT61 induced transient cellular accumulation at G(1)-S (24 hours) and in early S-phase (32 hours), with elevated p21(Cip1), cyclin E, and cyclin A in HT29 cells. GANT61 induced DNA damage within 24 hours, with the appearance of p-ATM and p-Chk2. Pharmacologic inhibition of Gli1 and Gli2 by GANT61 or genetic inhibition by transient transfection of the Gli3 repressor (Gli3R) downregulated Gli1 and Gli2 expression and induced γH2AX, PARP cleavage, caspase-3 activation, and cell death. GANT61 induced γH2AX nuclear foci, while transient transfection of Gli3R showed expression of Gli3R and γH2AX foci within the same nuclei in HT29, SW480, and HCT116. GANT61 specifically targeted Gli1 and Gli2 substantiated by specific inhibition of (i) direct binding of Gli1 and Gli2 to the promoters of target genes HIP1 and BCL-2, (ii) Gli-luciferase activity, and (iii) transcriptional activation of BCL-2. Taken together, these findings establish that inhibition of HH signaling at the level of the GLI genes downstream of Smo is critical in the induction of DNA damage in early S-phase, leading to cell death in human colon carcinoma cells.

Hedgehog signaling drives cellular survival in human colon carcinoma cells.

Aberrant activation of Hedgehog (HH) signaling is implicated in many human cancers. Classical HH signaling is characterized by Smoothened (Smo)-dependent activation of Gli1 and Gli2, which transcriptionally regulate target genes. A small molecule inhibitor of Gli1 and Gli2, GANT61, was used to block HH signaling in human colon carcinoma cell lines that express HH signaling components. GANT61 administration induced robust cytotoxicity in 5 of 6 cell lines and moderate cytotoxicity in the remaining 1 cell line. In comparison, the classical Smo inhibitor, cyclopamine, induced modest cytotoxicity. Further, GANT61 treatment abolished the clonogenicity of all six human colon carcinoma cell lines. Analysis of the molecular mechanisms of GANT61-induced cytotoxicity in HT29 cells showed increased Fas expression and decreased expression of PDGFRα, which also regulates Fas. Furthermore, DR5 expression was increased whereas Bcl-2 (direct target of Gli2) was downregulated following GANT61 treatment. Suppression of Gli1 by shRNA mimicked the changes in gene expression observed in GANT61-treated cells. Overexpression of dominant-negative FADD (to abrogate Fas/DR5-mediated death receptor signaling) and/or Bcl-2 (to block mitochondria-mediated apoptosis) partially rescued GANT61-induced cytotoxicity in HT29 cells. Thus, activated GLI genes repress DR5 and Fas expressions while upregulating Bcl-2 and PDGFRα expressions to inhibit Fas and facilitate cell survival. Collectively, these results highlight the importance of Gli activation downstream of Smo as a therapeutic target in models of human colon carcinoma.