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βIII-tubulin (encoded by TUBB3) expression is associated with therapeutic resistance and aggressive disease in non–small cell lung cancer (NSCLC), but the basis for its pathogenic influence is not understood. Functional and differential proteomics revealed that βIII-tubulin regulates expression of proteins associated with malignant growth and metastases. In particular, the adhesion-associated tumor suppressor maspin was differentially regulated by βIII-tubulin. Functionally, βIII-tubulin suppression altered cell morphology, reduced tumor spheroid outgrowth, and increased sensitivity to anoikis. Mechanistically, the PTEN/AKT signaling axis was defined as a critical pathway regulated by βIII-tubulin in NSCLC cells. βIII-Tubulin blockage in vivo reduced tumor incidence and growth. Overall, our findings revealed how βIII-tubulin influences tumor growth in NSCLC, defining new biologic functions and mechanism of action of βIII-tubulin in tumorigenesis. Cancer Res; 75(2); 415–25. ©2014 AACR.

Background:African Americans (AA) have a higher mortality associated with breast cancer (BC) when compared to Caucasians (CC).This has been attributed to diverse factors that include access to care, reproductive factors and different somatic genomic profiles. We aimed to compare the racial mutational landscape of 565 BC samples. Methods:DNA from formalin-fixed paraffin-embedded samples was sequenced using the Illumina NextSeq (Agilent SureSelect XT, 592 gene selected based on COSMIC database) and MiSeq (TruSeq, 47 gene) for mutation and gene amplification analyses. Protein expression was evaluated by Immunohistochemistry (IHC). The exome aggregation consortium database was assessed for known ethnicity associations. Results:Tumor samples from 118 AA and 447 CC female patients were included in this analysis. AA were younger (median age 56 vs58y, p0.005) and had higher proportion of triple negative BC (TNBC) (32% vs17%, p0.001). 50.3% of the samples were obtained from primary tumors and the remainder from metastatic sites. This was similar in AA and CC (48.8% vs51.8% primary tumors, pNS). The two genes with highest mutation prevalence were TP53 and PIK3CA. AA had fewer PIK3CA mutations (14.7% vs28.2%, p0.03). Within HR+/HER2+ and HR+/HER2- subtypes there was a similar trend in the number of PIK3CA mutations but it was no longer significant. The remainder mutation analysis did not differ between races. In terms of protein expression there were significant differences in the androgen receptor(AR), RRM1, EGFR and TS expression (table). AR positivity defined as ≥10% was less frequent in AA (40.0% vs60.4%, p0.0001 and when adjusted for age, p0.005) and associated with PIK3CA mutations in both AA and CC (p0.01 and p0.007). AR expression in TNBC was positive in 17.8% of CC and 5.4% of AA (pNS). Copy number variation (CNV) data assessed by NextGen revealed significantly higher gene copy number in AA compared to CC in CCND1 (16% vs4%, p0.04), FGF19 (16% vs1.3%, p0.01) and FGF4 (16% vs3%, p0.02). When only TNBC was considered, RRM1, TOPO1 and TUBB3 expression was significantly higher in AA than CC (table) and there were no differences in the mutational analyses. Evaluation of other BC subtypes (HER2, HR positive) is currently underway. Conclusions:In this large cohort of AA who underwent genomic profiling there were relatively few differences in the mutation analysis compared to CC. The only significant difference seen was the lower number of PIK3CA mutations in AA, which had been previously reported in a cohort of 105 AA from the TCGA data (Keenan et al. JCO 2015). Protein expression by IHC revealed lower expression of AR in AA, even after adjustment for age, which could have therapeutic implications. Some of the racial differences found in the molecular landscape of BC including PIK3CA mutations, AR, EGFR expression and CNV may contribute to a more aggressive tumor biology in AA. Protein expression by IHCBCTNBCAA %CC %pAA %CC %pHER2/neu912ns00nsAR40600.005180.07ER44620.0000nsPR35460.0900nsEGFR30160.036050nsERCC133460.063626nsMGMT6163ns6571nsPD-150340.066548nsPD-L106ns011nsPGP76ns1015nsPTEN5658ns4247nsRRM139270.0459270.01TLE350590.104357nsTOP2A7164ns86470.08TOPO16358ns69460.03TS44300.016349nsTUBB355410.0677510.03 Citation Format: Lynce F, Xiu J, Nunes MR, Swain SM, Gatalica Z, Isaacs C, Pohlmann P. Racial differences in the molecular landscape of breast cancer [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr PD8-04.

The supply of oxygen and nutrients to solid tumors is inefficient because cancer tissues have an inadequate number of microvessels, thus inducing the selective growth of the most aggressive cancer cells. This explains why many of the factors underlying a poor prognosis are induced in hypoxic/hypoglycemic conditions. Among these factors, a prominent role in several solid tumors is played by the class III β-tubulin gene (TUBB3). The study described here reveals that glucose deprivation enhances TUBB3 expression at both the gene and protein levels in A2780 ovarian cancer cells. In silico analysis of TUBB3 mRNA sequence predicted a putative binding site for the RNA-binding protein Hu antigen (HuR) in the 3′ flanking untranslated region. A hypoglycemic-dependent engagement of this site was shown using RNA pull-down and ribonucleoimmunoprecipitation techniques. Thereafter, HuR gene silencing revealed that TUBB3 translation is HuR dependent in hypoglycemia because HuR silencing inhibited the entry of TUBB3 mRNA into cytoskeletal and free polysomes. Finally, the clinical value of this finding was assessed in a clinical cohort of 46 ovarian cancer patients in whom it was found that HuR cytoplasmic staining was associated with high levels of TUBB3 and poor survival. Cancer Res; 70(14); 5891–900. ©2010 AACR.

Patupilone is an epothilone in advanced clinical development that has shown promising efficacy in heavily pretreated patients. This study aimed at characterizing the mechanisms of patupilone activity in resistant patients. To this end, we generated patupilone-resistant cells using two cellular models, the first characterized by high chemosensitivity and low class III β-tubulin (TUBB3) expression (A2780), and the second by low chemosensitivity and high TUBB3 expression (OVCAR-3). The obtained cell lines were named EPO3 and OVCAR-EPO, respectively. The same selection procedure was done in A2780 cells to generate a paclitaxel-resistant cell line (TAX50). Factors of resistance are expected to increase in the drug-resistant cell lines, whereas factors of drug sensitivity will be down-regulated. Using this approach, we found up-regulation of TUBB3 in TAX50, but not EPO3, cells, showing that TUBB3 mediates the resistance to paclitaxel but not to patupilone. Moreover, TUBB3 was a factor of patupilone sensitivity because OVCAR-EPO cells exhibited a dramatic reduction of TUBB3 and a concomitant sensitization to hypoxia and cisplatin-based chemotherapy. To identify the mechanisms underlying patupilone resistance, tubulin genes were sequenced, thereby revealing that a prominent mechanism of drug resistance is represented by point mutations in class I β-tubulin. Overall, these results suggest that paclitaxel and patupilone have nonoverlapping mechanisms of resistance, thus allowing the use of patupilone for those patients relapsing after paclitaxel-based chemotherapy. Furthermore, patupilone represents a promising first-line option for the treatment of high-risk ovarian cancer patients, who exhibit high TUBB3 levels and poor response to standard paclitaxel-platin chemotherapy. [Cancer Res 2008;68(24):10197–204]

Hypoxia selects the most aggressive and drug-resistant clones in solid malignancies. One of the pivotal transcription factors induced by hypoxia is Hif-1α. However, in serous ovarian cancer (SEOC), Hif-1α expression is not a prognostic biomarker. This study aims to assess the hypothesis that the serine-threonine kinase Nek6 functions as a downstream effector cooperating with Hif-1α in driving ovarian cancer aggressiveness. Nek6 was overexpressed and Hif-1α was silenced in A2780 cells. Nek6 was also stably silenced in Hey cells. The dependence of Nek6 expression on Hif-1α was assayed as a function of hypoxic growth conditions. Nek6 interaction with the cytoskeletal gateway of drug resistance was investigated with far western blot. The co-expression of NEK6, HIF1A, TUBB3 and GBP1 transcripts was quantified with qPCR in two cohorts of SEOC patients (346 locally treated patients and 344 from the TCGA dataset). Nek6 expression is induced by hypoxia in a Hif-1α dependent fashion. Nek6 directly interacts with GBP-1, thus being a component of the cytoskeletal gateway of drug resistance. Nek6 overexpression increases and silencing decreases the anchorage-independent growth of cultured cells. In SEOC patients, NEK6 expression is significantly correlated with HIF1A. Co-expression of NEK6, HIF1A, TUBB3 and GBP1 transcripts identifies a subset of SEOC patients characterized by poor outcome and drug resistance. This study demonstrates the functional relevance of Nek6 in the context of the adaptive response to hypoxia in SEOC. This finding may help identify a sub-population of patients at high risk of relapse to standard first-line chemotherapy. Citation Format: Marta De Donato, Mara Fanelli, Marisa Mariani, Giuseppina Raspaglio, Deep Pandya, Shiquan He, Paul Fiedler, Marco Petrillo, Giovanni Scambia, Cristiano Ferlini. Nek6 and Hif-1α cooperate with the cytoskeletal gateway of drug resistance to drive outcome in serous ovarian cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4327. doi:10.1158/1538-7445.AM2015-4327

Background: Because no single molecular marker have proven to be sufficiently powerful for routine estimation of prostate cancer prognosis as to yet, it is hoped that the combined analysis of multiple markers will allow for distinguishing between indolent and aggressive forms of prostate cancer. In fact, first generation commercial multiparametric test assays show promising results. We hypothesized that such tests may be further optimized. Design: A tissue microarray (TMA) containing 0.6 mm tissue cores from more than 12,000 prostate cancer patients with long-term follow-up data was analyzed with more than 100 molecular markers by immunohistochemistry or FISH. Markers with strong and independent prognostic value in uni- and multivariate analyses were combined to obtain panels of markers that yield additional prognostic information beyond the standard parameters (including pre-operative PSA, Gleason grade, tumor stage, nodal stage) in ROC analysis. For this purpose, a simple score (0-10 points) was build based on the number and type of adverse features per tumor. Results: Markers with strong independent prognostic relevance included deletions of 6q15 and PTEN, overexpression of EZH2, TUBB3, and nuclear accumulation of p53, indicating dominant-negative TP53 gene mutation. A total of 5,129 cancers harbored none of these alterations (score 0) and showed the best prognosis in a Kaplan-Meier plot using biochemical recurrence as the study endpoint. The worst prognosis was found for 610 tumors harboring ≥8 (score 8-10) of these alterations, while an intermediate prognosis with decreasing time to recurrence was found for cancers with scores 1-2 (n = 2,418), 3-4 (n = 1,367) and 5-7 (n = 1038). The differences between the groups were high significant (p Conclusion: Multiparametric classifiers have a high potential to improve the predictive accuracy of current models using established clinic-pathological models. Analyses of very large sets of patient tissues are instrumental to identify the best candidates for such molecular marker panels. It is likely, that continued analysis of our TMA will identify even better marker panels than the current 5 genes classifier. Citation Format: Maria Christina Tsourlakis, Martina Kluth, Thorsten Schlomm, Ronald Simon, Guido Sauter, Sarah Minner. Improved prediction of prostate cancer prognosis by using a multiparametric molecular classifier. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4353. doi:10.1158/1538-7445.AM2015-4353

Background: Non-small Cell Lung Cancer (NSCLC) survival rates are dismal and chemotherapy resistance is a significant clinical problem. βIII-tubulin (encoded by TUBB3 gene) is aberrantly expressed and is associated with chemoresistance and tumor aggressiveness in NSCLC (1), where it has been identified as a bona fide target for chemosensitisation (2,3). In order to understand how TUBB3/βIII-tubulin is regulated in NSCLC cells we sought to identify chemical small molecules that can modulate its expression. Methods: H460 cells expressing a TUBB3 or GAPDH promoter-luciferase reporter construct were generated and used in drug screening and promoter activity testing. To isolate modulators of TUBB3 promoter activity, a cell-based screen of libraries of diverse chemical small molecules (30K) and FDA-approved bioactives (3680) was performed. Cell viability, growth and proliferation were measured using standard methods. Cell cycle was assessed using flow cytometry, and gene and protein expression by RT-PCR and Western blotting, respectively. Microtubule morphology was assessed using immunostaining and confocal microscopy. Drug-treated clonogenic assays were used to quantitate changes in drug sensitivity. Results: Based upon their ability to modulate TUBB3 promoter activity, we identified two hit compounds, CCI01 and CCI02, as well as the bioactive compound, RITA. For all three leads we observed: 1) repression in TUBB3 promoter activity which was not a result of cell cytotoxicity; 2) no effects on cell cycle or viability, but instead cytostatic effects; 3) significantly enhanced TUBB3 expression in a time and dose-dependent manner. TUBB3 gene enhancement was translated at the protein level in CCI01 treated H460, but not in CCI02 or RITA treated cells. Additionally, CCI01 did not alter microtubule morphology but enhanced βIII-tubulin immunostaining in two independent NSCLC cell lines, H460 and H1299, compared to control. Importantly, CCI01 enhanced βIII-tubulin expression was functional and led to a significant decrease in in vitro sensitivity to DNA-damaging and tubulin-binding agents in H460 cells. Conclusion: A novel lead small molecule TUBB3/βIII-tubulin enhancer has been identified that is able to increase expression of βIII-tubulin in NSCLC and significantly reduce sensitivity to tubulin binding and DNA-damaging agents. Implications: Identification of a modulator of TUBB3/βIII-tubulin expression will provide a valuable research tool to probe βIII-tubulin regulation. (1) Kavallaris. Nature Rev Cancer, 10:194-204, 2010 (2) McCarroll et al., Cancer Res 70:4995-5003, 2010 (3) Gan et al., Cancer Res. 67:9356-9363, 2007 Citation Format: Felicity Chao Lin Kao, Tim Failes, Greg M. Arndt, Murray Norris, Maria Kavallaris. Discovery of a small molecule TUBB3/βIII-tubulin modulator in lung cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3660. doi:10.1158/1538-7445.AM2015-3660

Background. Despite the identification of MYCN-amplification as an adverse prognostic marker in neuroblastoma, no drugs that target MYCN have yet been developed. An alternative approach is to identify genes synthetically lethal to MYCN-amplified neuroblastoma. Through a whole genome shRNA screen, we have previously identified transcription factor AP4 (TFAP4) as a synthetic lethal gene of MYCN-amplified neuroblastoma. Silencing of TFAP4, both in vitro and in vivo inhibited the growth of MYCN-amplified neuroblastoma. TFAP4 has previously reported to repress neuronal specific genes in non-neuronal cells (Kim et al, 2006). Here, we investigate the function of TFAP4 in inhibiting differentiation of MYCN-amplified neuroblastoma. Methods: Master regulators specifically associated to MYCN amplified patients were identified by MARINA (Master Regulator Inference algorithm). TFAP4 expression was measured by real-time PCR, Western blot and immunohistochemistry. GAP43 expression was detected by Western blot. Gene expression changes after silencing of TFAP4 or retinoic acid treatment was determined by RNAseq. Results: TFAP4 was identified as a master regulator specifically in MYCN amplified neuroblastoma patients in both NCI-TARGET dataset and a published GEO dataset (Kocak et al, 2013). We determined that TFAP4 is a direct target of MYCN and TFAP4 expression was highly correlated with Stage 4, MYCN-amplified neuroblastoma in both patient datasets. Neuronal markers TUBB3 and GAP43 expression were inversely correlated with TFAP4 expression in TARGET (P = 9.77e-03 and P = 7.78e-08). Silencing TFAP4 in MYCN amplified cells induced a differentiated phenotype, as shown by markedly increased the number of neurites, and an increase in the neuronal differentiation marker GAP43. Forty hours after silencing TFAP4, we observed 415 genes that were significantly up-regulated and 457 genes down-regulated (P Conclusions. We demonstrated that TFAP4 is an important downstream effector of MYCN, that inhibits the neuronal differentiatin of MYCN-amplified neuroblastoma. This may provide novel targets for the treatment of neuroblastoma. Citation Format: Shuobo Zhang, Gonzalo Lopez, Jiyang Yu, Jose Silva, Angela Kadenhe-Chiweshe, Andrea Califano, Darrell Yamashiro. TFAP4 inhibits differentiation of MYCN-amplified neuroblastoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2083. doi:10.1158/1538-7445.AM2015-2083

Introduction: Breast Cancer (BCa) treatments often include taxanes in the treatment regimens and resistance to these drugs is common in a clinical setting. Several mechanisms were put forth to explain the resistance. These include induction of multidrug (MDR) resistance family of transporters, differential regulation of tubulin isotypes, microRNA (miR) mediated post-transcriptional regulation and autophagy. Aims: (i) to create a panel of breast cancer cell lines from SKBR3 (Her2+ and ER-ve) with varying levels of drug resistance to paclitaxel; (ii) to profile differentially expressed miRs in these panels of cell lines to understand the evolution of drug resistance with common and unique signatures associated with each of the cell lines in the panel and (iii) to assess β-tubulin (TUBB3) expression and its regulation by miRs. Methods: Drug resistant SKBR-3 cells were generated by a stepwise increase in drug concentration to the parental cells until a maximum tolerable dose was reached. Control cells were those without any treatment (wild type, WT) and serial passage of SKBR3 cells for the same numbers of generations as in each of the drug resistant lines. Total RNA extracted from the cell lines was subjected to small RNAome profiling (with focus on miRs) using Next Generation Sequencing (Illumina Genome Analyzer IIx). Partek Genomics Suite 6.6 was used for analyzing sequencing files. Data normalized (RPKM) from triplicate experiments (three independent cell growth experiments) were adjusted for potential batch effects. We report miRs showing statistically significant differences in expression levels (>2-fold and FDR adjusted p Citation Format: Sambasivarao Damaraju, Preethi Krishnan, Marc St George, Jack Tuszynski, Carol Cass, IngSwie Goping, Olga Kovalchuk. Post-transcriptional regulatory mechanisms contributing to the evolution of paclitaxel resistance in breast cancer model cell line, SKBR3. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3076. doi:10.1158/1538-7445.AM2015-3076

In order to identify determinants of cellular response to this new taxane, we derived a resistant variant from the MCF-7 human breast cancer cell line by stepwise selection in cabazitaxel. The 33-fold resistance to cabazitaxel in this variant, MCF-7/CTAX, was associated with ABCB1/P-glycoprotein (P-gp) activation, but also 3-fold residual resistance to taxanes after modulation with the P-gp inhibitor PSC-833, 2 μM. Another variant was established by co-selecting in the presence of PSC-833 (MCF-7/CTAX-P). This variant was negative for ABCB1 transcripts and accumulated parental levels of rhodamine-123, BODIPY-labeled paclitaxel and [3H]-docetaxel, indicating that the taxane resistance (9-fold) observed in this cell line is not mediated by transporters. However, we observed a 34% reduction in bound fluorescent-labeled paclitaxel in MCF-7/CTAX-P relative to parental controls by flow cytometry. These cells are hypersensitive to depolymerizing agents (vincas and colchicine) indicating a change in tubulin dynamic instability, and we observed reduced baseline tubulin polymer in untreated MCF-7/CTAX-P cells, and impaired tubulin polymerization in response to taxane exposure (cabazitaxel or docetaxel) relative to MCF-7 parental cells. Quantitative PCR and immunoblotting confirmed elevated levels of the class III (TUBB3) β-tubulin isotype in both MCF-7/CTAX and MCF-7/CTAX-P. Reduced BRCA1 content was observed in both MCF-7 variants, which could affect taxane response via the regulation of the mitotic spindle checkpoint. Gene silencing with specific siRNAs confirmed that reduced TUBB3 sensitizes cells to cabazitaxel, and reduced BRCA1 results in taxane resistance. In addition, altered epithelial-mesenchymal transition markers (elevated Vimentin, reduced E-cadherin) are associated with cabazitaxel resistance in these MCF-7 variants. In summary, cabazitaxel resistance mechanisms include MDR (although at a lower level than paclitaxel and docetaxel), and alterations in microtubule dynamicity, as manifested by higher expression of TUBB3, decreased BRCA1, and by the induction of EMT. Citation Format: George E. Duran, Yan C. Wang, E Brian Francisco, Francisco J. Martinez, Branimir I. Sikic. Resistance to cabazitaxel is associated with ABCB1/P-glycoprotein activation, alterations in β-tubulin content and dynamics, reduced BRCA1, and a mesenchymal phenotype in MCF-7 human breast cancer variants. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 763. doi:10.1158/1538-7445.AM2014-763

Breast cancer brain metastasis (BCBM) occur in about 10-30% of patients with breast cancer. It has been established that breast cancer is a heterogeneous disease with 3 predominant subtypes (1) ER/PR positive (3) HER2 positive (4) Triple negative breast cancer (TNBC)/Basal subtype. TNBC is associated with the highest incidence of metastasis to brain, mechanism of which is poorly understood. Therefore, in order to understand the proclivity of TNBC to the brain and identify potential targets for treatment BCMB, we employed two widely used paired cell model system, the MDA-MB-231/MDA-MB-231BrM2 and the CN-34/CN34BrM2. The levels of brain predominant markers [ST6GALNAC5 (ST6), Nestin, Tubulin β3 (TUBB3)] in brain-derived clones were assesed by qRT-PCR and found to be overexpressed in brain-derived MDA-MB-231BrM2 and CN34BrM2 in comparison with parental breast cancer cell lines. We also identified hyperactivated TGF-β signaling in brain derived clones in comparison with parental cells. Moreover, the treatment of parental cells with TGF-β increased the expression of ST6, Nestin and TUBB3 indicating that TGF-β plays role in the development of BCBM. To further delineate the role of these markers in BCBM, knockdown experiments using two different shRNA specific to TUBB3 (neuronal specific) were performed. TUBB3 knockdown resulted in a dramatic reduction in tumorspheres formation, migration, invasion and the ability to adhere to laminin in both MDA-MB-231BrM2. Furthermore, the downregulation of expression of β1 and β3 integrins in TUBB3 knockdown cells was discovered as assessed by qPCR and flow cytometry. These results implicate that overexpression of brain specific TUBB3 provides breast cancer cells with migration and invasion abilities and a possible survival advantage in the brain microenvironment. Citation Format: Deepak P. Kanojia, Purva Sarvaiya, Jian Qiao, Lingjiao Zhang, Irina Balyasnikova, Maciej S. Lesniak. The role of neuronal predominant gene expression in breast cancer brain metastasis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2006. doi:10.1158/1538-7445.AM2014-2006

MicroRNAs (miRNAs) in solid malignancies can behave as predictors of either good or poor outcome. This is the case with members of the miR-200 family, which are the primary regulators of the epithelial to mesenchymal transition and have been reported to act as both oncogenes and tumor suppressors in independent studies. Expression of miR-200c was assessed in a panel of ovarian cancer cell lines with inherent or acquired drug-resistance. Luciferase reporter assay and ribonucleic-immuno-precipitation assay was used to in vitro characterise the complexes between miR-200c, HuR and class III β-tubulin (TUBB3). Nanofluidic technology and immunohistochemistry were used to analyze the expression of HuR, TUBB3 and miR-200c in 220 ovarian cancer patients. Using such approach, we demonstrated that overexpression of miR-200c in ovarian cancer resulted in poor or good outcome depending on interaction with the RNA binding protein HuR. When the localization of HuR was confined in the nucleus, high expression of miR-200c suppressed class III β-tubulin (TUBB3) expression and resulted in a good prognosis, whereas when HuR occurred in cytoplasm, the same miRNA enhanced TUBB3 expression and produced a poor outcome. Indeed, in an ovarian cancer cell line model, the miR-200c increased HuR binding on TUBB3 mRNA in stressing microenvironmental conditions. This study demonstrated how the prognostic power of a single factor can depend on interactions with additional factors. Through a multidimensional analysis of all components, the actual mechanism(s) underlying the aggressive biological behavior of ovarian cancer can be unraveled, and the prognosis of ovarian cancer patients can be rendered based on rational explanations. Citation Format: Silvia Prislei, Enrica Martinelli, Marisa Mariani, Giuseppina Raspaglio, Steven Sieber, Gabriella Ferrandina, Shohreh Shahabi, Giovanni Scambia, Cristiano Ferlini. MiR-200c can mediate either poor or good outcome in ovarian cancer depending on HuR localization in the cell. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1957. doi:10.1158/1538-7445.AM2013-1957

Objectives: Eribulin mesylate (ERI) is a simplified synthetic macrocyclic ketone analog of the marine sponge natural product halichondrin B and an inhibitor of microtubule dynamics. In this study, we investigated whether selective signal pathways were associated with ERI activity compared to paclitaxel (PTX), which stabilize microtubules, based on GEP using 3 cancer cell line panels in vitro. Methods: In vitro anti-proliferation activity (IC50) was determined in 27 breast, 19 endometrial and 21 ovarian cancer cell lines treated with ERI and PTX. We performed GEP for the same cancer panels treated for 24 hours at conc. of 10 x IC50 using Affimetrix Human Genome U133 Plus 2.0 arrays. We applied paired t-test (p1.5) to identify genes significantly altered between treatments. Pathway enrichment was performed using Metacore and Ingenuity Pathway Analysis software. Cell lines were clustered by hierarchical clustering based on Pearson correlation. Results: We determined the sets of genes differentially altered between ERI and PTX treatments in 3 cancer panels. Regardless of cancer panels, several tubulins (TUBA1B, TUBA1C, TUBA3D, TUBA4A, TUBB2C, TUBBG1, TUBB3, TUBB2A, TUBB2C, TUBB and TUBB6) had significantly lower expression in cell lines treated with ERI, compared to PTX. Our analysis revealed that expression profiles of the gene sets correlated with drug sensitivity in cancer panels (p=0.004 for ERI in breast cancer, p=0.06 for PTX in endometrial cancer, p=0.05 for ERI in ovarian cancer). Pathway enrichment analysis of the gene revealed that the common pathways altered between 2 treatments in 3 cancer panels were related to cytoskeleton remodeling (p Conclusion: GEP analysis showed that gene sets altered with ERI and PTX were correlated to drug sensitivity in vitro anti-proliferative activity among 3 cancer panels. Breast cancer is the best target to differentiate ERI sensitivity based on GEP compared to PTX and EMT genes are selectively related to ERI sensitivity. Citation Format: Zoltan Dezso, Judith Oestreicher, Amy Weaver, Tadashi Kadowaki, Mamoru Yanagimachi, Junji Matsui, Sergei I. Agoulnik, Yasuhiro Funahashi. Gene expression profiling (GEP) reveals Epithelial Mesenchymal Transition (EMT) genes selectively differentiating eribulin sensitive breast cancer cell lines. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1522. doi:10.1158/1538-7445.AM2013-1522

Background: Epithelial ovarian cancer (EOC) accounts for the majority of deaths from gynecological malignancies, with a 5-year survival rate for women with advanced stage disease of only 46%. In EOC metastasis primarily occurs by a mechanism termed direct seeding, which involves shedding of tumor cells from the primary site into the peritoneal cavity. To allow survival in suspension before attachment at metastatic sites, ovarian cancer cells must acquire resistance to anoikis, a form of apoptosis initiated by detachment from native extracellular matrix. A targeted therapy that could simultaneously affect multiple factors critical for ovarian cancer progression such as anoikis resistance, chemoresistance and attachment to metastatic sites in the peritoneal cavity, would be a revolutionary breakthrough for this aggressive malignancy. Due to their ability to inhibit multiple targets involved in tumor survival, microRNAs (miRNAs) are attractive therapeutic candidates. We previously demonstrated that miR-200c is downregulated in ovarian cancer cell lines and restoration of miR-200c increased sensitivity to taxanes in vitro, by targeting TUBB3, a known mediator of chemoresistance. We tested the effect of miR-200c restoration in an intraperitoneal xenograft model of human ovarian cancer and observed dramatically decreased tumor formation. Further, even in established tumors, inducible restoration of miR-200c alone and in combination with paclitaxel, resulted in significantly reduced tumor burden. Hypothesis: Since restoration of miR-200c to multiple anoikis resistant EOC cell lines increased anoikis sensitivity and reduced adherence to biological substrates in vitro, we hypothesized that the decreased intraperitoneal tumor burden observed upon miR-200c restoration resulted from increased anoikis sensitivity. Methods and results: To elucidate mechanisms of anoikis resistance in ovarian cancer and identify targets involved in the ability of miR-200c to reverse anoikis resistance, we performed gene and miRNA expression profiling of attached Hey ovarian cancer cells as compared to the same cells grown under conditions of forced suspension for 24h. We identified miR-193 as downregulated in anoikis resistant cells in suspension. Among numerous genes predicted as miR-200c and/or miR-193 targets, SCLC7A11 (xCT), which encodes the light chain of the cystine/glutamate antiporter system xc, was increased by 10.7 fold in suspended versus attached cells. The xc- system is involved in glutathione (GSH) synthesis, mediating intracellular defense against oxidative stress. Reactive oxygen species (ROS) increased in anoikis-sensitive (OvCA 433) cells in suspension, but not in anoikis-resistant cells (Hey), suggesting that xCT might protect these cells from ROS-induced cell death. Consistent with this hypothesis, xCT mRNA and protein increased in anoikis resistant EOC cells in suspension, and transient xCT knockdown rendered cells anoikis sensitive. Interestingly, the xCT 3'UTR contains three predicted binding sites for miR-200c and two sites for miR-193b, and restoration of either miRNA blocked the suspension induced-increase in cXT protein, suggesting that both miRNAs target this gene. Conclusions: Our results indicate that the ability of miR-200c to reduce tumor burden results, at least in part, from its ability to restore anoikis sensitivity by inhibiting a suspension-induced increase in xCT that protects tumor cells from ROS. Thus, miR-200c could be a powerful candidate to block progression of ovarian cancer by enhancing anoikis sensitivity and restoring chemo-sensitivity to already attached tumor. Citation Format: Diana M. Cittelly, Dawn R. Cochrane, Erin N. Howe, Irina Dimitrova, Miriam D. Post, Broaddus R. Russell, Monique A. Spillman, Jennifer K. Richer. Decreasing ovarian cancer metastases and progression via restoration of microRNAs that target mediators of anoikis- and chemoresistance.. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr B70.

Colorectal cancer is still one of the deadliest diseases in western countries, particularly for those patients in which the diagnosis is performed at advanced stage. To identify useful predictive biomarkers of the outcome, we assessed using immunohistochemistry the expression of class III (TUBB3) and class V β-tubulin (TUBB6) in a retrospective clinical study on 180 colorectal cancer patients. When stratified by gender, we noticed that only in females a significant link between poor survival, metastatic disease and the expression of both TUBB3 and TUBB6 was found, while in males no relationship was detectable. In order to get insights into this phenomenon, we analyzed the expression levels of both tubulin isotypes in a panel of 22 colorectal cancer cell lines (13 from males and 9 from females) in basal conditions and after serum starvation, as a stimulus capable to activate the expression of this survival pathway. Male cell lines exhibited a relative resistance to serum starvation and higher levels of the tubulin isotypes, while only in female there was the activation of the pathway under stressing conditions. These findings indicate that in males the survival pathway related to TUBB3/TUBB6 is constitutively active, while in females it is activated by environmental stressors. In order to test if androgens could be responsible for the constitutive activation of this pathway in males, we analyzed the expression of androgen receptor (AR) in the 22 cancer cell lines. A statistically significant correlation was found between AR and TUBB3 (R= 0. 53, p= 0.0062) and TUBB6 (R=0.41; p=0.048) at the gene level. The pivotal role of androgen receptor in driving this pathway was demonstrated after constitutive silencing of AR in two models (SW480 and COLO-320) with high AR and TUBB3/TUBB6 expression. Knock-down of AR was associated to a significant reduction of the expression of both TUBB3 and TUBB6, along with decreased clonogenic activity and increased sensitivity to agents such as oxaliplatin and SN-38. Mortality for colorectal cancer is different by gender and higher in males. The molecular basis for such difference is unknown. In this study, we have demonstrated that in female cell lines (and probably patients) the activation of the survival pathway mediated by TUBB3 and TUBB6 is driven by microenvironmental stress like serum starvation. Those tumors which grow in the most compelling microenvironments will overexpress both proteins and will exhibit an aggressive behavior. In male this correlation is lost since the survival pathway is constitutively active and not dependent on microenvironmental conditioning. This makes that the disease intrinsically more aggressive and less sensitive to treatments. The involvement of AR in the activation of this survival pathway opens the avenue to clinical trials assessing the efficacy of antiandrogens in the management of advanced colorectal cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4522. doi:1538-7445.AM2012-4522

Background: MicroRNA-200c directly targets ZEB1, an epithelial to mesenchymal (EMT)-inducing transcriptional repressor of E-cadherin and other key determinates of epithelial identity and polarity. Therefore, restoration of miR-200c results in repression of ZEB1, dramatic reduction in migration and invasion, and increased sensitivity to microtubule targeting chemotherapeutics. We hypothesized that in addition to ZEB1, miR-200c targets additional genes that contribute to its myriad effects on maintaining the epithelial phenotype by repressing mesenchymal genes. Results: We performed microarray analysis following restoration of miR-200c to aggressive endometrial cancer cells and identified 18 genes that were both significantly repressed and predicted to be direct targets of miR-200c. These genes, including fibronectin 1 (FN1), class III beta-tubulin (TUBB3), neurotrophic tyrosine receptor kinase type 2 (NTRK2 or TrkB), leptin receptor (LEPR), moesin (MSN1) and Rho GTPase activating protein 19 (ARHGAP19), are all typically expressed only in non-epithelial cells, such as fibroblasts or neurons. Utilizing luciferase reporter assays and mutational analysis, we validate each of these genes as direct targets of miR-200c. We also demonstrate that restoration of miR-200c leads to a dramatic decrease in mRNA and protein levels of these targets. Further we find that restoration of miR-200c restores anoikis sensitivity to suspended cells, leading to over a 1.5 fold increase in cell death. TrkB is a potent suppressor of anoikis in breast and ovarian cancer models. Addition of TrkB lacking the miR-200c binding site reversed the ability of miR-200c to suppress anoikis. Interestingly, neurotrophin 3 (NTF3), a ligand for TrkB, contains 2 putative miR-200c binding sites and is down regulated upon restoration of miR-200c. Thus, loss of miR-200c in carcinoma cells allows inappropriate translation of both TrkB and NTF3, setting up an autocrine loop that results in anoikis resistance. Conclusions: Our data demonstrate that miR-200c serves as a “guardian of the epithelial phenotype” and suppressor of EMT by directly targeting and repressing a program of genes that should not be on in differentiated epithelial cells. We identify a novel function of miR-200c, the ability to suppress anoikis resistance, an important yet understudied step in the metastatic cascade. Funding source: DOD Breast Cancer Program Idea Award BC084162 to JKR Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 137. doi:10.1158/1538-7445.AM2011-137

Objective: Class III β-tublin (TUBB3) is a potent prognostic biomarker for ovarian cancer and other solid tumors. In this work we have discovered a novel role for TUBB3 as a gateway for cytoskeletal prosurvival signals. Methods: Through a genomic screening of a cell line made resistant to a drug whose activity is TUBB3 dependent, we have identified two GTPases (GBP1 and GNAI1) whose expression is strictly related to TUBB3 expression. Using the technique of Far Western Blotting, we have demonstrated that these GTPases physically interact with TUBB3. At variance of TUBB3, the two GTPAses can be overexpressed in vitro. Therefore, they served as prey in a high density protein microarray to identify protein kinases capable to interact with them. Results: Using transient overexpression the functional role of the two GTPases was ascertained, with GBP1 capable to activate the TUBB3 pathway and GNAI1 with an inhibitory function on its. The physical interaction between the two GTPases and cytoskeletal proteins were proven with Far Western Blot. At variance with TUBB3, the two GTPases can be expressed as recombinant proteins. Taken advantage of this fact, the two proteins served as prey on high density protein array containing over 9,000 human proteins. At least 18 protein kinases were capabe to interact with either GBP1 or GNAI1 or both. Among these kinases, we focused our attention on PIM1. PIM1:TUBB3 correlation was proven in ovarian cancer cell lines, both sensitive and resistant to chemotherapeutics, using coimmunoprecipiation experiments and again far western blotting. For the first time, we discovered that PIM1 is expressed in the microtubules and its expression is directly related to resistance to Paclitaxel and Cisplatin, but not to Epothilone-B. Importantly, PIM1 is overexpressed in cytoskeleton in microtubules when TUBB3 pathway is activated in hypoxic conditions. Finally, to analyze the translational value of our findings, we performed immunohistochemical analysis of TUBB3 and PIM1 expression in a clinical subset of 98 ovarian cancer patients. This approach revealed that double negative patients (TUBB3 and PIM1) have an overall survival (median 76 months) better than that noticeable in double positive (median 29 months). Conclusions: TUBB3 is commonly believed as a simple mechanism of Paclitaxel-resistance. Instead, this work demonstrates that TUBB3 is a gateway for prosurvival signals like PIM1 into the cytoskeleton. Ovarian cancer patients exhibiting expression of both TUBB3 and PIM1 have high probability of drug resistance and extreme short survival and should be carefully monitored for response to first line chemotherapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4055. doi:10.1158/1538-7445.AM2011-4055

Background: Class III β tubulin (TUBB3) reduces microtubule stability and confers resistance to microtubule stabilizing taxanes, including paclitaxel and docetaxel. Expression of vascular endothelial growth factor (VEGF) is highly regulated by hypoxia and the regulation of VEGF expression by hypoxia is mediated by a hypoxia inducible factor-1a(HIF-1a), which increase transcription of the VEGF gene. Recent report showed hypoxia induces expression of TUBB3 by HIF-1a. Inhibition of VEGF system may decrease the expression of HIF-1a and TUBB3. This study was undertaken to investigate the synergistic antitumor effects of bevacizumab and palitaxel on TUBB3 expressing gastric cancer cells. Methods: We evaluated the expressions of TUBB3 and HIF-1a under normoxic and hypoxic conditions in human gastric cancer cells. The chemoresistance of TUBB3 to paclitaxel was checked through knockdown of TUBB3. Expressions of TUBB3 and HIF-1a were measured under blocking of VEGFR1/2 with anti-FLT1 and anti-KDR antibodies. The effects of bevacizumab and paclitaxel on expressions of TUBB3 and HIF1a were monitored by western blot and MTT assay. Results: The chemosensitivities to paclitaxel were increased by siTUBB3. Elevated expressions of TUBB3 and HIF-1a in gastric cancer cells under hypoxic conditions were reduced in the prescence of anti-KDR and anti-FLT1 antibodies. When we added the bevacizumab to paclitaxel, the protein expressions of TUBB3 and HIF-1a in gastric cancer cells under hypoxic conditions were significantly decreased as compared with paclitaxel alone. Apoptosis of gastric cancer cells were more increased in a combined treatment with bevacizumab and paclitaxel than paclitaxel alone. Tyrosine phosphorylation of Erk and Akt was also measured in gastric cancer cells under hypoxic conditions in the absence or prescence of anti-KDR, anti FLT-1 and paclitaxels. Hypoxia induced phosphorylation of Erk and Akt in gastric cancer cells was more decreased by addition of anti FLT-1 and anti KDR antibodies to paclitaxel as compared with paclitaxel alone. Conclusion: Our experiments show that a VEGF/VEGFR/pERK and, or pAKT pathway is activated by hypoxic gastric cancer cells to induce HIF-1a and TUBB3 expressions. Combined treatment with paclitaxel and bevacizumab, a recombinant humanized monoclonal antibody that binds to VEGF, synergistically decreased the expressions of HIF-1a and TUBB3 in gastric cancer cells in hypoxic conditions. With increasing use of taxanes in the treatment of gastric cancer, this study showed the potential synergistic antitumor effects of bevacizumab and paclitaxel in a TUBB3, a marker of resistance to paclitaxel, overexpressing gastric cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4252. doi:10.1158/1538-7445.AM2011-4252

Microtubules are a component essential of the cytoskeleton and mediate a complex network of functions, including the assembly of mitotic spindle but also the intracellular trafficking of signaling proteins. In order to achieve their functions, composition of microtubules is tightly regulated, particularly at the (+) end, exposing the β-tubulin subunit. At least seven independent genes encode for β-tubulin, whose expression is diverse in several tissues. Functionally β-tubulin isotypes can be divided in constitutive isotypes and regulatory isotypes. Regulatory isotypes include class III β-tubulin (TUBB3), whose role as factor of resistance to chemotherapy has been demonstrated in a large number of solid tumors. Some structural motifs of TUBB3 are shared with class V β-tubulin (TUBB6), but since no commercial antibodies are available for this isotype its role as an additional factor of chemoresistance is not known. In this work we have developed a polyclonal anti-TUBB6 antibody. Since the highest degree of dishomology between the β-tubulin isotypes is concentrated at the C-terminus, all the tails of the β-tubulin isotypes were cloned in E. coli in a carrier vector encoding MBP (maltose-binding protein). Recombinant proteins were expressed in E. coli and then purified through columns filled with Amylose resin column, able to entrap MBP. The negative control was represented by MBP alone. The developed anti-TUBB6 antibody was validated since it demonstrated specific reactivity only against TUBB6. Therefore, the profile of expression of TUBB6 was assessed in a panel of normal and cancer tissues. In normal tissues, immunostaining of TUBB6 was confined to given cellular elements and in a focal pattern such as in ciliary cells of bronchus, salpinx, thereby suggesting that class V is focally associated to the cilium complex. In contrast to the focal expression noticed in normal tissues, in cancer specimens TUBB6 exhibited a diffuse staining pattern, with the highest expression detectable in colon and prostate carcinoma. In order to have some insights on the role of TUBB6 as a biomarker, we studied TUBB6 expression in a cohort of 56 ovarian cancer patients with a median follow up of 54 months. There was no statistically significant difference in terms of Time to Progression (TTP) in cases with high versus low TUBB6 expression (p value= 0.4). On the other hand, cases with high TUBB6 expression showed a worse Overall Survival (OS) with respect to cases with low TUBB6 expression (median OS= 50 months versus median OS= 96 months, respectively, p value=0.047). In summarizing these results, patients expressing high levels of TUBB6 are featured by biological aggressiveness and poor response in terms of OS to standard chemotherapy. These results suggest that TUBB6 could be useful as a biomarker for ovarian cancer patients and possibly other tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 645.

BAL27862 is a synthetic small molecule that induces apoptosis due to the inhibition of tubulin polymerization via a potentially new mechanism of action. The activity of this novel compound was evaluated in human breast and ovarian cancer cell line variants selected for resistance by continuous exposure to taxanes. BAL27862 retained activity against MDR1 (ABCB1)/P-glycoprotein-mediated resistance variants, unlike vinca alkaloids and taxanes. Activity was also observed in the taxane-resistant ovarian 1A9/TxTP50 tumor cell line, which overexpresses TUBB3 and was approximately 35x resistant to taxanes. Moreover, BAL27862 retained activity in ovarian cancer cells with acquired mutations in the class I (M40) β-tubulin isotype (1A9/PTX10, Phe270->Val; 1A9/PTX22, Ala364->Thr) which confer taxane resistance. The MCF-7/TxTP50 breast tumor variant was 9x resistant to taxanes, and 5x resistant to epothilone B, yet retained sensitivity to BAL27862, the vinca alkaloids (vinblastine and vincristine), colchicine and anthracyclines. These cells have significantly reduced levels of the taxane-binding target, the class I β-tubulin isotype, and reduced levels of total α- and β-tubulin relative to wild-type cells, alterations which may be responsible for a 34% reduction in bound BODIPY-paclitaxel as detected by FACS analysis. Evidence of resistance to BAL27862 (3x) was only detected in the ovarian OVCAR-3/TxTP5 variant, which was also resistant to taxanes (8x), the vinca alkaloids (4x) and colchicine (2x) and ixabepilone (2x). Finally, silencing TUBB3 using a specific siRNA sensitized cells to taxanes but did not affect BAL27862 activity in both wild-type MCF-7 and OVCAR-3 cells relative to appropriate controls, suggesting that TUBB3 status may not influence BAL27862 response. Thus, BAL27862 is a new agent with a distinct in vitro activity profile against a broad panel of human breast and ovarian tumor cell lines resistant to standard tubulin-interacting agents. These data strongly support further development of BAL27862 as a novel anticancer agent. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4412.