Tanya Dwarte

Activation of tissue transglutaminase transcription by histone deacetylase inhibition as a therapeutic approach for Myc oncogenesis

Histone deacetylase (HDAC) inhibitors reactivate tumor suppressor gene transcription; induce cancer cell differentiation, growth arrest, and programmed cell death; and are among the most promising new classes of anticancer drugs. Myc oncoproteins can block cell differentiation and promote cell proliferation and malignant transformation, in some cases by modulating target gene transcription. Here, we show that tissue transglutaminase (TG2) was commonly reactivated by HDAC inhibitors in neuroblastoma and breast cancer cells but not normal cells and contributed to HDAC inhibitor-induced growth arrest. TG2 was the gene most significantly repressed by N-Myc in neuroblastoma cells in a cDNA microarray analysis and was commonly repressed by N-Myc in neuroblastoma cells and c-Myc in breast cancer cells. Repression of TG2 expression by N-Myc in neuroblastoma cells was necessary for the inhibitory effect of N-Myc on neuroblastoma cell differentiation. Dual step cross-linking chromatin immunoprecipitation and protein coimmunoprecipitation assays showed that N-Myc acted as a transrepressor by recruiting the HDAC1 protein to an Sp1-binding site in the TG2 core promoter in a manner distinct from its action as a transactivator at E-Box binding sites. HDAC inhibitor treatment blocked the N-Myc-mediated HDAC1 recruitment and TG2 repression in vitro. In neuroblastoma-bearing N-Myc transgenic mice, HDAC inhibitor treatment induced TG2 expression and demonstrated marked antitumor activity in vivo. Taken together, our data indicate the critical roles of HDAC1 and TG2 in Myc-induced oncogenesis and have significant implications for the use of HDAC inhibitor therapy in Myc-driven oncogenesis.

Effective delivery of siRNA into cancer cells and tumors using well-defined biodegradable cationic star polymers.

Cancer is one of the most common causes of death worldwide. Two types of cancer that have high mortality rates are pancreatic and lung cancer. Despite improvements in treatment strategies, resistance to chemotherapy and the presence of metastases are common. Therefore, novel therapies which target and silence genes involved in regulating these processes are required. Short-interfering RNA (siRNA) holds great promise as a therapeutic to silence disease-causing genes. However, siRNA requires a delivery vehicle to enter the cell to allow it to silence its target gene. Herein, we report on the design and synthesis of cationic star polymers as novel delivery vehicles for siRNA to silence genes in pancreatic and lung cancer cells. Dimethylaminoethyl methacrylate (DMAEMA) was polymerized via reversible addition-fragmentation transfer polymerization (RAFT) and then chain extended in the presence of both cross-linkers N,N-bis(acryloyl)cistamine and DMAEMA, yielding biodegradable well-defined star polymers. The star polymers were characterized by transmission electron microscopy, dynamic light scattering, ζ potential, and gel permeation chromatography. Importantly, the star polymers were able to self-assemble with siRNA and form small uniform nanoparticle complexes. Moreover, the ratios of star polymer required to complex siRNA were nontoxic in both pancreatic and lung cancer cells. Treatment with star polymer-siRNA complexes resulted in uptake of siRNA into both cell lines and a significant decrease in target gene mRNA and protein levels. In addition, delivery of clinically relevant amounts of siRNA complexed to the star polymer were able to silence target gene expression by 50% in an in vivo tumor setting. Collectively, these results provide the first evidence of well-defined small cationic star polymers to deliver active siRNA to both pancreatic and lung cancer cells and may be a valuable tool to inhibit key genes involved in promoting chemotherapy drug resistance and metastases.

TUBB3/βIII-tubulin acts through the PTEN/AKT signaling axis to promote tumorigenesis and anoikis resistance in non-small cell lung cancer.

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.

Enhancing the anti-angiogenic action of histone deacetylase inhibitors

BackgroundHistone deacetylase inhibitors (HDACIs) have many effects on cancer cells, such as growth inhibition, induction of cell death, differentiation, and anti-angiogenesis, all with a wide therapeutic index. However, clinical trials demonstrate that HDACIs are more likely to be effective when used in combination with other anticancer agents. Moreover, the molecular basis for the anti-cancer action of HDACIs is still unknown. In this study, we compared different combinations of HDACIs and anti-cancer agents with anti-angiogenic effects, and analysed their mechanism of action.ResultsTrichostatin A (TSA) and α-interferon (IFNα) were the most effective combination across a range of different cancer cell lines, while normal non-malignant cells did not respond in the same manner to the combination therapy. There was a close correlation between absence of basal p21WAF1 expression and response to TSA and IFNα treatment. Moreover, inhibition of p21WAF1 expression in a p21WAF1-expressing breast cancer cell line by a specific siRNA increased the cytotoxic effects of TSA and IFNα. In vitro assays of endothelial cell function showed that TSA and IFNα decreased endothelial cell migration, invasion, and capillary tubule formation, without affecting endothelial cell viability. TSA and IFNα co-operatively inhibited gene expression of some pro-angiogenic factors: vascular endothelial growth factor, hypoxia-inducible factor 1α and matrix metalloproteinase 9, in neuroblastoma cells under hypoxic conditions. Combination TSA and IFNα therapy markedly reduced tumour angiogenesis in neuroblastoma-bearing transgenic mice.ConclusionOur results indicate that combination TSA and IFNα therapy has potent co-operative cytotoxic and anti-angiogenic activity. High basal p21WAF1 expression appears to be acting as a resistance factor to the combination therapy.

The cyclin-dependent kinase inhibitor, p21WAF1, promotes angiogenesis by repressing gene transcription of thioredoxin-binding protein 2 in cancer cells

The cyclin-dependent kinase inhibitor, p21(WAF1), induces cell-cycle arrest and can act as a tumor suppressor. However, increasing evidence indicates that p21(WAF1) can also increase resistance to some anticancer therapies and thus promote tumor growth. The mechanisms explaining this paradox have not been explained. We found that conditioned media from MCF-7 breast cancer cells transfected with a p21(WAF1)-specific small interfering RNA (siRNA) significantly reduced endothelial cell migration, invasion and vascular sprouting. Liquid chromatography/mass spectrometry analysis of the conditioned media revealed that p21(WAF1) knockdown significantly reduced secretion of thioredoxin (Trx), a redox protein known to promote tumor angiogenesis. p21(WAF1) knockdown decreased Trx enzymatic activity in cancer cells, by effects on the expression levels of intracellular thioredoxin-binding protein 2 (TBP2), known to bind and inactivate Trx. Consistent with these findings, media from cancer cells transfected with TBP2 siRNA promoted endothelial cell invasion and blocked the anti-angiogenic effect of p21(WAF1) siRNA. Addition of Trx siRNA blocked the pro-angiogenic effects of TBP2 siRNA. Chromatin immunoprecipitation assays showed p21(WAF1) bound TBP2 gene promoter. Taken together, our data suggests that p21(WAF1) can induce Trx secretion and angiogenesis in cancer cells, by direct transcriptional repression of the TBP2 promoter.

Correction to "effective delivery of siRNA into cancer cells and tumors using well-defined biodegradable cationic star polymers".

Published: February 18, 2015 Figure 1. Synthesis of poly(DMAE-MA) biodegradable cationic star polymers. (A) A schematic diagram showing the synthesis steps required to produce the cationic star polymer: (i) synthesis of P(DMAE-MA) homopolymer arm; (ii) chain extension of P(DMAE-MA) in the presence of N,N′-bis(acryloyl)cystamine and DMAE-A. (B) H NMR spectrum of the purified star polymer after quaternization with HCl (recorded in D2O)

Abstract 2076: βIII-tubulin is required for the tumorigenic phenotype and resistance to anoikis via the PTEN/AKT signaling axis in non-small cell lung cancer

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA BACKGROUND: Non-small cell lung cancer (NSCLC) has a dismal prognosis and remains the most common cause of cancer death worldwide. Expression of βIII-tubulin, encoded by the TUBB3 gene, is associated with clinical resistance and aggressive disease in NSCLC1. Herein, we interrogated the mechanistic role of βIII-tubulin in regulating the tumorigenic potential of NSCLC. METHODS: Functional studies involved independent clones of NSCLC H460 cells stably expressing shRNA targeting βIII-tubulin, H460 controls stably expressing (non-functional) shRNA and βIII-tubulin rescue clones established in our laboratory2. Differential proteomics was conducted using fluorescence based 2D-DIGE and mass spectrometry. Gene and protein expression performed by qRT-PCR and western blotting respectively. To assess effects on tumor growth and incidence we used metastatic (tail vein) and subcutaneous models of NSCLC. Tumors monitored by CT or Xenogen imaging. RESULTS: Functional and differential proteomics revealed that βIII-tubulin regulates expression of tumor growth- and metastases-associated proteins. In particular, the tumor suppressor maspin, associated with adhesion and metastasis, was differentially regulated by βIII-tubulin. Functionally, βIII-tubulin suppression led to altered cell morphology, increased cell adhesion and increased sensitivity to anoikis. Mechanistically, we identified PTEN and AKT kinase as a key signaling axis mediating anoikis and regulated by βIII-tubulin levels in NSCLC cells. Finally, βIII-tubulin suppression was shown to reduce NSCLC tumor growth and incidence in vivo. Collectively, these data identified βIII-tubulin as a regulator of tumor growth and metastasis through regulation of PTEN and AKT signaling. We conclude that suppressing βIII-tubulin may reduce tumor growth in NSCLC. SIGNIFICANCE: This is the first study to show that silencing βIII-tubulin alters the expression of proteins involved in promoting tumorigenicity and increases sensitivity to anoikis, leading to reduced tumor incidence. Targeting βIII-tubulin could be a promising strategy for inhibiting tumor growth and metastasis in NSCLC. 1 Kavallaris, M. Nature Rev Cancer, 10:194-204, 2010 2 McCarroll et al. Cancer Res 70 :4995-5003, 2010 Citation Format: Joshua A. McCarroll, Pei Pei Gan, Rafael B. Erlich, Marjorie Liu, Tanya Dwarte, Mia C. Akerfeldt, Melissa Chang, Michael S. Shum, Frances Byrne, Maria Kavallaris. βIII-tubulin is required for the tumorigenic phenotype and resistance to anoikis via the PTEN/AKT signaling axis in non-small cell lung cancer. [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 2076. doi:10.1158/1538-7445.AM2014-2076

Abstract 1969: Silencing αIII-tubulin by RNA interfering nanoparticles in non-small cell lung cancer

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Advanced non-small cell lung cancer (NSCLC) has a dismal prognosis and remains the most common cause of cancer death worldwide. One of the primary reasons for its poor survival rate is its high level of resistance to chemotherapy. Expression of the microtubule protein αIII-tubulin is associated with an aggressive and drug resistant disease in NSCLC. Recently, we demonstrated for the first time that targeting αIII-tubulin using siRNA sensitizes NSCLC cells to chemotherapy and reduces tumour growth in mice. However, therapeutic delivery of siRNA to cancer cells in vivo remains problematic. Previously our group has designed novel non-viral nanoparticles (iNOP-7) which can complex and deliver siRNA in vivo. However, whether these nanoparticles can deliver siRNA targeting αIII-tubulin to NSCLC cells is unknown. Therefore, the aims of this study were to determine whether iNOP-7 could deliver siRNA targeting αIII-tubulin to lung cancer cells in vitro and in vivo. Methods: Two NSCLC cell lines (H460 and Calu-6) were treated with increasing amounts of iNOP-7 complexed to siRNA. Cell viability was assessed using a colorimetric cell counting assay. Cellular uptake of iNOP-7-siRNA complexes was determined by confocal microscopy and flow cytometry. Gene silencing efficiency of iNOP-7-αIII-tubulin siRNA complexes was measured by real time PCR and Western blotting. To determine whether iNOP-7 could deliver siRNA to lung tumors, 1x106 stably expressing luciferase H460 cells were orthotopically implanted into the lungs of Balb/c nude mice. Tumor growth was monitored weekly using the Xenogen IVIS System. Twelve days post-tumor implantation mice were treated systemically with iNOP-7 complexed to fluorescently labelled siRNA or αIII-tubulin siRNA (1.5mg/kg once a day for three days). Twenty four hours after the final injection tumor tissue was collected and uptake and gene silencing efficiency was evaluated using confocal microscopy and real-time PCR. Results: iNOP-7 did not affect cell viability and was able to effectively deliver high levels of siRNA to both cell lines. Importantly, iNOP-7-αIII-tubulin siRNA complexes potently silenced αIII-tubulin mRNA and protein levels by >85% (n=3, p 65% (n=2 animals per group) when compared to controls. Conclusions: This is the first study to show that RNA interfering nanoparticles can silence αIII-tubulin expression in NSCLC cells both in vitro and in vivo. Future studies will address the therapeutic potential of iNOP-7-αIII-tubulin siRNA complexes alone and in combination with chemotherapeutic agents in an orthotopic lung cancer mouse model. 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 1969. doi:1538-7445.AM2012-1969

Abstract 5026: βIII-Tubulin regulates expression of proteins involved in tumorigenesis and metastasis in non-small cell lung cancer

Survival rates for advanced non-small cell lung cancer (NSCLC) remain dismal. Recently, we showed that silencing the microtubule protein βIII-tubulin using siRNA sensitizes NSCLC cells to chemotherapy drugs 1 . We have also recently shown that βIII-tubulin plays a role in regulating tumor growth both in vitro and in vivo 2 . Importantly, high levels of βIII-tubulin are correlated with more aggressive and drug refractory tumors in the clinic 3 . To date, the broader role of βIII-tubulin in NSCLC has not been determined and the aims of this study were to examine the role of βIII-tubulin on the expression of proteins involved in regulating tumorigenesis and metastasis. Methods: Using a functional and differential proteomics approach we examined NSCLC clones stably expressing shRNA against βIII-tubulin (pRS/βIII SH4 and pRS/βIII SH59 ) or control (pRS/Ctrl SH1 and pRS/Ctrl SH2 ). Cytosolic and nuclear protein fractions were prepared and 2-D DIGE was performed over broad (pI 4-7) and narrow (pI 4.5-5.5) pI ranges. Differences in protein expression between pRS/βIII SH4 and pRS/βIII SH59 and pRS/Ctrl SH1 and pRS/Ctrl SH2 cells were assessed using Decyder software. Protein spots of interest were excised and identified by mass spectrometry. Those identified as being significantly altered were then validated by western blot. Results: Eleven out of a total of 963 proteins (1.1%; pI 4-7) and 53 out of 753 proteins (7%; pI 4.5-5.5) were found to be significantly altered in the cytoplasmic fractions of pRS/βIII SH4 and 59 cells when compared to their controls (pRS/Ctrl SH1 and SH2 ). In addition, 33 out of 1331 proteins (2.5%; pI 4-7) and 42 out of 502 proteins (8.4%; pI 4.5-5.5) were significantly altered in the nuclear fractions of pRS/βIII SH4 and SH59 cells compared to controls. Importantly, a number of proteins which are involved in regulating tumor growth and metastasis were identified as being differentially expressed in the βIII-tubulin knockdown cells. A significant decrease (greater than 2 fold) in heat shock protein 60 expression (promotes metastasis) was observed in the pRS/βIII SH4 and SH59 nuclear fractions when compared to controls. Furthermore, a greater than 6 fold increase in the tumor suppressor protein Tropomyosin 1 and a greater than 2 fold increase in the serpin B5 precursor protein Maspin (inhibitor of metastasis) was identified in the pRS/βIII SH4 and SH59 cytoplasmic fractions when compared to controls. These changes were confirmed by western blotting. Conclusions: This is the first study to show that silencing βIII-tubulin significantly alters the expression of proteins involved in regulating tumorigenesis and metastasis in lung cancer. Targeting βIII-tubulin could be a promising strategy for inhibiting tumor growth and metastasis in lung cancer. 1 Gan et al. Cancer Res, 67:9356-63, 2007 2 McCarroll et al. 100 th Annual AACR meeting. A3337, 2009 3 Seve D 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5026.