Gitanjali Jayachandran

Phase I Clinical Trial of Systemically Administered TUSC2(FUS1)-Nanoparticles Mediating Functional Gene Transfer in Humans

Background Tumor suppressor gene TUSC2/FUS1 (TUSC2) is frequently inactivated early in lung cancer development. TUSC2 mediates apoptosis in cancer cells but not normal cells by upregulation of the intrinsic apoptotic pathway. No drug strategies currently exist targeting loss-of–function genetic abnormalities. We report the first in-human systemic gene therapy clinical trial of tumor suppressor gene TUSC2. Methods Patients with recurrent and/or metastatic lung cancer previously treated with platinum-based chemotherapy were treated with escalating doses of intravenous N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTAP):cholesterol nanoparticles encapsulating a TUSC2 expression plasmid (DOTAP:chol-TUSC2) every 3 weeks. Results Thirty-one patients were treated at 6 dose levels (range 0.01 to 0.09 milligrams per kilogram). The MTD was determined to be 0.06 mg/kg. Five patients achieved stable disease (2.6–10.8 months, including 2 minor responses). One patient had a metabolic response on positron emission tomography (PET) imaging. RT-PCR analysis detected TUSC2 plasmid expression in 7 of 8 post-treatment tumor specimens but not in pretreatment specimens and peripheral blood lymphocyte controls. Proximity ligation assay, performed on paired biopsies from 3 patients, demonstrated low background TUSC2 protein staining in pretreatment tissues compared with intense (10–25 fold increase) TUSC2 protein staining in post-treatment tissues. RT-PCR gene expression profiling analysis of apoptotic pathway genes in two patients with high post-treatment levels of TUSC2 mRNA and protein showed significant post-treatment changes in the intrinsic apoptotic pathway. Twenty-nine genes of the 82 tested in the apoptosis array were identified by Igenuity Pathway Analysis to be significantly altered post-treatment in both patients (Pearson correlation coefficient 0.519; p<0.01). Conclusions DOTAP:chol-TUSC2 can be safely administered intravenously in lung cancer patients and results in uptake of the gene by human primary and metastatic tumors, transgene and gene product expression, specific alterations in TUSC2-regulated pathways, and anti-tumor effects (to our knowledge for the first time for systemic DOTAP:cholesterol nanoparticle gene therapy). Trial Registration ClinicalTrials.gov NCT00059605

Tumor-specific activation of human telomerase reverses transcriptase promoter activity by activating enhancer-binding protein-2β in human lung cancer cells

The up-regulated expression and telomerase activity of human telomerase reverse transcriptase (hTERT) are hallmarks of tumorigenesis. The hTERT promoter has been shown to promote hTERT gene expression selectively in tumor cells but not in normal cells. However, little is known about how tumor cells differentially activate hTERT transcription and induce telomerase activity. In this study, we identified activating enhancer-binding protein-2β (AP-2β) as a novel transcription factor that specifically binds to and activates the hTERT promoter in human lung cancer cells. AP-2β was detected in hTERT promoter DNA-protein complexes formed in nuclear extracts prepared only from lung cancer cells but not from normal cells. We verified the tumor-specific binding activity of AP-2β for the hTERT promoter in vitro and in vivo and detected high expression levels of AP-2β in lung cancer cells. We found that ectopic expression of AP-2β reactivated hTERT promoter-driven reporter green fluorescent protein (GFP) gene and endogenous hTERT gene expression in normal cells, enhanced GFP gene expression in lung cancer cells, and prolonged the life span of primary lung bronchial epithelial cells. Furthermore, we found that inhibition of endogenous AP-2β expression by AP-2β gene-specific small interfering RNAs effectively attenuated hTERT promoter-driven GFP expression, suppressed telomerase activity, accelerated telomere shortening, and inhibited tumor cell growth by induction of apoptosis in lung cancer cells. Our results demonstrate the tumor-specific activation of the hTERT promoter by AP-2β and imply the potential of AP-2β as a novel tumor marker or a cancer therapeutic target.

Tumor Suppressor 101F6 and Ascorbate Synergistically and Selectively Inhibit Non–Small Cell Lung Cancer Growth by Caspase-Independent Apoptosis and Autophagy

101F6 is a candidate tumor suppressor gene harbored on chromosome 3p21.3, a region with frequent and early allele loss and genetic alterations in many human cancers. We previously showed that enforced expression of wild-type 101F6 by adenoviral vector-mediated gene transfer significantly inhibited tumor cell growth in 3p21.3-deficient non-small cell lung cancer (NSCLC) cells in vitro and in vivo. The molecular mechanism of 101F6-mediated tumor suppression is largely unknown. A computer-aided structural and functional model predicts the 101F6 protein to be a member of the cytochrome b561 protein family that is involved in the regeneration of the antioxidant ascorbate. 101F6 protein is expressed in normal lung bronchial epithelial cells and fibroblasts but is lost in most lung cancers. Treatment with 101F6 nanoparticle-mediated gene transfer in combination with a subpharmacologic dose (200-500 micromol/L) of ascorbate synergistically and selectively inhibited lung cancer cell growth in vitro. Systemic injection of 101F6 nanoparticles plus the i.p. injection of ascorbate synergistically inhibited both tumor formation and growth in human NSCLC H322 orthotopic lung cancer mouse models (P<0.001). Furthermore, exogenous expression of 101F6 enhanced intracellular uptake of ascorbate, leading to an accumulation of cytotoxic H(2)O(2) and a synergistic killing of tumor cells through caspase-independent apoptotic and autophagic pathways. The antitumor synergism showed by the combination treatment with systemic administration of 101F6 nanoparticles and ascorbate on lung cancer offers an attractive therapeutic strategy for future clinical trials in cancer prevention and treatment.

Synergistic Tumor Suppression by Coexpression of FUS1 and p53 Is Associated with Down-regulation of Murine Double Minute-2 and Activation of the Apoptotic Protease-Activating Factor 1–Dependent Apoptotic Pathway in Human Non–Small Cell Lung Cancer Cells

FUS1 is a novel tumor suppressor gene identified in human chromosome 3p21.3 region. Loss of expression and deficiency of posttranslational modification of FUS1 protein have been found in a majority of human lung cancers. Restoration of wild-type FUS1 in 3p21.3-deficient human lung cancer cells exhibited a potent tumor suppression function in vitro and in vivo. In this study, we evaluated the combined effects of FUS1 and tumor suppressor p53 on antitumor activity and explored the molecular mechanisms of their mutual actions in human non-small cell lung cancer (NSCLC) cells. We found that coexpression of FUS1 and p53 by N-[1-(2,3-dioleoyloxyl)propyl]-NNN-trimethylammoniummethyl sulfate:cholesterol nanoparticle-mediated gene transfer significantly and synergistically inhibited NSCLC cell growth and induced apoptosis in vitro. We also found that a systemic treatment with a combination of FUS1 and p53 nanoparticles synergistically suppressed the development and growth of tumors in a human H322 lung cancer orthotopic mouse model. Furthermore, we showed that the observed synergistic tumor suppression by FUS1 and p53 concurred with the FUS1-mediated down-regulation of murine double minute-2 (MDM2) expression, the accumulation and stabilization of p53 protein, as well as the activation of the apoptotic protease-activating factor 1 (Apaf-1)-dependent apoptotic pathway in human NSCLC cells. Our results therefore provide new insights into the molecular mechanism of FUS1-mediated tumor suppression activity and imply that a molecular therapy combining two or more functionally synergistic tumor suppressors may constitute a novel and effective strategy for cancer treatment.

Fragile histidine triad-mediated tumor suppression of lung cancer by targeting multiple components of the Ras/Rho GTPase molecular switch.

The fragile histidine triad (FHIT) gene has been shown to function as a tumor suppressor gene in vitro and in vivo. However, the mechanism of its action is still largely unknown. To elucidate the molecular mechanism and biological pathway in FHIT-mediated tumor suppression, we used a complementary gene and protein expression profiling with DNA microarray and ProteinChip technologies to quantitatively monitor cellular changes in gene and protein expression and discover the molecular targets of FHIT in non-small cell lung carcinoma (NSCLC) cells. The Ras/Rho signaling pathway was identified as one of the unique biological pathways associated with FHIT activity. A significantly down-regulated expression of genes and proteins of multiple key components in the Ras/Rho GTPases molecular switch, including Ran, Rab, Rac, Rap, and Ral, was observed on gene and protein expression profiles and further validated by Western blot analysis. Ectopic activation of FHIT in FHIT-deficient H1299 cells also significantly reduced the invasive potential of tumor cells by down-regulating expression of RhoC, a potential marker of tumor cell invasion and metastases. A simultaneous knockdown of the expression of several key Ras/Rho signaling molecules using gene-specific small interfering RNAs (RHO-siRNA) targeting selected Rab11, Rac1, and Rap1 genes significantly inhibited tumor cell growth and induced apoptosis in NSCLC cells in vitro, and a local injection of RHO-siRNAs complexed with N-[1-(2,3-dioleoyloxyl)propyl]-N,N,N-trimethylammoniummethyl sulfate:cholesterol nanoparticles inhibited tumor growth in A549 tumor xenografts in mice, mimicking the AdFHIT-mediated tumor-suppressing effect. These results suggest a new role of FHIT in down-regulating the Ras/Rho GTPase-associated oncogenic signaling pathway.

NPRL2 Sensitizes Human Non-Small Cell Lung Cancer (NSCLC) Cells to Cisplatin Treatment by Regulating Key Components in the DNA Repair Pathway

NPRL2, one of the tumor suppressor genes residing in a 120-kb homozygous deletion region of human chromosome band 3p21.3, has a high degree of amino acid sequence homology with the nitrogen permease regulator 2 (NPR2) yeast gene, and mutations of NPRL2 in yeast cells are associated with resistance to cisplatin-mediated cell killing. Previously, we showed that restoration of NPRL2 in NPRL2-negative and cisplatin-resistant cells resensitize lung cancer cells to cisplatin treatment in vitro and in vivo. In this study, we show that sensitization of non-small cell lung cancer (NSCLC) cells to cisplatin by NPRL2 is accomplished through the regulation of key components in the DNA-damage checkpoint pathway. NPRL2 can phosphorylate ataxia telangiectasia mutated (ATM) kinase activated by cisplatin and promote downstream γ-H2AX formation in vitro and in vivo, which occurs during apoptosis concurrently with the initial appearance of high-molecular-weight DNA fragments. Moreover, this combination treatment results in higher Chk1 and Chk2 kinase activity than does treatment with cisplatin alone and can activate Chk2 in pleural metastases tumor xenograft in mice. Activated Chk1 and Chk2 increase the expression of cell cycle checkpoint proteins, including Cdc25A and Cdc25C, leading to higher levels of G2/M arrest in tumor cells treated with NPRL2 and cisplatin than in tumor cells treated with cisplatin only. Our results therefore suggest that ectopic expression of NPRL2 activates the DNA damage checkpoint pathway in cisplatin-resistant and NPRL2-negative cells; hence, the combination of NPRL2 and cisplatin can resensitize cisplatin nonresponders to cisplatin treatment through the activation of the DNA damage checkpoint pathway, leading to cell arrest in the G2/M phase and induction of apoptosis. The direct implication of this study is that combination treatment with NPRL2 and cisplatin may overcome cisplatin resistance and enhance therapeutic efficacy.

Exogenous restoration of TUSC2 expression induces responsiveness to erlotinib in wildtype epidermal growth factor receptor (EGFR) lung cancer cells through context specific pathways resulting in enhanced therapeutic efficacy

Expression of the tumor suppressor gene TUSC2 is reduced or absent in most lung cancers and is associated with worse overall survival. In this study, we restored TUSC2 gene expression in several wild type EGFR non-small cell lung cancer (NSCLC) cell lines resistant to the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor erlotinib and analyzed their sensitivity to erlotinib in vitro and in vivo. A significant inhibition of cell growth and colony formation was observed with TUSC2 transient and stable expression. TUSC2-erlotinib cooperativity in vitro could be reproduced in vivo in subcutaneous tumor growth and lung metastasis formation lung cancer xenograft mouse models. Combination treatment with intravenous TUSC2 nanovesicles and erlotinib synergistically inhibited tumor growth and metastasis, and increased apoptotic activity. High-throughput qRT-PCR array analysis enabling multi-parallel expression profile analysis of eighty six receptor and non-receptor tyrosine kinase genes revealed a significant decrease of FGFR2 expression level, suggesting a potential role of FGFR2 in TUSC2-enhanced sensitivity to erlotinib. Western blots showed inhibition of FGFR2 by TUSC2 transient transfection, and marked increase of PARP, an apoptotic marker, cleavage level after TUSC2-erlotinb combined treatment. Suppression of FGFR2 by AZD4547 or gene knockdown enhanced sensitivity to erlotinib in some but not all tested cell lines. TUSC2 inhibits mTOR activation and the latter cell lines were responsive to the mTOR inhibitor rapamycin combined with erlotinib. These results suggest that TUSC2 restoration in wild type EGFR NSCLC may overcome erlotinib resistance, and identify FGFR2 and mTOR as critical regulators of this activity in varying cellular contexts. The therapeutic activity of TUSC2 could extend the use of erlotinib to lung cancer patients with wildtype EGFR.

Abstract 2741: Synergistic inhibition of tumor growth and overcoming chemo-resistance by simultaneously targeting key components in DNA damage/repair, epigenetic, and putative cancer stem cell signaling pathways using novel dual-functional DNA-alkylating/HDAC inhibitor and tumor suppressor gene nanoparticles in lung cancer

In this study we explored a novel therapeutic strategy aimed to simultaneously target multiple key components in DNA damage/repair, epigenetic, and putative cancer stem cell (CSC) signaling pathways using a novel dual-target DNA-alkylating nitrogen mustard/HDAC inhibitor (DANM/HDACi) and tumor suppressor gene (TSG) nanoparticles to promote antitumor synergy and overcome drug resistance in lung cancer. We evaluated therapeutic effects of a new class DNAM/HDACi, NL10, as a single agent or in combination with novel TSGs NPRL2, a regulator of the DNA damage checkpoint pathway, and p53, a regulator of apoptosis and drug resistance in the DNA damage/repair pathway in more than 50 human NSCLC and SCLC cell lines. We detected a high anti-tumor potency by NL101 treatment across lung cancer cell lines, with IC 50 doses ranging from nanomolar to lower micromolar levels. A 20 to 100-fold higher level of cytotoxicity was observed across these cell lines, particularly in SCLC cell lines, treated by NL101 than in those treated by bendamustine, a nitrogen mustard that is currently in Phase II/III clinical trials in SCLC patients. We analyzed the NL101-induced DNA damage in NSCLC H1299 cells and found that NL101 induced a significantly higher degree of DNA damage than that induced by either bendamustine or cisplatin at the same IC 20 dose level for each agent. We analyzed the DNA-damage-induced apoptosis in NSCLC cells treated by NL101 alone or in combination with DOTAP:Cholesterol (DC)-complexed NPRL2- or p53-expressing plasmid DNA nanoparticles. We detected a significantly enhanced apoptosis in both combination treatments with NL101+DC-NPRL2 and NL101+DC-p53. We also found that combination treatment with NL101 and DC-NPRL2 nanoparticles promoted a synergistic inhibitory effect on tumor cell-induced clonogenecity in H1299 cells and on tumor cell growth in SCLC H69 and GLC16 cells at effective dose levels of NL101 from IC 10 to IC 75 . Furthermore, we found that the sensitivity of lung cancer cells to NL101 treatment showed a significantly negative correlation with expression levels of the HDAC family proteins HDAC1, HDAC2, HDAC3, and HDAC5 and the putative lung cancer stem cell (CSC) marker ALDH1 protein. We found a dose-dependent inhibition of multiple HDACs and CSC markers in both NSCLC and SCLC cells. Our results suggest that a combination treatment using a novel DANM/HDACi with pro-apoptotic TSGs targeting the DNA damage/repair, epigenetic, and CSC signaling pathways will enhance chemotherapeutic sensitivity, promote anti-cancer synergism, overcome drug resistance, and block tumor progression and relapse by targeting putative CSCs. (Supported by grants P50CA70907, RO1 CA-116322, and W81XWH0920139) 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 2741. doi:1538-7445.AM2012-2741

Abstract 4988: The S-nitrosylation-mediated modification of p53 leads to altered DNA binding and associates with inactivity of p53 in human melanoma cells

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Melanoma represents one of the cancers most resistant to radiation, DNA-damaging chemotherapy, and biotherapy. Notably, melanoma expresses high levels of wild-type tumor suppressor p53, while mutational inactivation of p53 is very rare. The mechanism for inactivating wild-type p53 function in melanoma remains unknown. It is known that melanoma secrets high levels of proinflammatory cytokines and produces reactive oxygen (ROS) and nitrogen species (RNS). Chronic inflammation is strongly associated with the progression of melanoma. Our previous studies showed that constitutive expression of inducible nitric oxide synthase (iNOS) has been found in human melanoma, and was highly correlated with poor patient survival. Further studies showed that iNOS inhibitor L-NIL could significantly inhibit melanoma growth and extended the survival of tumor-bearing mice. Moreover, in melanoma A375 cells, which express wild-type p53, the depletion of nitric oxide (NO) by scavenger c-PTIO enhanced cisplatin-induced apoptosis in a p53-dependent manner. Thus, we hypothesize that the aberrant high levels of NO in melanoma cells inhibit p53 transcriptional activity through posttranslational modification of important cysteines of p53 by S-nitrosylating the thiol group. Our biotin switch assay data confirmed that p53 was highly S-nitrosylated in A375 cells. Furthermore, the NOS inhibitor, L-NAME, significantly reduced S-nitrosylated p53 levels in A375 cells as compared with controls. ChIP assay studies showed that manipulation of NO levels by NOS inhibitor or NO donor substantially affected p53 binding to the promoters of downstream genes, such as p21, Pig3, and Mdm2, in A375 cells. In the initial Mass spectrometry studies on protein samples derived from biotin switch assays, we identified that Cys277 of p53, which directly contacts DNA in the DNA-binding domain, was highly S-nitrosylated in A375 cells. Molecular modeling to simulate p53-DNA complex was performed based on p53 crystal structure. The structural analysis showed that the S-nitrosylation of Cys277 reduced three hydrogen bonds between p53 and the conserved DNA site of p21Waf/Cip1 as compared with non-S-nitrosylated p53. However, a new hydrogen bond was also observed between Lys120 of S-nitrosylated p53 with DNA. It indicates that the S-nitrosylation of Cys277 can reduce the affinity and specificity of p53 binding to p21Waf/Cip1 promoter resulting in inhibiting transcription. Therefore, our studies represent a novel mechanism for S-nitrosylation-mediated regulation of wild-type p53 activity in melanoma cells as well as other cancer cells with aberrant high levels of RNS. Supported by MDACC SPORE in Melanoma P50CA093459. 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 4988. doi:1538-7445.AM2012-4988

Abstract 5391: Systemic gene therapy with tumor suppressor TUSC2/FUS1 nanoparticles for recurrent/metastatic lung cancer

Background: The tumor suppressor gene TUSC2/FUS1 (TUSC2) is frequently inactivated early in lung cancer development. TUSC2 mediates apoptosis in cancer cells but not normal cells by upregulation of the Apaf-1-dependent apoptotic pathway. DOTAP:cholesterol nanoparticles encapsulating a TUSC2 expression plasmid (DOTAP:chol-TUSC2) showed preferential uptake by cancer cells and prolonged survival in mouse xenograft metastatic lung cancer models. Methods: Patients with recurrent and/or metastatic lung cancer previously treated with platinum-based chemotherapy were treated with escalating doses of intravenous DOTAP:chol-TUSC2 every 3 weeks. The primary end point was assessment of DOTAP:chol-TUSC2 toxicity and determination of the MTD. TUSC2 plasmid expression in pretreatment and 24 hour posttreatment tumor specimens from subjects consenting to tumor biopsies was analyzed with quantitative RT-PCR analysis using a TUSC2 plasmid sequence-specific probe, in situ proximity ligation assay for TUSC2 protein, and apoptosis signaling nanoscale PCR array. Results: Thirty-one patients were treated at 6 dose levels ranging from 0.01 to 0.09 mg/kg and 7 had paired pre- and posttreatment biopsies. RT-PCR analysis detected high TUSC2 plasmid expression in 6 of 7 posttreatment tumor specimens but not in pretreatment specimens and negative controls. Immunohistochemical staining has been performed on 1 paired specimen, demonstrating low background TUSC2 protein staining in the pretreatment tissue compared with high intense TUSC2 protein staining in the posttreatment tissue. RT-PCR gene expression profiling analysis of apoptotic pathway genes in one paired specimen demonstrated significant upregulation and downregulation of genes involved in both the intrinsic and extrinsic apoptotic pathways. Among 4 patients treated without premedications, all 4 developed grade 2 or 3 fever. Among the 27 patients premedicated with dexamethasone and diphenhydramine, the highest fever was grade 2, which occurred in 2 subjects. The only dose-limiting toxicities were 2 episodes of transient grade 3 hypophosphatemia, resulting in an MTD of 0.06 mg/kg. Twenty-three patients who received 2 or more doses were evaluable for response, with 5 achieving stable disease (2.6-10.8 months) and 18 progressing. One patient with stable disease had evidence of a durable metabolic response on positron emission tomography imaging. The pretreatment apoptotic index was predictive of disease stability. Median survival time was 9.1 months. Conclusions: DOTAP:chol-TUSC2 can be safely administered intravenously in lung cancer patients and results in demonstrable gene and protein expression in tumors. It appears to regulate apoptotic pathway genes with evidence of antitumor activity. The MTD for phase II testing is 0.06 mg/kg every 3 weeks. Based on promising preclinical data, a trial in combination with erlotinib is planned. 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 5391. doi:10.1158/1538-7445.AM2011-5391