Leah Gandee

Mitochondrial DNA depletion sensitizes cancer cells to PARP inhibitors by translational and post-translational repression of BRCA2

Previous studies have shown that pharmacologic inhibition of poly (ADP-ribose) polymerase (PARP), a nuclear protein that is crucial in signaling single-strand DNA breaks, is synthetically lethal to cancer cells from patients with genetic deficiency in the DNA repair proteins BRCA1 and BRCA2. Herein, we demonstrate that depletion of the mitochondrial genome (mtDNA) in breast, prostate and thyroid transformed cells resulted in elevated steady-state cytosolic calcium concentration and activation of calcineurin/PI3-kinase/AKT signaling leading to upregulation of miR-1245 and the ubiquitin ligase Skp2, two potent negative regulators of the tumor suppressor protein BRCA2, thus resulting in BRCA2 protein depletion, severe reduction in homologous recombination (HR) and increased sensitivity to the PARP inhibitor rucaparib. Treatment of mtDNA-depleted cells with the PI3-kinase inhibitor LY294002, the calmodulin antagonist W-7, the calcineurin inhibitor FK506, the calcium chelator BAPTA-AM, or suppression of AKT activity by AKT small-interfering RNA (siRNA) enhanced BRCA2 protein levels as well as HR. Decreasing the intracellular calcium levels using BAPTA, or direct reconstitution of BRCA2 protein levels either by recombinant expression or by small molecule inhibition of both Skp2 and miR-1245 restored sensitivity to rucaparib to wild-type levels. Furthermore, by studying prostate tissue specimens from prostate carcinoma patients we found a direct correlation between the presence of mtDNA large deletions and loss of BRCA2 protein in vivo, suggesting that mtDNA status may serve as a marker to predict therapeutic efficacy to PARP inhibitors. In summary, our results uncover a novel mechanism by which mtDNA depletion restrains HR, and highlight the role of mtDNA in regulating sensitivity to PARP inhibitors in transformed cells.

Nanoindentation of Pseudomonas aeruginosa bacterial biofilm using atomic force microscopy

Bacterial biofilms are a source of many chronic infections. Biofilms and their inherent resistance to antibiotics are attributable to a range of health issues including affecting prosthetic implants, hospital-acquired infections, and wound infection. Mechanical properties of biofilm, in particular, at micro- and nano-scales, are governed by microstructures and porosity of the biofilm, which in turn may contribute to their inherent antibiotic resistance. We utilize atomic force microscopy (AFM)-based nanoindentation and finite element simulation to investigate the nanoscale mechanical properties of Pseudomonas aeruginosa bacterial biofilm. This biofilm was derived from human samples and represents a medically relevant model.

Dual-Drug Containing Core-Shell Nanoparticles for Lung Cancer Therapy

Late-stage diagnosis of lung cancer occurs ~95% of the time due to late manifestation of its symptoms, necessitating rigorous treatment following diagnosis. Existing treatment methods are limited by lack of specificity, systemic toxicity, temporary remission, and radio-resistance in lung cancer cells. In this research, we have developed a folate receptor-targeting multifunctional dual drug-loaded nanoparticle (MDNP) containing a poly(N-isopropylacrylamide)-carboxymethyl chitosan shell and poly lactic-co-glycolic acid (PLGA) core for enhancing localized chemo-radiotherapy to effectively treat lung cancers. The formulation provided controlled releases of the encapsulated therapeutic compounds, NU7441 - a potent radiosensitizer, and gemcitabine - an FDA approved chemotherapeutic drug for lung cancer chemo-radiotherapy. The MDNPs showed biphasic NU7441 release and pH-dependent release of gemcitabine. These nanoparticles also demonstrated good stability, excellent hemocompatibility, outstanding in vitro cytocompatibility with alveolar Type I cells, and dose-dependent caveolae-mediated in vitro uptake by lung cancer cells. In addition, they could be encapsulated with superparamagnetic iron oxide (SPIO) nanoparticles and visualized by MRI in vivo. Preliminary in vivo results demonstrated the low toxicity of these particles and their use in chemo-radiotherapy to effectively reduce lung tumors. These results indicate that MDNPs can potentially be used as nano-vehicles to provide simultaneous chemotherapy and radiation sensitization for lung cancer treatment.

The efficacy of immediate versus delayed antibiotic administration on bacterial growth and biofilm production of selected strains of uropathogenic Escherichia coli and Pseudomonas aeruginosa

Purpose The treatment of urinary tract infections (UTI) with antibiotics is commonly used, but recurrence and antibiotic resistance have been growing and concerning clinicians. We studied whether the rapid onset of a protective biofilm may be responsible for the lack of effectiveness of antibiotics against selected bacteria. Materials and Methods Two established uropathogenic Escherichia coli strains, UTI89 and CFT073, and two Pseudomonas aeruginosa strains, PA01 and Boston-41501, were studied to establish a reliable biofilm formation process. Bacterial growth (BG) was determined by optical density at 600 nm (OD 600) using a spectrophotometer, while biofilm formation (BF) using crystal violet staining was measured at OD 550. Next, these bacterial strains were treated with clinically relevant antibiotics, ciprofloxacin HCl (200 ng/mL and 2 μg/mL), nitrofurantoin (20 μg/mL and 40 μg/mL) and ampicillin (50 μg/mL) at time points of 0 (T0) or after 6 hours of culture (T6). All measurements, including controls (bacteria -1% DMSO), were done in triplicates and repeated three times for consistency. Results The tested antibiotics effectively inhibited both BG and BF when administered at T0 for UPEC strains, but not when the antibiotic administration started 6 hours later. For Pseudomonas strains, only Ciprofloxacin was able to significantly inhibit bacterial growth at T0 but only at the higher concentration of 2 μg/mL for T6. Conclusion When established UPEC and Pseudomonas bacteria were allowed to culture for 6 hours before initialization of treatment, the therapeutic effect of selected antibiotics was greatly suppressed when compared to immediate treatment, probably as a result of the protective nature of the biofilm.

Development of a locally advanced orthotopic prostate tumor model in rats for assessment of combined modality therapy.

The purpose of this study was to develop an aggressive locally advanced orthotopic prostate cancer model for assessing high-dose image-guided radiation therapy combined with biological agents. For this study, we used a modified human prostate cancer (PCa) cell line, PC3, in which we knocked down a tumor suppressor protein, DAB2IP (PC3-KD). These prostate cancer cells were implanted into the prostate of nude or Copenhagen rats using either open surgical implantation or a minimally invasive procedure under ultrasound guidance. We report that: i) these DAB2IP-deficient PCa cells form a single focus of locally advanced aggressive tumors in both nude and Copenhagen rats; ii) the resulting tumors are highly aggressive and are poorly controlled after treatment with radiation alone; iii) ultrasound-guided tumor cell implantation can be used successfully for tumor development in the rat prostate; iv) precise measurement of the tumor volume and the treatment planning for radiation therapy can be obtained from ultrasound and MRI, respectively; and v) the use of a fiducial marker for enhanced radiotherapy localization in the rat orthotopic tumor. This model recapitulates radiation-resistant prostate cancers which can be used to demonstrate and quantify therapeutic response to combined modality treatments.

Abstract 2873: Identification of a new mechanism of microRNA turnover from miR-106a-363 cluster leading to epithelial-to-mesenchymal transition in prostate cancer

Background: Prostate cancer (PCa) is the second leading cause of cancer mortality in US. The majority of PCa mortality is due to the recurrent of metastatic castration resistant PCa. The acquisition of epithelial-to-mesenchymal transition (EMT) signifies PCa metastasis. In particular, altered microRNAs (miRNAs) expression is closely associated with PCa progression from prostatic intraepithelial neoplasia to metastatic adenocarcinoma. miRNAs are small noncoding RNAs regulating approximately 60% protein-coding genes by post-transcriptional suppression or translational inhibition. miRNA gene expression at post-transcriptional level becomes more complicated when multiple miRNAs derived from the same cluster generate a polycistronic primary transcript, and each individual miRNA displays different expression profile and functional role. miRNA-363 (miR-363) belongs to the miR-106a-363 cluster containing miR-106a, miR-18b, miR-20b, miR-19b-2, miR-92a-2 and miR-363. Unlike the other five miRNAs which are closely resemble to the oncogenic miR-17-92 cluster in their seed sequence and function, miR-363 has been implicated to play a tumor suppressor role in several types of cancer, indicating a different regulatory mechanism of miR-363 from the miR-106a-363 cluster. Interferon-induced tetratricopeptide repeat 5 (IFIT5) is first characterized as a viral RNA binding protein and has been shown to directly bind to cellular tRNA, which partially shared a structural similarity with precursor miRNAs (pre-miRNAs). However, until now, there is no report indicating the RNA recognition role of IFIT5 in miRNA biogenesis machinery. Results: In the present study, miR-363 was clearly identified as a tumor suppressor miRNA by inhibiting EMT in PCa cells via targeting slug/SNAI2. More importantly, we have demonstrated, for the first time, that IFIT5 is able to recognize a unique structure at the 5′ end of precursor miR-363, which facilitates pre-miR-363 degradation by the recruitment of a 5′-3′exoribonuclease, XRN1. Meanwhile, we have also shown that the significant elevation of IFIT5 is detected in several PCa cells undergone EMT leading to highly metastatic potential. In addition, an inverse correlation between miR-363 and IFIT5 mRNA level was found in human PCa specimens. Conclusion: We unveil IFIT5 complex as a new post-transcriptional regulatory mechanism specific for miR-363 turnover at the precursor stage, which determines the stability and unique functional role of miR-363 distinct from other oncogenic miRNA members in the miR-106a-363 cluster. Overall, this study provides an insight of miRNA biogenesis machinery in cancer metastasis and new strategies of therapeutic intervention of PCa. Citation Format: U-Ging Lo, Rey-Chen Pong, Diane Yang, Jiancheng Zhou, Leah Gandee, Shu-Fen Tseng, Jer-Tsong Hsieh. Identification of a new mechanism of microRNA turnover from miR-106a-363 cluster leading to epithelial-to-mesenchymal transition in prostate 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 2873. doi:10.1158/1538-7445.AM2015-2873

IFN-γ induces epithelial-to-mesenchymal transition of cancer cells via an unique microRNA processing

Interferon-γ (IFNγ) is a potent cytokine in modulating tumor immunity and tumoricidal effects. We demonstrate a new function of IFNγ in inducing epithelial-to-mesenchymal transition (EMT) in normal and cancer cells from different cell types. IFNγ activates JAK-STAT signaling pathway leading to the transcription of IFN-stimulated genes (ISGs), such as interferon-induced tetratricopeptide repeat 5 (IFIT5). We unveil a new function of IFIT5 complex in degrading precursor microRNAs (pre-miRNA) that include pre-miR-363 from the miR-106a-363 cluster, as well as pre-miR-101 and pre-miR-128 with a similar 5’-end structure with pre-miR-363. Noticeably, these suppressive miRNAs have similar functions by targeting EMT transcription factors in prostate cancer (PCa) cells. We further demonstrated that IFIT5 plays a critical role in IFNγ-induced cell invasiveness in vitro and lung metastasis in vivo. Clinically, IFIT5 is highly elevated in high-grade PCa and its expression inversely correlates with these suppressive miRNAs. Altogether, this study unveils pro-tumorigenic role of the IFN pathway via a new mechanism of action, which certainly raises concern about its clinical application.

Abstract 1466: Interferon-induced microRNA turnover leading to epithelial-to-mesenchymal transition (EMT) in cancer

Introduction: Interferon-γ (IFN-γ) is a cytokine that has been shown to be associated with antitumor mechanisms during tumor immune surveillance. However, a steady flow of reports suggests that it has pro-tumorigenic effects. In this study, we have investigated the role of IFN-γ in cancer progression and its mechanism of action in regulating microRNA turnover. Methods: Three cell lines from different cancer types (prostate PC-3, renal 786-0 and hepatoma, HepG2) were employed. Luciferase reporter gene assay and real-time RT-PCR were used for examining the IFN-γ-induced gene transcription. Site-directed mutagenesis, in vitro transcription, RNA pull down, and in vitro RNA degradation assays were used for determining miRNA biogenesis. Western blot assay was used to determine EMT marker expression. Transwell assay was used for determining cell migration and invasion. Results: In the presence of IFN-γ, both the migration and invasion of PC-3 increased, which was accompanied with elevated EMT marker (such as Vimentin) as well as factors (such as Slug and ZEB1). IFN-γ is known to induce transcriptional activation of IFN-stimulated genes (ISGs) via JAK-STAT signaling pathway. Subsequently, we identified that IFN-γ could induce interferon-induced tetratricopeptide repeat 5 (IFIT 5) mRNA and protein levels in these cell lines. IFIT5 is first identified as viral RNA binding protein and we recently demonstrate its new function as a post-transcriptional machinery for miRNAs turnover. We therefore screened the potential IFIT5-regulated miRNAs based on microRNA microarray screening and identified several candidates such as miR-101, miR-335, miR-203, miR-128, miR-363, miR-153, miR-146a, miR-125b and miR-200a. We further examine the functional roles of these miRNAs contributing to cancer metastasis. We demonstrated that miR-363 could target Slug to suppress EMT as well as cell migration and invasion. Also, the seed regions of miR-101, miR-335, miR-203 and miR-128 have sequence-matched target sites at the 3’UTR of ZEB1, and Slug mRNA, and we further showed that they could indeed suppress the expression of these EMT factors. Clinically, IFIT5 mRNA level is elevated in higher-grade prostate cancer (PCa), and positively correlated with ZEB1, ZEB2 and Slug in PCa. By knocking down IFIT5, we observed suppression of both ZEB1 and Slug, along with decreased migration motility in these cells. Taken together, our data demonstrate the pro-progression role of IFN-γ in different cancer types. Conclusion: IFN-γ can potentiate cancer progression by inducing EMT, which is mediated through IFIT5-mediated miRNA turnover. IFIT5 complex represents unique machinery for the turnover of a specific population of tumor suppressor miRNAs. Citation Format: U-Ging Lo, Rey-Chen Pong, Jer-Tsong Hsieh, Leah Gandee. Interferon-induced microRNA turnover leading to epithelial-to-mesenchymal transition (EMT) in cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1466.