Cationic polyurethanes-short branch PEI-mediated delivery of Mir145 inhibited epithelial–mesenchymal transdifferentiation and cancer stem-like properties and in lung adenocarcinoma

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Abstract

The high invasiveness and frequent recurrence of lung adenocarcinoma (LAC) are major reasons for treatment failures and poor prognoses. Alterations in microRNAs (miRNAs) expression have been shown in lung cancers. Recent reports have demonstrated that tumors contain a small subpopulation of cancer stem cells (CSCs) that possesses self-renewing capacity and is responsible for tumor malignancy including metastasis, relapse, and chemoradioresistance. However, a miRNAs-based therapeutic approach in LAC-associated CSCs (LAC-CSCs) is still blurred. Using miRNA/mRNA-microarray and Quantitative RT-PCR, we found that the expression of miR145 is negatively correlated with the levels of Oct4/Sox2/Fascin1 in LAC patient specimens, and an Oct4highSox2highFascin1highmiR145low phenotype predicted poor prognosis. We enriched LAC-CSCs by side population sorting or identification of CD133 markers and found that LAC-CSCs exhibited low miR145 and high Oct4/Sox2/Fascin1 expression, CSC-like properties, and chemoradioresistance. To clarify the role of miR145, we used a polyurethane-short branch-polyethylenimine (PU-PEI) as the vehicle to deliver miR145 into LAC-CSCs. PU-PEI-mediated miR145 delivery reduced CSC-like properties, and improved chemoradioresistance in LAC-CSCs by directly targeting Oct4/Sox2/Fascin1. Importantly, the repressive effect of miR145 on tumor metastasis was mediated by inhibiting the epithelial–mesenchymal transdifferentiation (EMT) and metastastic ability, partially by regulating Oct4/Sox2/Fascin1, Tcf4, and Wnt5a. Finally, in vivo study showed that PU-PEI-mediated miR145 delivery to xenograft tumors reduced tumor growth and metastasis, sensitized tumors to chemoradiotherapies, and prolonged the survival times of tumor-bearing mice. Our results demonstrated that miR145 acts as a switch regulating lung CSC-like and EMT properties, and provide insights into the clinical prospect of miR145-based therapies for malignant lung cancers.

Introduction

Lung cancer is one of the leading causes of cancer-related deaths worldwide [1], and the most common histological type of lung cancer is lung adenocarcinoma (LAC). Its high invasiveness and frequent recurrence are major reasons for treatment failures and poor prognoses. Depending on their main target genes, microRNAs (miRNAs) may function as either oncogenes or tumor suppressors in lung cancer progression [2]. Recent studies have shown that specific miRNA expression profiles may distinguish between lung cancer subtypes [3] and predict prognosis and disease recurrence in early-stage lung cancers [4]. The miR17-92 cluster, for example, is overexpressed in small cell lung cancer and, in conjunction with c-Myc, promotes tumor development and neovascularization [4]. Other miRNAs, such as miR31, miR185, miR125, and miR210, are also considered oncogenes because of their elevated expression in non-small cell lung carcinoma (NSCLC) compared to normal lung tissue [5]. On the other hand, miR143, miR145, mir181a and miR182 are downregulated in NSCLC compared to non-cancerous lung tissue and are therefore considered tumor suppressors [6], [7]. Chemically synthesized miR34 was recently shown to block tumor growth in a mouse NSCLC model, implying its potentially therapeutic application in lung cancer [8]. In addition, recent studies have pointed out that cancer stem cells (CSCs) play a critical role in regulating tumorigenicity, treatment failure, tumor relapse, and metastasis in lung cancer [9], [10], [11]. However, the miRNA-dependent mechanisms regulating tumor initiation and metastasis in lung cancer stem cells remain unclear.

Epithelial–mesenchymal transdifferentiation (EMT), a de-differentiation program that converts adherent epithelial cells to individual migratory cells, is critical for embryonic development, oncogenic progression, and cancer metastasis [12]. EMT promotes stemness in normal breast tissue and breast cancer cells [12], and on the other hand, embryonic stem cell (ESC)-genes, including Oct4 and Nanog, positively regulate tumor metastasis by enhancing EMT in LAC [9]. miR145, a tumor-suppressive miRNA, was recently linked to the ESC signature [13]. miR145-mediated inhibition of Sox2 was discovered in Ewing sarcoma, in which suppression of miR145 resulted in elevated Sox2 levels and an enhanced cancer stem cell (CSC) phenotype [14]. However, so far the role of miR145 in LAC tumorigenesis is not fully understood and whether miR145 regulates EMT remains unclear.

Non-viral gene delivery, such as polymer-based gene delivery system, has been considered to be potentially safer than viral-mediated delivery [15]. The polycations/DNA complexes can protect DNA from nuclease degradation, and are nanoscale in size and small enough to enter the cell through endocytosis. In addition, cationic polymers can provide a pH-buffering ability and behave as a “proton sponge” which assists the escape of the complexes from lysosome and improves the transfection efficiency [16]. Although cytotoxicity and non-biodegradability might be problematic in long-term safety considerations, biodegradable polycations have been synthesized and used as a safe gene transfer [17]. Polyurethanes (PUs) are conventionally used as biomaterials in tissue engineering and in hydrogels because of their biocompatibility and physio-chemical properties [18], [19], [20]. It has been shown that cationic polyurethane-short branch polyethylenimine (PU-PEI) exhibits high transfection efficiency with relatively low cytotoxicity [21], [22]. Therefore, it is encouraging to investigate whether such non-viral vectors will be helpful to in vivo gene delivery in the intractable malignant cancers like GBM. Developing such materials is in urgent need for tackling cancers.

Our previous study indicated that overexpression of Oct4 and SirT1 delivered by PU-PEI reprogrammed aged retinal pigmented epithelium cells into progenitor-like cells and rescued the light-induced retinal cell loss and dysfunction [23]. In the present report, we analyzed a cohort of LAC patient samples and found that miR145low, Oct4high, Sox2high, or Fascin1high phenotype serves as markers of poor prognosis and outcome in LAC, within which miR145 is the most relevant marker. Therefore, we further assessed the utility of PU-PEI as a vehicle for miR145 delivery, and evaluated the therapeutic efficacy of PU-PEI-based miR145 delivery on LAC tumorigenesis and metastasis. PU-PEI-mediated miR145 overexpression inhibits the CSC-like properties of LAC, metastasis and sensitizes highly tumorigenic CSC-like cells to chemoradiotreatment by repressing its downstream targets, Oct4, Sox2, and Fascin1. Our findings suggested that restoration of miR145 expression may provide a novel miRNA-based approach for the treatment of LAC.

Section snippets

Cell culture, isolation of primary cells from LAC tissues and reagents

All procedures of tissues acquirements are following the tenets of the Declaration of Helsinki and all samples were obtained after patients had given informed consent. In brief, after surgical removal of the LAC tissues described in Table 1, the tissues were washed 3 times in glucose containing HBSS and then the sample were sliced at thickness of 300 mm and the sliced tissues were immersed in 0.1% (w/w) collagenase containing glucose containing HBSS for 15 min at 37 °C and rotation shaker shaking

The expression level of the tumor suppressor miR145 negatively correlates with the expression levels of Oct4, Sox2, and Fascin1 in clinical LAC patient specimens

Recent studies have demonstrated that tumors contain a small subpopulation of CSCs, which possess self-renewing capacities and are responsible for tumor maintenance and metastasis [25], [26]. Lung CSCs can be identified by side population (SP) sorting or by the presence of the surface marker CD133 [27], [28]. Ho and colleagues used flow cytometry and Hoechst 33342 dye efflux assay to isolate and characterized SP cells from six human lung cancer cell lines, and showed that SP cells were enriched

Discussion

LAC bears a dismal prognosis with rapid evolution and relapse within the first year, which has been attributed to the persistence of CSC, also known as tumor-initiating cells (TIC). Several lines of evidence have highlighted the involvements of miRNAs in the development and/or maintenance of CSC-like properties [43–46]. However, the roles of miRNA in regulating of CSC-like, metastatic and chemoradioresistant properties in LAC and lung CSC still remain unclear. In this study, expression of

Conclusion

Collectively, the present study demonstrated an inhibitory effect of miR145 on tumor initiation, EMT, tumor metastasis and chemoradioresistance capacities, in part by repressing Oct4/Sox2/Fascin1 expression in LAC cells. We suggest that the cellular levels of miR145 act as a switch that modulates the CSC properties in LAC. The promising therapeutic potential of PU-PEI-mediated miR145 delivery in an animal model suggests that it may provide an approach to improve cancer treatments, especially in

Acknowledgments

This study was funded by the NSC-(97-3111-B-075-773 001-MY3, 100-2811-B-010-046, 100-2325-B-010-010, 100-2314-B-040-010-MY3), Taipei Veterans General Hospital (Stem Cell Project E96-100), Yen-Tjing-Ling Medical Foundation (CI-100-14/32), The Genomic Center Project of National Yang-Ming University (Ministry of Education, Aim for the Top University Plan), and Center of Excellence for Cancer Research at Taipei Veterans General Hospital (DOH99-TD-C-111-007), Taiwan.

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