Systemic delivery of siRNA nanoparticles targeting RRM2 suppresses head and neck tumor growth

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Abstract

Systemic delivery of siRNA to solid tumors remains challenging. In this study, we investigated the systemic delivery of a siRNA nanoparticle targeting ribonucleotide reductase subunit M2 (RRM2), and evaluated its intratumoral kinetics, efficacy and mechanism of action. Knockdown of RRM2 by an RNAi mechanism strongly inhibited cell growth in head and neck squamous cell carcinoma (HNSCC) and non-small cell lung cancer (NSCLC) cell lines. In a mouse xenograft model of HNSCC, a single intravenous injection led to the accumulation of intact nanoparticles in the tumor that disassembled over a period of at least 3 days, leading to target gene knockdown lasting at least 10 days. A four-dose schedule of siRNA nanoparticle delivering RRM2 siRNA targeted to HNSCC tumors significantly reduced tumor progression by suppressing cell proliferation and inducing apoptosis. These results show promise for the use of RRM2 siRNA-based therapy for HNSCC and possibly NSCLC.

Introduction

RNA interference (RNAi) has advanced to clinical trials with great promise following the Nobel-prize winning discovery in 1998 [1]. The pursuit of siRNA-based therapeutics designed to engage RNAi pathways has the potential to provide new, effective ways of imparting therapy to patients [2], [3], [4]. To treat most cancers and other disseminated diseases, it is necessary to administer the siRNA systemically. However, the systemic administration of siRNA in vivo faces a series of hurdles such as kidney filtration, uptake by phagocytes, aggregation with serum proteins, and enzymatic degradation by endogenous nucleases before reaching the cytoplasm of the target cell. As a class of new delivery vehicles, cell-specific targeted nanoparticles have the potential to provide increased efficacy and reduced toxicity relative to conventional therapeutics [5]. Several promising strategies have been adopted to improve the systemic delivery of siRNA in animal models [2], [3], [6], [7], [8], but their safe and effective delivery still remain challenging, particularly in the clinical setting.

Ribonucleotide reductase (RR) is the rate-limiting enzyme in the conversion of ribonucleotide 5′-diphosphates into 2′-deoxyribonucleotides, which are essential for DNA synthesis and replication. RR enzymatic activity is modulated by the level of its M2 subunit (RRM2), which is expressed only in the late G1/early S phase of the cell cycle when DNA replication occurs [9], [10], [11], [12], [13]. Overexpression of RRM2 plays an active role in tumor progression and in the cellular response to DNA damage [14], [15]. Elevated RR activity and RRM2 overexpression significantly increase the drug-resistant properties and the angiogenic and invasive potential of human cancer cells [12], [16], [17], [18]. RRM2 was identified as a diagnostic marker and an indicator of poor patient outcome of several cancers [19], [20], suggesting that RRM2 contributes to malignant progression and is a potential therapeutic target. In our study, we used a targeted nanoparticle for systemic delivery of RRM2 siRNA. The clinical version of this delivery system has been denoted CALAA-01, and is now in a Phase I clinical trial for systemic delivery of siRNA to treat patients with solid cancers [7], [21].

Human transferrin protein (Tf) ligand is displayed on the exterior of CALAA-01 as a targeting moiety to engage the Tf receptor (TfR), which has long been known to be upregulated in malignant cells [22] and is a common ligand used to target tumor cells [23], [24], [25], [26]. It has been reported that CALAA-01 can deliver a higher fraction of siRNA within tumor cells by targeting TfR to achieve intracellular localization compared to nontargeted analogs [27]. This system recently provided the first example of dose-dependent accumulation of targeted nanoparticles in human tumors and also the first example of delivering a siRNA that was proven to mediate an RNAi mechanism of action [7].

Evaluation of CALAA-01 is further extended in the present work to demonstrate the intratumoral kinetics of the nanoparticle system and to observe its efficacy in tumor growth inhibition in head and neck cancer, which is one of the leading causes of cancer death worldwide [28]. Such studies are critical for understanding the effective dosing and prolonged knockdown of the targeted protein to achieve better therapeutic effect. Here, we explored the role of RRM2 by utilizing siRNA-mediated gene silencing that leads to growth suppression in vivo and in vitro. This could impact treatment of several other cancers. To our knowledge, this is the first animal study in head and neck cancer confirming that systemically delivered siRNA can produce specific gene inhibition in a mouse model and can contribute to the inhibition of tumor progression by suppressing cell proliferation and by induction of apoptosis.

Section snippets

Cell lines

HNSCC cell lines Tu177 and Tu212 (provided by Dr. Gary L. Clayman, University of Texas M.D. Anderson Cancer Center), Tu686, 686LN and 886LN (provided by Dr. Peter G. Sacks, New York University College of Dentistry), SQCCY1 (provided by Dr. Shi-Yong Sun, Emory University) and M4e cells were derived from a metastasis xenograft mouse model, [29] and were cultured in DMEM/F12 (1:1) with 10% heat inactivated fetal bovine serum (FBS). A549, H292, H460 and H1299 lung cancer cell lines were kindly

RRM2 and TfR proteins are expressed in HNSCC and NSCLC cells and siR2 efficiently suppresses RRM2 protein

Several HNSCC and NSCLC cell lines were tested for RRM2 and TfR expression levels in order to identify a suitable model system to evaluate the efficacy of the CALAA-01 nanoparticle, which has Tf on the surface as a targeting ligand and encapsulates siRNA targeted to RRM2 [7]. HNSCC (Tu177, Tu212, Tu686, 686LN, 886LN, M4e and SqCCY1) and NSCLC (A549, H292 and H460) cells displayed various degrees (moderate to high level) of expression of TfR and RRM2 proteins compared to normal cells such as BJ

Discussion

We describe findings that are highly significant and relevant in the context of RRM2-mediated tumor progression and provide vital clues for the development of novel cancer therapies. The role of RRM2 in HNSCC and NSCLC was explored, which could have profound impact on both understanding and targeting these diseases. Our results suggest that RRM2 plays a critical role in cell growth inhibition in both HNSCC and NSCLC cells. We have demonstrated the therapeutic potential of

Conclusions

Our study offers promise in siRNA-based therapy for HNSCC and possibly NSCLC. RRM2-specific siRNA silences RRM2 by an RNAi mechanism and strongly inhibits cell growth in HNSCC and NSCLC cell lines. We have demonstrated the therapeutic potential of CALAA-01 in HNSCC. In a mouse xenograft model, a single intravenous injection shows intact nanoparticles in the tumor sections up to 72 h, leading to target gene knockdown lasting at least 10 days. A four-dose schedule of CALAA-01 delivering siRNA

Acknowledgments

This work was supported by NIH/NCI grants U01CA151802 and U54CA119347, P50 CA128613 (Head and Neck Cancer SPORE) and, P30 CA138292. We thank Dr. Anthea Hammond for her critical and editorial review of this article. Dr. Davis has founders stock in Calando Pharmaceuticals and there is no financial conflict of interest to other authors.

References (37)

  • A. Fire et al.

    Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans

    Nature

    (1998)
  • D. Bumcrot et al.

    RNAi therapeutics: a potential new class of pharmaceutical drugs

    Nat. Chem. Biol.

    (2006)
  • D. Castanotto et al.

    The promises and pitfalls of RNA-interference-based therapeutics

    Nature

    (2009)
  • F. Takeshita et al.

    Therapeutic potential of RNA interference against cancer

    Cancer Sci.

    (2006)
  • M.E. Davis et al.

    Nanoparticle therapeutics: an emerging treatment modality for cancer

    Nat. Rev. Drug Discov.

    (2008)
  • D.W. Bartlett et al.

    Physicochemical and biological characterization of targeted, nucleic acid-containing nanoparticles

    Bioconjug. Chem.

    (2007)
  • M.E. Davis et al.

    Evidence of RNAi in humans from systemically administered siRNA via targeted nanoparticles

    Nature

    (2010)
  • K.A. Whitehead et al.

    Knocking down barriers: advances in siRNA delivery

    Nat. Rev. Drug Discov.

    (2009)
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