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Ann Thorac Surg 2006;82:249-253
© 2006 The Society of Thoracic Surgeons

Original article: General thoracic

Inhibition of Retinoblastoma Tumor Suppressor Activity by RNA Interference in Lung Cancer Lines

Michael F. Reed, MD a , * , William A. Zagorski, BS a , John A. Howington, MD a , Jack T. Zilfou, PhD c , Erik S. Knudsen, PhD b

a Division of Thoracic Surgery, Department of Surgery, University of Cincinnati College of Medicine, Cincinnati, Ohio
b Department of Cell Biology, University of Cincinnati College of Medicine, Cincinnati, Ohio
c Cold Spring Harbor Laboratory, Cold Spring Harbor, New York

Accepted for publication February 13, 2006.

* Address correspondence to Dr Reed, Division of Thoracic Surgery, Department of Surgery, University of Cincinnati College of Medicine, 231 Albert B. Sabin Way, PO Box 670558, Cincinnati, OH 45267-0558 (Email: michael.reed{at}uc.edu).

Presented at the Poster Session of the Fifty-second Annual Meeting of the Southern Thoracic Surgical Association, Orlando, FL, Nov 10–12, 2005.


   Abstract
Top
 Abstract
Introduction
 Material and Methods
 Results
 Comment
 Acknowledgments
 References
 
BACKGROUND: Inactivation of retinoblastoma (RB) tumor suppressor function occurs frequently in lung cancer. Short-hairpin RNA can be constructed to target specific sequences and efficiently knock down protein expression. We developed a short-hairpin RNA approach to specifically target Rb in lung cancer cells to determine the influence of RB knockdown on proliferation.

METHODS: NCI-H520 human lung cancer cells (wild-type Rb) were transfected with pMSCVpuro-Rb3C, a plasmid containing a short-hairpin sequence targeted to human Rb. Transfectants harboring the construct were selected with puromycin. Loss of RB expression in selected cell populations was determined by immunoblotting. Proliferating cells were counted to establish growth rates. Retinoblastoma-proficient and RB-deficient tumor growth was monitored in nude mice.

RESULTS: Transfection with pMSCVpuro-Rb3C dramatically diminished RB expression and led to aberrant expression of RB-regulated genes. Cells harboring pMSCVpuro-Rb3C grew at an increased rate compared with control cells: 480.6 ± 37.7 versus 159.4 ± 36.2 (relative cell count at 12 days). Tumor growth in nude mice also increased with RB knockdown compared with control mice: 135.2 ± 73.6 mm3 versus 40.0 ± 17.0 mm3 (tumor volume at 10 days).

CONCLUSIONS: Inhibition of RB expression is efficiently achieved in lung cancer cells with short-hairpin RNA. Genetic targets of RB are deregulated with RB knockdown. Retinoblastoma depletion increases growth in vitro and in murine xenografts. These studies indicate that even in the context of an established tumor cell line, RB limits tumorigenic proliferation. Additionally, this model will serve as an ideal system to evaluate the role of RB activity on therapeutic response.


   Introduction
Top
 Abstract
 Introduction
Material and Methods
 Results
 Comment
 Acknowledgments
 References
 
The identification of the retinoblastoma (RB) susceptibility gene as the first tumor suppressor gene was a milestone in understanding cell cycle control and oncogenesis [1, 2]. Loss of RB activity has subsequently been described in the majority of human tumors through mutation or loss of the Rb gene, as well as functional inactivation through various mechanisms [3–6]. Similarly, RB is inactivated in the majority of lung cancers through loss of p16INK4A–cyclin D–CDK4–RB pathway function [7]. In the case of non–small cell lung cancer (NSCLC), RB is inactivated through disparate mechanisms including mutation [8–10], deregulated phosphorylation through abnormal CDK4–cyclin D expression, and loss of p16INK4A activity by aberrant promoter methylation [11, 12] or homozygous deletions or point mutations [13–16]. Thus, NSCLC provides a unique system to evaluate the effect of discrete mechanisms of RB inactivation on therapeutic response.

A critical function of RB is control of cellular proliferation by means of regulation of the cell cycle. In its active hypophosphorylated state, RB serves as a transcriptional corepressor, inhibiting the function of the E2F family of transcription factors and thereby preventing expression of specific E2F target genes required for cell cycle progression [17–19]. We have identified numerous other targets of RB-mediated control through an unbiased screen using RNA microarray analysis of cells engineered with inducible ectopic expression of a constitutively active RB allele [20]. The majority of the genetic targets of RB participate in cell cycle control. Furthermore, a distinct class of genes repressed by RB includes the molecular targets of chemotherapeutic agents. Topoisomerase II alpha (TopoII{alpha}), the target of etoposide, and thymidylate synthase, the target for 5-fluorouracil, were among the RB target genes [21, 22]. This finding suggested that the deregulation of specific RB targets could contribute to altered chemosensitivity.

Appropriate coordination of the cell cycle maintains genomic integrity by ensuring faithful replication and partitioning of the genome [23, 24]. Inactivation of cell cycle control mechanisms predisposes cells to the development of genomic instability and cancer [3, 25]. With the inactivation of RB occurring frequently in cancer, and its apparent control of the molecular targets of chemotherapeutics, experimental manipulation of RB might serve to determine mechanisms of chemosensitivity. Yet, shortcomings exist in prior experimental approaches to RB control when applied to human cancer. The use of murine embryonic fibroblasts is a requirement when Rb –/– cells are studied because of embryonic lethality after germline loss of both Rb alleles. We have therefore exploited the Cre/LoxP system to achieve conditional Rb knockout in murine adult fibroblasts [26]. Although adult fibroblasts provide an excellent tool for studying certain mechanisms of RB activity, such as checkpoint responses [26, 27], they are not the ideal model for assessing the role of RB in human malignancies. Ideally, one would devise a system to specifically manipulate RB activity in human cancer cells.

The recently elucidated mechanism of RNA interference, in which fragments of RNA block the expression of endogenous genes in a sequence-specific manner, can be exploited to inhibit the expression of specific proteins [28–33]. We hypothesized that RB activity in lung cancer cells can be precisely targeted for inhibition using small interfering RNA (siRNA), thereby deregulating cell growth. The first aim was to establish knockdown of RB expression in lung cancer cells using the technique of RNA interference. Second, we aimed to then determine whether diminished RB activity causes increased cellular proliferation in vitro and in an in vivo murine xenograft model.


   Material and Methods
Top
 Abstract
 Introduction
 Material and Methods
Results
 Comment
 Acknowledgments
 References
 
Cell Culture, Plasmids, and Transfection
The NCI-H520 human NSCLC line (ATCC, Manassas, VA) is RB proficient. Cells were plated in tissue culture dishes and maintained in RPMI 1640 (Mediatech, Inc, Herndon, VA) supplemented with 10% fetal bovine serum, 100 U/mL penicillin–streptomycin, and 2 mmol/L glutamine at 37°C in 5% CO2. A validated siRNA was used to ablate RB expression. The short hairpin sequence, Rb3C (5'-TTCATAACACAGTCCTAACTGGAGTGTGTGGAAGCTTGCGCATACTCCGGTTAGGACTGTTATGAATTTTTTT-3') is targeted to the A/B pocket of human RB. It is cloned into the HpaI site in the multiple cloning site of the 6.3-kilobase plasmid pMSCVpuro, which harbors the puromycin-resistance gene (Clontech Laboratories, Inc, Mountain View, CA). Transfections with pMSCVpuro-Rb3C, or the empty control (pMSCVpuro), were performed using FuGene6 lipid-based reagent (Roche, Indianapolis, IN) according to the manufacturer's protocol. Transfectants harboring the construct were selected with 2.5 µg/mL puromycin (Calbiochem, San Diego, CA). Exponentially expanding cells were counted every 48 hours to establish growth curves.

Immunoblotting
Protein expression was determined by immunoblot (Western) analysis using standard techniques [26]. Briefly, cells were harvested by trypsinization and lysed in radioimmunoprecipitation buffer. Equal amounts of protein were resolved by sodium dodecylsulfate–polyacrylamide gel electrophoresis. Specific proteins were detected using the following primary antibodies: RB (851 polyclonal antisera [34]), ß-tubulin (D-10, Santa Cruz Biotechnology, Inc, Santa Cruz, CA), TopoII{alpha} (H-231, Santa Cruz Biotechnology), thymidylate synthase (gift from M. Fukushima), CDK2 (M-2, Santa Cruz Biotechnology), proliferating cell nuclear antigen (PC-10, Santa Cruz Biotechnology), and cyclin F (C-20, Santa Cruz Biotechnology).

Xenografts
The protocol was approved by the University of Cincinnati Institutional Animal Care and Use Committee and complied with the 1996 "Guide for the Care and Use of Laboratory Animals" recommended by the US National Institutes of Health. Tumors were grown as xenografts in 6- to 8-week-old female athymic mice (BALB/c strain, Harlan Sprague-Dawley Inc, Indianapolis, IN) by subcutaneous flank injection of 5 x 106 cells in 150 µL of phosphate-buffered saline solution mixed with 50 µL of Matrigel (BD Biosciences, Bedford, MA). Tumors were measured with calipers daily. Tumor volume was calculated as v = {pi}(width2 x length)/6. Mice were euthanized by CO2 anesthetization followed by cervical dislocation before the tumor volume measured 3,000 mm3.

Statistics
Cellular proliferation was calculated as a relative increase in cell count compared with the number of cells plated at the start of the experiment. Tumor growth in the murine xenografts was calculated as a relative increase in tumor volume compared with the first day that the tumors could be palpated and accurately measured (day 5 after injection). Each experiment in the cell growth and tumor growth studies was performed in (at minimum) triplicate, and results are presented as the mean ± standard deviation. Graphs showing cell counts and tumor volumes were created with Excel 2002 (Microsoft Corp, Redmond, WA), with error bars representing one standard deviation.


   Results
Top
 Abstract
 Introduction
 Material and Methods
 Results
Comment
 Acknowledgments
 References
 
Downregulation of Retinoblastoma by RNA Interference is Efficiently Achieved in Lung Cancer Cell Lines
We used RNA interference to evaluate the efficiency of RB knockdown. The plasmid clone pMSCVpuro-Rb3C produces a short-hairpin RNA (shRNA), which is processed into a double-stranded siRNA that has been documented to virtually eliminate RB expression in another human cell line (MCF7, human breast carcinoma; data not shown). We transfected the shRNA construct, or the empty control, into NCI-H520 cells. This cell line was chosen as human NSCLC cells that possess wild-type Rb. We then subjected the transfectants to selection in puromycin. The degree of RB inhibition was determined by immunoblot (Western) analysis of whole cell lysates. The population (Rb3C) exhibited a decrease in RB level (Fig 1) compared with the control cells (MSCV).


Figure 1
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  		Fig 1. Transfection of short-hairpin RNA targeted to retinoblastoma gene effectively diminishes expression of the retinoblastoma (RB) tumor suppressor (immunoblot analysis). ß-Tubulin serves as a protein loading control. (MSCV = NCI H520 pMSCVpuro; Rb3C = NCI H520 pMSCVpuro-Rb3C).

 
Targets of Retinoblastoma Control Are Deregulated With Retinoblastoma Knockdown by Small Interfering RNA
With the successful inhibition of RB expression in human lung cancer cells using RNA interference, we next sought to demonstrate that RB function was similarly blocked. Retinoblastoma acts as a transcriptional corepressor [17–19]. As such, active RB inhibits a diversity of gene products, including a number of cell cycle control proteins. We have previously identified a variety of genes that are under RB control, using microarray analysis of RNA isolated from cells engineered to express RB in an inducible manner [20]. We determined whether selected genes that are under RB control were deregulated with inhibition of RB expression. Immunoblot analysis of whole cell lysates obtained from the Rb3C line and the MSCV control lines were probed with antibodies to various cell cycle control proteins. The cell cycle control proteins CDK2, PCNA, and cyclin F, each of which has been shown to be under RB control, exhibited increased expression when RB expression was abrogated by RNA interference (Fig 2). We also probed for TopoII{alpha} (the molecular target of etoposide) and thymidylate synthase (the target of 5-fluorouracil), both of which are under RB control. The expression of TopoII{alpha} and thymidylate synthase increased with reduced RB levels. Thus, downregulation of RB level by siRNA also results in inhibition of RB activity.


Figure 2
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  		Fig 2. Downregulation of retinoblastoma (RB) by means of small interfering RNA results in increased expression of retinoblastoma target genes (immunoblot analysis). (CDK2 = cyclin-dependent kinase 2; PCNA = proliferating-cell nuclear antigen; MSCV = NCI H520 pMSCVpuro; Rb3C = NCI H520 pMSCVpuro-Rb3C; TopoII{alpha} = topoisomerase II alpha; TS = thymidylate synthase).

 
Depletion of Retinoblastoma Increases Cell Growth In Vitro
Loss of normal activity of the p16INK4A–cyclin D–CDK4–RB pathway is believed to occur in the vast majority of human cancers. We therefore asked whether inhibition of RB expression using RNA interference in human lung cancer cells would alter proliferation. Exponentially expanding populations of the H520-Rb3C cells were compared with the H520-MSCV controls. Cells were not allowed to achieve confluence, to prevent contact inhibition. Three plates of each line were initially established, and growth curves were plotted. The lung cancer line with decreased RB level (Rb3C) showed more rapid in vitro growth (Fig 3). Specifically, the relative increase in number of cells harboring the shRNA targeting RB (Rb3C) was 10.2 ± 2.0 at 4 days, 82.6 ± 9.9 at 8 days, and 480.6 ± 37.7 at 12 days, compared with 4.6 ± 0.2 at 4 days, 23.6 ± 5.6 at 8 days, and 159.4 ± 36.2 at 12 days in the control cells (MSCV). Hence, inhibition of RB by RNA interference led to increased proliferation in vitro.


Figure 3
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  		Fig 3. Inhibition of retinoblastoma activity by small inhibitory RNA increases in vitro growth rates in non–small cell lung cancer cells. NCI H520 cells harboring the retinoblastoma short-hairpin RNA (Rb3C), or the empty vector control (MSCV), were counted every 48 hours. The relative cell count is in comparison to the cell number at day 0. Error bars represent one standard deviation.

 
Depletion of Retinoblastoma Increases Tumor Growth in a Murine Xenograft Model
Neoplastic human cells can be grown as tumors in nude mice. This system permits study of the effects of genetic manipulations of cancer cells. H520 cells (Rb3C or MSCV control) were injected subcutaneously into the flanks of BALB/c athymic mice. Tumors were measured with calipers daily, and tumor volumes were calculated. Tumors were palpable and measurable by day 5 after injection. The tumor volume for cells containing shRNA targeting RB (Rb3C) was 135.2 ± 73.6 mm3 when the animals were euthanized at 10 days compared with 40.0 ± 17.0 mm3 for the control cells (MSCV). Therefore, tumors with decreased RB activity (Rb3C) exhibited more rapid growth than the controls (Fig 4).


Figure 4
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  		Fig 4. Non–small cell lung cancer xenografts with reduced retinoblastoma expression by small interfering RNA (Rb3C) exhibit increased growth in nude mice, compared with the empty vector control (MSCV). Tumor volumes were calculated once the tumor was measurable. Error bars represent one standard deviation.

 

   Comment
Top
 Abstract
 Introduction
 Material and Methods
 Results
 Comment
Acknowledgments
 References
 
The RB tumor suppressor plays a critical role in choreographing progression through the cell cycle, maintaining appropriate checkpoint control, and thereby protecting genomic integrity. It is believed that disruption of RB activity results in deregulation of proliferation and contributes to the development of malignancy. Alteration of RB function occurs in the majority of human tumors, including NSCLC. Retinoblastoma also controls the expression of a variety of target genes, such as cell cycle control genes and targets of chemotherapeutic agents [20]. Thus, NSCLC provides a unique system in which to evaluate the effects of discrete mechanisms of RB action in a human malignancy.

We have previously demonstrated the requirement of intact RB function for appropriate checkpoint responses to therapeutic stimuli using murine adult fibroblasts [26, 27]. We thus sought to elucidate the role of RB in control of proliferation in human NSCLC. Short-hairpin RNA can be constructed to target specific sequences and efficiently knock down protein expression. We hypothesized that RB activity in NSCLC can be precisely targeted for inhibition using shRNA, thereby deregulating growth. We have previously achieved significant inhibition of RB expression in breast cancer cells with RNA interference using a short-hairpin sequence, Rb3C, targeted to the A/B pocket of human RB, cloned into the plasmid pMSCVpuro. In the current study we demonstrate that inhibition of RB is efficiently achieved in NSCLC lines by siRNA.

Active RB regulates the expression of numerous target genes [20]. Moreover, inhibition of RB activity by overexpression of the CDK inhibitor p16INK4A recapitulated this. In this study we demonstrate that downregulation of RB levels by siRNA similarly results in loss of RB activity, as observed by deregulation of certain RB targets, including TopoII{alpha}, thymidylate synthase, CDK2, PCNA, and cyclin F. The development of malignancy, as well as the immortalization of cells in vitro, typically includes disruption of RB activity through one of the many aforementioned mechanisms that disrupt the p16INK4A–CDK4–cyclin D–RB pathway [7]. Yet here, in the setting of a human cancer line containing wild-type Rb, another alteration in RB pathway function must be present to render RB functionally inactive. The arrest induced by p16INK4A requires intact RB, indicating that manipulation of one cell cycle control protein might require that the remainder of the RB pathway be undamaged for an effect to occur. In studies of human cancer, loss of RB and p16INK4A appear to be mutually exclusive events [4, 6, 14, 35]. Thus RB knockdown by siRNA might have no effect on targets of RB transcriptional repression in certain human tumor cells; however, we show that introduction of siRNA lowers RB protein level and deregulates RB target genes. Although functional inactivation of RB contributes to the development of neoplasia, we demonstrate that further disruption of RB activity can still occur.

Our results also demonstrate that inhibition of RB activity, in the setting of human cancer cells, can increase proliferation. We show that depletion of RB increases growth rates of NSCLC cells in vitro and tumor growth in a murine xenograft model. Altered cell cycle responses are typical when cell cycle control mechanisms are disrupted in vitro. For example, acute loss of RB leads to chemoresistance, as well as aneuploidy, in fibroblasts [36]. The finding of increased cellular proliferation with RB knockdown would not necessarily be predicted in a human cancer cell system. As noted earlier, in this study a cell line with wild-type Rb was used, implying that another mechanism for achieving functional inactivation of RB was presumably involved in the evolution of malignancy. On the other hand, the RB pathway may not have been targeted during tumorigenesis. Here, forced loss of RB activity through knockdown of expression by RNA interference increased proliferation. Thus, even in the background of transformed human cancer cells, further disruption of the RB pathway through a specific downregulation of RB protein levels can achieve deregulation of proliferation. This might represent a direct cell cycle effect of decreased RB level. Another possibility is that increased cell number is attributable to a change in other growth-regulating mechanisms, such as apoptosis. Future studies will address the mechanism of growth deregulation with decreased RB expression in lung cancer cells.

In conclusion, RNA interference is a useful technique for specifically manipulating the level of a tumor suppressor in human cancer cells. Retinoblastoma is dramatically depleted by siRNA, and the expression of E2F targets under RB control is unrestrained. Loss of RB activity results in increased in vitro proliferation of NSCLC cells. This is mirrored in vivo in which RB-depleted NSCLC cells form tumors more rapidly than control cells. This will serve as an ideal system to further evaluate the role of RB activity on proliferation and checkpoints, such as those invoked in response to chemotherapeutic agents.


   Acknowledgments
Top
 Abstract
 Introduction
 Material and Methods
 Results
 Comment
 Acknowledgments
References
 
This study was supported by a grant to MFR from the Dean's Discovery Fund, University of Cincinnati College of Medicine. ESK is funded by a grant from the National Cancer Institute (CA106471). Christopher N. Mayhew, PhD, gave valuable assistance with critical review of the manuscript.


   References
Top
 Abstract
 Introduction
 Material and Methods
 Results
 Comment
 Acknowledgments
 References
 

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