Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Intratumoral delivery of shRNA targeting cyclin D1 attenuates pancreatic cancer growth

Cancer Gene Therapy volume 17, pages 325–333 (2010)Cite this article

Abstract

The aim of this study was to assess the biological consequences of cyclin D1 silencing in pancreatic cancer cells. A replication-defective lentivirus based small hairpin RNA (shRNA) system targeting cyclin D1 caused a marked reduction in cyclin D1 protein levels in ASPC-1 and BxPC3 pancreatic cancer cell lines in conjunction with decreased cell growth and invasiveness in vitro. Moreover, a single intratumoral injection of the recombinant lentivirus targeting cyclin D1 attenuated the growth of pre-existing tumors arising from two distinct cell lines. This attenuated growth correlated with decreased proliferation and angiogenesis, as well as attenuated vascular endothelial growth factor expression. It is concluded that lentivirus-delivered shRNA targeting cyclin D1 suppresses the growth, invasiveness, tumorigenicity and pro-angiogenic potential of human pancreatic cancer cells, thereby raising the possibility that intratumoral injections of viruses targeting cyclin D1 could provide a new therapeutic approach in pancreatic ductal adenocarcinoma.

Download PDF

Introduction

Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of death due to cancer in the United States of America.1 In spite of recent therapeutic advances, long-term survival in PDAC is often limited to patients who underwent surgery during early stages of the disease.2 The biological aggressiveness of PDAC is due, in part, to the tumor's resistance to chemotherapy and to its propensity to metastasize even when the primary tumor is small.3 PDAC is also characterized by a high frequency of mutations in the Kras gene, as well as by the inactivation of several tumor suppressor genes, such as p16Ink4a p53 and Smad4.4 PDAC also displays abnormal upregulation of multiple mitogenic and angiogenic growth factors and their cognate high-affinity receptors.5 Together, these alterations serve to enhance the biological aggressiveness of PDAC.

Cyclin D1, a cell-cycle regulator, is known to be overexpressed in several human cancers including PDAC.6 In response to mitogenic growth factors, normal cells exhibit cyclin D1 accumulation by early to mid phase of G1. Cyclin D1 then activates its cognate cyclin-dependent kinases, cyclin-dependent kinase 4 and 6. Activated cyclin-dependent kinase 4/6 initiates the phosphorylation-dependent inactivation of the retinoblastoma protein, RB, which then promotes progression through the G1–S phase of the cell cycle.7 Cyclin D1 also has cyclin-dependent kinase independent functions.8 Thus, it can associate with and enhance the activity of transcription factors by recruiting cofactors that govern chromatin remodeling,9 and contribute to the regulation of cell metabolism, differentiation and migration.7

Pancreatic ductal adenocarcinomas express high levels of cyclin D1, and this overexpression has been correlated with decreased patient survival.10 Moreover, suppression of cyclin D1 expression by an antisense-based strategy resulted in marked inhibition of pancreatic cancer cell growth in vitro and in vivo.11, 12 However, such an antisense strategy is not readily amenable for use in therapeutic regimens. RNA interference has rapidly become a powerful tool for gene silencing, drug discovery and target validation,13 and vectors that synthesize small hairpin (sh) RNA permit sustained gene silencing.14 These vectors allow the synthesis of 50 bp-long ssRNAs that fold into 21–23 bp dsRNA with a small hairpin in the middle, and are subsequently processed to small interfering RNAs (siRNAs) by the cellular machinery. These shRNA-expression vectors can be engineered to contain selectable markers to generate stable transfectants, to co-express reporter genes and to be incorporated into viral vectors such as a lentiviral vector.15 This virus is modified to be non-replicative and is efficiently transduced into human cells, including non-dividing cells.15, 16 It then integrates into the host genome, resulting in long-term and stable expression of the shRNA.17

In this study we used third generation lentiviral vectors to integrate the sequence of a cyclin D1 shRNA in two human pancreatic carcinoma cell lines, thereby allowing for stable expression of the shRNA and a sustained suppression of cyclin D1 expression. This downregulation resulted in attenuated growth of the cells in anchorage-dependent and -independent conditions, as well as decreased invasiveness in vitro. Moreover, a single intratumoral injection of these recombinant lentiviruses targeting cyclin D1 attenuated the growth of pre-existing tumors by decreasing cancer cell proliferation and angiogenesis.

Materials and methods

Cell growth and invasion assays

ASPC-1 and BxPC3 human pancreatic cancer cell lines from ATCC (Manassas, VA) were grown in RPMI 1640 (Mediatech, Herndon, VA). The medium was supplemented with 10% fetal bovine serum (FBS) from Omega Scientific (Tarzana, CA) and with 100 U ml−1 penicillin and 100 μg ml−1 streptomycin (complete medium).

Doubling time

To assess the doubling time, cells were seeded in 6-well plates at a density of 1.0 × 104 cells per plate and the doubling time was calculated over 5 days. All doubling time experiments were repeated three separate times, each time on separate days.

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) growth assay

Cells (3000 per well) were plated in 96-well microtiter plates (Corning, Acton, MA). MTT was added to each well (final concentration 0.5 mg ml−1) 72 h later. The plates were incubated at 37 °C for 3 h, and the medium replaced with 100 μl of acidified isopropanol followed by gentle shaking for 15 min to solubilize the formazan blue crystals. Absorbance at 570 nm was measured using a microtiter plate reader. Cell viability was expressed as the percentage of growth of parental cells. Each experimental condition was assayed in six wells, and each experiment was repeated at least thrice.

Soft agar assay

Soft agar assays were carried out as reported previously.18 Cells (8 × 103) were suspended in complete medium containing 0.3% agar and seeded in triplicate in 6-well plates onto a base layer of complete medium containing 1% agar. Complete medium containing 0.3% agar was added every 5 days for 15 days, and colony counting was then carried out.

Invasion assays

Invasion assays were carried out as reported previously.19 Briefly, cells were suspended in 500 μl RPMI with 0.1% bovine serum albumin and placed onto the upper compartment of Matrigel-coated Transwell chambers (8 μm pore size, BioCoat Matrigel Invasion Chambers; BD Biosciences, San Jose, CA). The lower compartment was filled with 750 μl RPMI with 5% serum. After 18–20 h, cells on the upper surface of the filter were carefully removed with a cotton swab and the membranes were fixed in methanol. The cells that had migrated through the membrane to the lower surface of the filter were stained with toluidine blue (Fisher Scientific, Pittsburgh, PA) and counted using a light microscope.

Immunoblotting

Immunoblotting was done as described previously.20 Briefly, polyvinylidene fluoride membranes were incubated overnight with a 1:1000 dilution of anti-cyclin D1 human anti-mouse monoclonal antibody (Cell Signaling Technology, Danvers, MA), washed and incubated for 30 min with a secondary horseradish peroxidase-conjugated antibody. Bound antibodies were visualized using enhanced chemiluminescence (Pierce, Rockford, IL). To confirm equal loading of lanes, membranes were stripped for 30 min at 50°C in buffer containing 2% SDS, 62.5 mM Tris (pH 6.7) and 100 mM 2-mercaptoethanol and blotted with a 1:10 000 dilution of a rat anti-tubulin antibody (Abcam, Cambridge, MA).

siRNA transient transfection and lentivirus shRNA gene transduction

Small interfering RNA sequences directed against human cyclin D1 (Dharmacon, Lafayette, CO) were initially transfected into ASPC-1 cells using Jet PEI (Qbiogene, Solon, OH) according to the manufacturer's protocol. Oligonucleotides corresponding to the shRNA sequence-of-interest were annealed and cloned into the lentivirus vector, pLentiLox 3.7 (pll3.7) (Addgene, Cambridge, MA). Virus stocks were prepared by co-transfecting pll3.7 with three packaging plasmids (pMDLg/pRRE, CMV-VSVG and RSV-Rev) into 293 T cells.21 The viral supernatants were harvested 36–48 h later, filtered and centrifuged (90 min at 25 000 g). Viral titers were determined by fluorescence-activated cell sorting analysis.

In vivo tumorigenicity assay

To assess the effects of cyclin D1 suppression on established tumors, 24 mice were injected subcutaneously into the dorsal flank area with 200 μl of complete medium containing 1 × 106 ASPC-1 cells. Separately, 24 mice were similarly injected with 200 μl of complete medium containing 1 × 106 BxPC3 cells. The mice were divided randomly into three groups of eight mice per cell line. Once tumors reached a volume of 30–40 mm3 (usually 8–10 days after injection of the cells), the tumors were injected with 50 μl (4 × 107 viral particles) of Optimem (Invitrogen, Carlsbad, CA), with virus containing the shRNA against luciferase, or with virus containing the shRNA against cyclin D1. The tumors were measured every 3 days. Tumor volumes were calculated as π/4 × width × height × length of the tumor.22 When the tumor diameter reached 15 mm, the mice were killed.

Immunohistochemistry

After rapid tumor removal, tissues were cryo-embedded in cryo-OCT compound (Fisher Scientific). All immunohistochemistry experiments were done as previously described.23 The following antibodies were used: a rabbit polyclonal anti-Ki-67 (1:50 dilution; Abcam, Cambridge, MA) antibody to assess proliferation, a rat anti-mouse monoclonal antibody targeting CD31 as an endothelial cell marker (1:100 dilution; PharMingen, San Jose, CA) and a polyclonal anti-VEGF (1:50 dilution; Santa Cruz, Santa Cruz CA) antibody. Quantitative morphometric analysis of Ki-67, CD31 and VEGF positive cells in tumor samples was carried out as previously reported.24 Briefly, 10 areas per slide were captured using Olympus DP70 camera ( × 100 magnification) and quantified with the Image-Pro plus program (Version 4.51, Media cybernetics, L.P., Silver Spring, MD). Three tumors per group were analyzed. Blood vessel densities were calculated as the ratio of CD31 positive areas to the total area of the image, whereas proliferation was calculated as the ratio of Ki-67 area to total cell area.

Statistical analysis

Data were analysed using the Kruskall and Wallis test for mean comparisons using the Dunn–Benferroni test for multiple comparisons, with P<0.05 taken as significant.

Results

shRNA selection and knock down of cyclin D1

To select the shRNA sequence to be introduced into the lentivirus, a pool of different siRNA sequences directed against human cyclin D1 were tested in ASPC-1 cells. After siRNA transfection, cell lysates were subjected to immunoblotting using anti-cyclin D1 antibodies. The siRNA pool efficiently silenced cyclin D1 expression at the protein level (data not shown). Each sequence of this pool was then tested to select the two most efficient siRNA sequences, which were then used to design the oligonucleotides for the shRNA targeting cyclin D1. These oligonucleotides were then annealed and cloned into the pll3.7 vector.

Lentivirus infectivity, determined after infection with the green fluorescence protein-expressing lentivirus at a multiplicity of infection of 10, revealed that 90% of ASPC-1 and BxPC3 human pancreatic cancer cells exhibited strong fluorescence, and these results were confirmed by fluorescence-activated cell sorting (data not shown). Next, ASPC-1 and BxPC3 were infected at different multiplicity of infection with shRNA-lentiviruses targeting cyclin D1, or with shRNA-lentiviruses targeting luciferase (shLuc-LV) that was used as a control. We tested the processing and subsequent functionality of the control shRNA by infecting 293 cells that constitutively express luciferase with shLuc-LV. A decrease in luciferase activity in these cells demonstrated that the shRNA targeting luciferase is processed by the silencing pathway (data not shown).

As determined by immunoblotting, shRNA sequences 8 and 10 were the most effective at suppressing cyclin D1 levels in both cell lines, as was the combination of both shRNAs (Figure 1). These viruses were therefore used in all subsequent experiments.

Figure 1
figure 1

Cyclin D1 silencing. ASPC-1 (a) and BxPC3 (b) cells that were either not infected (parental cells: P) or infected with the mock-infected lentivirus pll37 (M), or with pll37 containing sequences encoding luciferase (L), cyclin D1 specific small hairpin (sh) RNA 8 (8) or 10 (10), or their combination (K). Cell lysates, prepared 72 h after infection, were subjected to immunoblotting using anti-cyclin D1 antibodies. Membranes were re-probed with anti-tubulin antibody to assess equivalent lane loading.

Full size image

Cyclin D1 downregulation results in reduced proliferation and invasion

Initial experiments were carried out to determine the effects of cyclin D1 downregulation on cell doubling times. Parental and mock-infected cells had similar doubling times of 31±0.5 h, whereas ASPC-1 cells infected with lentivirus expressing shRNA 8 (sh8-LV) and shRNA 10 (sh10-LV) displayed doubling times of 39 h (P<0.05) and 40±1.34 h (P<0.01), respectively. Similarly, parental and mock infected BxPC3 cells exhibited doubling times of 25±0.7 h, whereas infection of these cells with sh8-LV and sh10-LV prolonged the doubling times to 34±1.4 h (P<0.05) and 30±1.2 h (P<0.05), respectively.

Cyclin D1 downregulation was also associated with a significant inhibitory effect on cell proliferation, as determined by the MTT assay (Figure 2a), as well as by a significant decrease in colony formation in soft agar (Figure 2b). Thus, as shown in the parenthesis by means±s.e. and respective P-values, by comparison with control, the number of colonies formed was decreased by 54% (197±5; P<0.01) and 51% (203±2; P<0.01) in ASPC-1 cells infected with sh8-LV and sh10-LV, respectively, and by 54% (190±4; P<0.01) after infection with both viruses. Moreover, in BxPC3 cells, colony number was decreased by 33% (297±4; P<0.05) and 36% (287±9; P<0.05) after infection with sh8-LV and sh10-LV, respectively, and by 43% (252±9; P<0.01) after infection with both viruses.

Figure 2
figure 2

In vitro consequences of cyclin D1 silencing. ASPC-1 and BxPC3 cells that were either not infected (parental cells: P) or infected with the mock-infected lentivirus pll37 (E), or pll37 containing sequences encoding luciferase (L), or cyclin D1 specific small hairpin (sh) RNA 8 (8) or 10 (10), or their combination (K). (a) Anchorage-dependent growth was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Data are the means ± s.e. of triplicate determinations from three independent experiments. *P<0.05, **P<0.01 when compared with Luc. (b) Anchorage-independent growth was determined in soft agar. Data are the means±s.e. of triplicate determinations from three independent experiments. **P<0.01 when compared with Luc. (c) Cell invasion was determined in Transwell chambers. Data are the means±s.e. of triplicate determinations from three independent experiments. **P<0.01 when compared with Luc.

Full size image

The effects of cyclin D1 knockdown on cell invasion were examined next using a Matrigel-based invasion assay. In the presence of serum (5%), cell invasion was enhanced in ASPC-1 and BxPC3 cells. In both cell lines, infection with sh8-LV, sh10-LV or the combination significantly attenuated the stimulatory effects of serum by comparison with values observed in mock-infected cells (Figure 2c). Thus, invasion was attenuated by 39% (41.5±1.3; P<0.01) and 50% (34.2±1.5; P<0.01) in ASPC-1 cells infected with sh8-LV and sh10-LV, respectively, and by 57% (29.3±1.3; P<0.01) after infection with both viruses. Similarly, invasion was attenuated by 56% (59.2±4.9; P<0.01) and 47% (70.7±4.7; P<0.01) in BxPC3 cells infected with sh8-LV and sh10-LV, respectively, and by 54% (61.2±4.2; P<0.01) after infection with both viruses. These observations indicate that in both cell lines, the combination of sh8-LV and sh10-LV was, at least, as effective as either lentivirus alone.

Cyclin D1 knockdown attenuates the growth of established tumors

To determine whether the lentivirus could efficiently infect tumors and attenuate their growth, ASPC-1 and BxPC3 cells, which express high cyclin D1 levels (data not shown), were used next. Tumors were generated by subcutaneous injections of ASPC-1 or BxPC3 cells (1 × 106 cells per injection). Upon reaching a volume of 30–40 mm3, intratumoral injections were initially carried out with a green fluorescence protein-expressing pll3.7 virus. After 1 week, strong green fluorescence protein expression was observed in the dissected tumors, which was similar to that observed after the injection of cancer cells that were infected ex-vivo with the green fluorescence protein-expressing pll3.7 virus (Supplementary data, Figure S1). These observations indicate that after being injected into an established tumor, the virus was able to diffuse and infect cells throughout the tumor mass. The same procedure was used next for shRNA experiments.

Each tumor was injected once with 50 μl of a combination of sh8-LV and sh10-LV (shD1-LV), or with 50 μl of a control lentivirus expressing shRNA directed against luciferase (shLuc-LV). When experiments were terminated for tumor harvesting, the ASPC-1- and BxPC3-derived tumors that were infected with shD1-LV exhibited a 44% (115±18; P<0.05) and a 65% (230±18; P<0.05) decrease in volume, respectively, by comparison with corresponding controls (Figure 3, and Supplementary data, Figure S2). A statistically significant difference in tumor volumes between shLuc-LV-injected tumors and shD1-LV-injected tumors was seen as early as 9 and 18 days after the viral injection of ASPC-1- and BxPC3-derived tumors, respectively. Moreover, the subsequent growth of all shD1-LV-injected tumors remained significantly attenuated.

Figure 3
figure 3

Effects of cyclin D1-directed shRNA on tumor growth. ASPC-1 or BxPC3 cells were injected subcutaneously in athymic nude mice. After tumors reached a volume of 30–40 mm3, they were either injected with buffer (ASPC-1) or with luciferase-directed (sh-Luc) or cyclin D1-directed (sh-D1) small hairpin (sh) RNA-lentiviruses. Tumor volumes were calculated in mm3 as described in the Methods section. Data are expressed as means±s.e.m. from eight mice in each group at each time point. *P<0.05 when compared with respective controls.

Full size image

ASPC-1-derived tumors injected with shD1-LV exhibited a 47% decrease (P<0.01) in Ki-67 immunoreactivity (a marker of cell proliferation), and a 56% decrease (P<0.05) in CD31 immunoreactivity (an endothelial cell marker), in comparison with respective controls (Figure 4). Similarly, BxPC3-derived tumors exhibited a 42% decrease (P<0.05) in Ki-67 immunoreactivity and a 45% decrease in CD31 immunoreactivity (P<0.05), in comparison with respective controls (Figure 5). In addition, VEGF immunoreactivity in ASPC-1- and BxPC3-derived tumors injected with shD1-LV was decreased by 52% (P<0.01) and 58% (P<0.01), respectively, in comparison with corresponding controls (Figures 4 and 5).

Figure 4
figure 4

Effects of cyclin D1-directed small hairpin (sh) RNA on proliferation and angiogenesis in ASPC-1-derived tumors. (a) Immunohistochemical analysis. Representative immunostaining for Ki-67, CD31 and VEGF in ASPC-1-derived tumors that were injected in vivo with buffer (Control), a luciferase-directed shRNA-lentivirus (sh-Luc), or a cyclin D1-directed shRNA-lentivirus (sh-D1). Original magnification, × 20. (b) Quantitative morphometry for Ki-67, CD31 and VEGF immunoreactivity from the tumors shown in (a). Data are the means±s.e.m. from three tumors per group, with 10 random fields analyzed for each tumor. *P<0.05, **P<0.01 when compared with respective controls.

Full size image
Figure 5
figure 5

Effects of cyclin D1-directed small hairpin (sh) RNA on cell proliferation and angiogenesis in BxPC3-derived tumors. (a) Immunohistochemical analysis. Representative immunostaining for Ki-67, CD31 and VEGF in tumors BxPC3-derived tumors that were injected in vivo with buffer (control), a luciferase-directed shRNA-lentivirus (sh-Luc), or a cyclin D1-directed shRNA-lentivirus (sh-D1). Original magnification, × 20. (b) Quantitative morphometry for Ki-67, CD31 and VEGF immunoreactivity. Data are the means±s.e.m. from three tumors per cell line per group. *P<0.05, **P<0.01 when compared with respective controls.

Full size image

Discussion

The three D-type cyclins that are encoded by distinct genes exhibit considerable amino-acid sequence homology, and have both overlapping and separate functions. The cyclin D1 gene, in particular, has been associated with neoplastic transformation. This gene is located on chromosome 11q13, a region that is amplified in breast, bladder, head and neck, lung and esophageal cancers25, 26 and that exhibits chromosomal translocations and rearrangements in parathyroid adenomas and B-cell lymphomas.25, 26 The associated increase in cyclin D1 levels has been correlated with a shortened G1 phase of the cell cycle and reduced mitogen dependency27, 28, 29 Cyclin D1 inhibition by antibody microinjection or by transfection with a cyclin D1 antisense expression construct prevents normal fibroblasts from entering the S phase of the cell cycle and markedly inhibits the proliferation of human esophageal and colon cancer cells30, 31, 32 and murine lung cancer cells.33 Moreover, increased cyclin D1 expression correlates with decreased survival in PDAC6, 10 as well as in esophageal34 and breast35 cancers. Although there are fewer functional data for the two other members of the cyclin D family, cyclin D3 expression is also upregulated in PDAC,36 whereas cyclin D2 expression was reported to be reduced as a result of promoter methylation.37

Lentiviruses are a subclass of retroviruses that allow for efficient gene transduction into cells and tissues, infecting both proliferating and non-proliferating cells, and resulting in stable integration of specific genes into the cellular genome.38 In contrast to adenoviruses, lentiviruses do not trigger a potentially dangerous immune response. They are ideally suited, therefore, for use in silencing strategies in cancer cells.14 In this study we determined that a single exposure of ASPC-1 and BxPC3 human pancreatic cancer cell lines to lentiviruses encoding two different specific shRNA targeting cyclin D1, resulted in stable integration of the virus and high level of expression of the shRNAs, along with subsequent marked reduction in cyclin D1 protein levels. Thus, lentiviral delivery of shRNA is an efficient way to markedly downregulate cyclin D1 in pancreatic cancer cells.

Cyclin D1 downregulation was associated with decreased cell proliferation, attenuated anchorage-independent growth and blunted invasiveness in vitro. This is the first direct demonstration that cyclin D1 promotes invasion in pancreatic cancer cells. This finding is in agreement with the observation that cyclin D1 overexpression in glioma cells is associated with increased matrix metalloproteinase-2 and matrix metalloproteinase-9 activity and upregulation of Rac1, which modulates the formation of ruffling membranes and cell motility.39 Moreover, in mouse embryo fibroblasts, mammary epithelial cells and macrophages derived from cyclin D1−/− mice, migration is greatly attenuated.40, 41 These observations may explain why patients with cyclin D1-positive pancreatic tumors tend to experience perineural and lymphatic invasion more often than patients with cyclin D1-negative tumors42, 43 and suggest that cyclin D1 may contribute to the invasiveness and metastatic potential of pancreatic cancer cells.

To test the therapeutic potential of the cyclin D1 shRNA expressing lentivirus approach, direct injection of the recombinant lentivirus was carried out in established tumors. A single injection of the recombinant lentivirus was sufficient to inhibit the growth of established xenografts arising from either ASPC-1 or BxPC3 cells, indicating that the downregulation of cyclin D1 by the shRNA lentiviral strategy resulted in a sustained biological effect in vivo. This conclusion is also supported by the observation that Ki-67 staining was significantly decreased in these tumors. In as much as Ki-67 is a marker of actively proliferating cells, this observation indicates that cyclin D1 levels directly affect proliferation status in vivo. This decrease in proliferation is consistent with known effects of cyclin D1 on cell cycle, with our in vitro observations with respect to cell growth, as well as with our observation, that cyclin D1 antisense expression can block the mitogenic action of epidermal growth factor, fibroblast growth factor-2 and insulin growth factor-1 in pancreatic cancer cells.11

Downregulation of cyclin D1 was also associated with a significant attenuation in tumor angiogenesis, as evidenced by decreased CD31 immunoreactivity and measured by quantitative morphometry. Given our finding that vascular endothelial growth factor (VEGF) levels were decreased in these tumors, it is possible that the silencing of cyclin D1 led to decreased VEGF expression, which may have contributed to decreased tumor angiogenesis. In support of this conclusion, cyclin D1 downregulation in a colorectal cancer cell line resulted in lower levels of VEGF expression in vitro, which was associated with decreased tumor angiogenesis in vivo.12 It is also possible, however, that the intratumoral injection of our lentiviruses led to direct downregulation of cyclin D1 in tumor endothelial cells and a direct suppressive effect on angiogenesis in addition to the lowered expression of VEGF. Irrespective of the mechanisms, given the importance of tumor angiogenesis in PDAC,23 our results raise the possibility that targeting cyclin D1 in PDAC may have the additional benefit of suppressing angiogenesis in this malignancy.

Our observations also point to the exciting possibility that this recombinant lentivirus could eventually be delivered by endoscopic ultrasonography into established tumors in patients with PDAC, thereby yielding a new therapeutic approach for this deadly malignancy. Several lines of evidence suggest that such an approach may have multiple beneficial effects. First, increased cyclin D1 expression is observed relatively early during tumorigenesis, in pre-malignant lesions termed pancreatic intraepithelial neoplasia, raising the possibility that targeting cyclin D1 could prevent the progression toward higher-grade lesions and cancer. This is a potentially important aspect of such therapy, as the presence of pancreatic intraepithelial neoplasia lesions in the vicinity of the tumor resection margins may serve as a source for cancer recurrence in PDAC. Second, cyclin D1 is required for carcinogenesis in some cancer models, as cyclin D1-deficient mice do not form mammary carcinomas despite the induction by c-neu and v-Ha-ras.43 Therefore, targeting cyclin D1 may block the actions of oncogenic Kras, which is commonly mutated in PDAC, and the actions of multiple tyrosine kinase receptors that are often overexpressed in PDAC.5 Third, PDAC is characterized by the presence of an abundant stroma that contains proliferating cancer associated fibroblasts and pancreatic stellate cells, and this stroma has been proposed to confer a growth advantage to pancreatic cancer cells within the tumor mass.44 Direct injections of the virus into the pancreatic tumor mass would result in a downregulation of cyclin D1 in non-cancer cells as well, which would suppress the proliferation of stromal elements. Fourth, suppression of cyclin D1 levels could result in decreased angiogenesis through direct actions on the endothelial cells, and, indirectly, by decreasing VEGF expression.

Elevated cyclin D1 levels contribute to the chemoresistance of pancreatic cancer cells, as shown by the increased chemosensitivity to cis-platinum of PDAC cells when cyclin D1 expression is inhibited11, 45 Therefore, targeting cyclin D1 may allow for suppression of tumor growth and angiogenesis, while allowing for more effective responses to chemotherapeutic agents. Taken together, these observations raise the possibility that intratumoral injections of viruses targeting cyclin D1, in conjunction with standard chemotherapy, could provide a new therapeutic approach in PDAC.

References

  1. Jemal A, Siegel R, Ward E, Hao Y, Xu J, Murray T et al. Cancer statistics, 2008. CA Cancer J Clin 2008; 58: 71–96.

    Article  Google Scholar 

  2. Abbruzzese JL . Adjuvant therapy for surgically resected pancreatic adenocarcinoma. JAMA 2008; 299: 1066–1067.

    CAS  Article  Google Scholar 

  3. Xiong HQ, Carr K, Abbruzzese JL . Cytotoxic chemotherapy for pancreatic cancer: Advances to date and future directions. Drugs 2006; 66: 1059–1072.

    CAS  Article  Google Scholar 

  4. Kern SE . Molecular genetic alterations in ductal pancreatic adenocarcinomas. Med Clin North Am 2000; 84: 691–695.

    CAS  Article  Google Scholar 

  5. Korc M . Role of growth factors in pancreatic cancer. Surg Oncol Clin N Am 1998; 7: 25–41.

    CAS  Article  Google Scholar 

  6. Gansauge S, Gansauge F, Ramadani M, Stobbe H, Rau B, Harada N et al. Overexpression of cyclin D1 in human pancreatic carcinoma is associated with poor prognosis. Cancer Res 1997; 57: 1634–1637.

    CAS  PubMed  Google Scholar 

  7. Fu M, Wang C, Li Z, Sakamaki T, Pestell RG . Minireview: Cyclin D1: normal and abnormal functions. Endocrinology 2004; 145: 5439–5447.

    CAS  Article  Google Scholar 

  8. Bernards R . CDK-independent activities of D type cyclins. Biochim Biophys Acta 1999; 1424: M17–M22.

    CAS  PubMed  Google Scholar 

  9. Chan HM, Krstic-Demonacos M, Smith L, Demonacos C, La Thangue NB . Acetylation control of the retinoblastoma tumour-suppressor protein. Nat Cell Biol 2001; 3: 667–674.

    CAS  Article  Google Scholar 

  10. Kornmann M, Ishiwata T, Itakura J, Tangvoranuntakul P, Beger HG, Korc M . Increased cyclin D1 in human pancreatic cancer is associated with decreased postoperative survival. Oncology 1998; 55: 363–369.

    CAS  Article  Google Scholar 

  11. Kornmann M, Arber N, Korc M . Inhibition of basal and mitogen-stimulated pancreatic cancer cell growth by cyclin D1 antisense is associated with loss of tumorigenicity and potentiation of cytotoxicity to cisplatinum. J Clin Invest 1998; 101: 344–352.

    CAS  Article  Google Scholar 

  12. Yasui M, Yamamoto H, Ngan CY, Damdinsuren B, Sugita Y, Fukunaga H et al. Antisense to cyclin D1 inhibits vascular endothelial growth factor-stimulated growth of vascular endothelial cells: implication of tumor vascularization. Clin Cancer Res 2006; 12: 4720–4729.

    CAS  Article  Google Scholar 

  13. Nishikura K . A short primer on RNAi: RNA-directed RNA polymerase acts as a key catalyst. Cell 2001; 107: 415–418.

    CAS  Article  Google Scholar 

  14. Grimm D, Kay MA . RNAi and gene therapy: a mutual attraction. Hematology Am Soc Hematol Educ Program 2007; 2007: 473–481.

    Article  Google Scholar 

  15. Hannon GJ, Conklin DS . RNA interference by short hairpin RNAs expressed in vertebrate cells. Methods Mol Biol 2004; 257: 255–266.

    CAS  PubMed  Google Scholar 

  16. Amado RG, Chen IS . Lentiviral vectors—the promise of gene therapy within reach? Science 1999; 285: 674–676.

    CAS  Article  Google Scholar 

  17. Sikorski R, Peters R . Gene therapy. Treating with HIV. Science 1998; 282: 1438.

    CAS  Article  Google Scholar 

  18. Kleeff J, Ishiwata T, Maruyama H, Friess H, Truong P, Büchler MW et al. The TGF-beta signaling inhibitor Smad7 enhances tumorigenicity in pancreatic cancer. Oncogene 1999; 18: 5363–5372.

    CAS  Article  Google Scholar 

  19. Neupane D, Korc M . 14-3-3sigma Modulates pancreatic cancer cell survival and invasiveness. Clin Cancer Res 2008; 14: 7614–7623.

    CAS  Article  Google Scholar 

  20. Kleeff J, Korc M . Up-regulation of transforming growth factor (TGF)-beta receptors by TGF-beta1 in COLO-357 cells. J Biol Chem 1998; 273: 7495–7500.

    CAS  Article  Google Scholar 

  21. Naldini L, Blomer U, Gallay P, Ory D, Mulligan R, Gage FH et al. In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector. Science 1996; 272: 263–267.

    CAS  Article  Google Scholar 

  22. Rowland-Goldsmith MA, Maruyama H, Matsuda K, Idezawa T, Ralli M, Ralli S et al. Soluble type II transforming growth factor-beta receptor attenuates expression of metastasis-associated genes and suppresses pancreatic cancer cell metastasis. Mol Cancer Ther 2002; 1: 161–167.

    CAS  Article  Google Scholar 

  23. Fukasawa M, Korc M . Vascular endothelial growth factor-trap suppresses tumorigenicity of multiple pancreatic cancer cell lines. Clin Cancer Res 2004; 10: 3327–3332.

    CAS  Article  Google Scholar 

  24. Luo J, Guo P, Matsuda K, Truong N, Lee A, Chun C et al. Pancreatic cancer cell-derived vascular endothelial growth factor is biologically active in vitro and enhances tumorigenicity in vivo. Int J Cancer 2001; 92: 361–369.

    CAS  Article  Google Scholar 

  25. Hall M, Peters G . Genetic alterations of cyclins, cyclin-dependent kinases, and Cdk inhibitors in human cancer. Adv Cancer Res 1996; 68: 67–108.

    CAS  Article  Google Scholar 

  26. Motokura T, Arnold A . Cyclins and oncogenesis. Biochim Biophys Acta 1993; 1155: 63–78.

    CAS  PubMed  Google Scholar 

  27. Jiang W, Kahn SM, Zhou P, Zhang YJ, Cacace AM, Infante AS et al. Overexpression of cyclin D1 in rat fibroblasts causes abnormalities in growth control, cell cycle progression and gene expression. Oncogene 1993; 8: 3447–3457.

    CAS  PubMed  Google Scholar 

  28. Quelle DE, Ashmun RA, Shurtleff SA, Kato JY, Bar-Sagi D, Roussel MF et al. Overexpression of mouse D-type cyclins accelerates G1 phase in rodent fibroblasts. Genes Dev 1993; 7: 1559–1571.

    CAS  Article  Google Scholar 

  29. Resnitzky D, Gossen M, Bujard H, Reed SI . Acceleration of the G1/S phase transition by expression of cyclins D1 and E with an inducible system. Mol Cell Biol 1994; 14: 1669–1679.

    CAS  Article  Google Scholar 

  30. Zhou P, Jiang W, Zhang YJ, Kahn SM, Schieren I, Santella RM et al. Antisense to cyclin D1 inhibits growth and reverses the transformed phenotype of human esophageal cancer cells. Oncogene 1995; 11: 571–580.

    CAS  PubMed  Google Scholar 

  31. Arber N, Doki Y, Han EK, Sgambato A, Zhou P, Kim NH et al. Antisense to cyclin D1 inhibits the growth and tumorigenicity of human colon cancer cells. Cancer Res 1997; 57: 1569–1574.

    CAS  PubMed  Google Scholar 

  32. Baldin V, Lukas J, Marcote MJ, Pagano M, Draetta G . Cyclin D1 is a nuclear protein required for cell cycle progression in G1. Genes Dev 1993; 7: 812–821.

    CAS  Article  Google Scholar 

  33. Schrump DS, Chen A, Consoli U . Inhibition of lung cancer proliferation by antisense cyclin D. Cancer Gene Ther 1996; 3: 131–135.

    CAS  PubMed  Google Scholar 

  34. Naitoh H, Shibata J, Kawaguchi A, Kodama M, Hattori T . Overexpression and localization of cyclin D1 mRNA and antigen in esophageal cancer. Am J Pathol 1995; 146: 1161–1169.

    CAS  PubMed  PubMed Central  Google Scholar 

  35. McIntosh GG, Anderson JJ, Milton I, Steward M, Parr AH, Thomas MD et al. Determination of the prognostic value of cyclin D1 overexpression in breast cancer. Oncogene 1995; 11: 885–891.

    CAS  PubMed  Google Scholar 

  36. Ebert MP, Hernberg S, Fei G, Sokolowski A, Schulz HU, Lippert H et al. Induction and expression of cyclin D3 in human pancreatic cancer. J Cancer Res Clin Oncol 2001; 127: 449–454.

    CAS  Article  Google Scholar 

  37. Matsubayashi H, Sato N, Fukushima N, Yeo CJ, Walter KM, Brune K et al. Methylation of cyclin D2 is observed frequently in pancreatic cancer but is also an age-related phenomenon in gastrointestinal tissues. Clin Cancer Res 2003; 9: 1446–1452.

    CAS  PubMed  Google Scholar 

  38. Zufferey R, Dull T, Mandel RJ, Bukovsky A, Quiroz D, Naldini L et al. Self-inactivating lentivirus vector for safe and efficient in vivo gene delivery. J Virol 1998; 72: 9873–9880.

    CAS  PubMed  PubMed Central  Google Scholar 

  39. Arato-Ohshima T, Sawa H . Over-expression of cyclin D1 induces glioma invasion by increasing matrix metalloproteinase activity and cell motility. Int J Cancer 1999; 83: 387–392.

    CAS  Article  Google Scholar 

  40. Li Z, Wang C, Prendergast GC, Pestell RG . Cyclin D1 functions in cell migration. Cell Cycle 2006; 5: 2440–2442.

    CAS  Article  Google Scholar 

  41. Neumeister P, Pixley FJ, Xiong Y, Xie H, Wu K, Ashton A et al. Cyclin D1 governs adhesion and motility of macrophages. Mol Biol Cell 2003; 14: 2005–2015.

    CAS  Article  Google Scholar 

  42. Lebe B, Sagol O, Ulukus C, Coker A, Karademir S, Astarcioglu H et al. The importance of cyclin D1 and Ki67 expression on the biological behavior of pancreatic adenocarcinomas. Pathol Res Pract 2004; 200: 389–396.

    CAS  Article  Google Scholar 

  43. Sutherland RL, Musgrove EA . Cyclin D1 and mammary carcinoma: new insights from transgenic mouse models. Breast Cancer Res 2002; 4: 14–17.

    CAS  Article  Google Scholar 

  44. Korc M . Pancreatic cancer-associated stroma production. Am J Surg 2007; 194: S84–S86.

    CAS  Article  Google Scholar 

  45. Kornmann M, Danenberg KD, Arber N, Beger HG, Danenberg PV, Korc M . Inhibition of cyclin D1 expression in human pancreatic cancer cells is associated with increased chemosensitivity and decreased expression of multiple chemoresistance genes. Cancer Res 1999; 59: 3505–3511.

    CAS  PubMed  Google Scholar 

Download references

Acknowledgements

This study was supported, in part, by US Public Health Service Grant CA-101306 to MK; SD was the recipient of a post-doctoral fellowship award from the SASS Foundation for Medical Research.

Author information

Affiliations

  1. Departments of Medicine, Pharmacology and Toxicology, Norris Cotton Center and Dartmouth Medical School, Lebanon, NH, USA

    S J Deharvengt, J R Gunn, S B Pickett & M Korc

Authors
  1. S J Deharvengt
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. J R Gunn
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. S B Pickett
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. M Korc
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to M Korc.

Ethics declarations

Competing interests

The authors declare no conflict of interest.

Additional information

Supplementary Information accompanies the paper on Cancer Gene Therapy website

Supplementary information

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Deharvengt, S., Gunn, J., Pickett, S. et al. Intratumoral delivery of shRNA targeting cyclin D1 attenuates pancreatic cancer growth. Cancer Gene Ther 17, 325–333 (2010). https://doi.org/10.1038/cgt.2009.75

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/cgt.2009.75

Keywords

  • cyclin D1
  • pancreatic cancer
  • shRNA
  • lentivirus

Further reading

Search

Advanced search

Quick links