An oncolytic adenovirus expressing soluble transforming growth factor-β type II receptor for targeting breast cancer: in vitro evaluation Free

In the last several years, there has been a significant interest in using adenoviral vectors for high-level gene expression in the mammalian cells (1–4). Majority of the studies have employed replication-deficient (E1 deleted) adenoviruses expressing the genes of interest (1–4). Recently, adenoviruses that selectively replicate in the tumor cells with certain genetic backgrounds (e.g., tumor cells expressing mutant p53 protein) have been developed (5–7). The selective replication of oncolytic adenovirus in tumor cells amplifies the viral titer, resulting in cell lysis. The released viral particles in turn can infect the neighboring tumor cells, resulting in tumor cytolysis and regression (5–7). Although replicating adenoviruses have an advantage over the replication-deficient adenoviruses (which do not lyse the infected cells and can not spread from cell to cell in a tumor mass), in vivo efficacy of even oncolytic viruses is generally not sufficient for cancer therapy. Therefore, there is a tremendous need to enhance the effectiveness of the oncolytic viruses for cancer therapy.

The long-term goal of this project is to investigate if a conditionally replicating adenovirus (dl01/07) armed with the soluble form of transforming growth factor-β type II receptor (sTGFβRII) will have an advantage as an antitumor agent for treating breast cancer. We chose to use the dl01/07 mutant because the dl01/07 virus has two deletions in E1A region, one deletion is 4 to 25 amino acids (dl01), and the second deletion is 111 to 123 amino acids (dl07). The resultant E1A proteins can not bind with p300/CBP or pRb proteins (8). Therefore, in primary cells, dl01/07 is ineffective for S-phase induction, and the adenovirus can not replicate. However, cancer cells are able to progress to S phase, thus permitting virus replication in these cells.

We chose to target TGF-β because TGF-β plays an important role in late-stage tumorigenesis by stimulating tumor invasion, promoting neoangiogenesis, inducing bone metastasis, and helping cancer cells to escape immunosurveillance (9–19). TGF-βs have three mammalian isoforms (TGF-β1, TGF-β2, and TGF-β3), each with distinct functions in vivo. After TGF-β binding to TGFβRII, TGF-β type I receptor is recruited to the complex. This allows for the constitutively active TGFβRII kinase to transphosphorylate and activate the TGF-β type I receptor kinase. In breast cancer cells, this initiates the downstream response by various pathways that include SMADs, extracellular signal-regulated kinase-1, extracellular signal-regulated kinase-2, mitogen-activated protein kinases (MAPK), phosphatidylinositol 3-kinase pathways and induces transcriptional modulation of target genes (20, 21). Given the integral role of TGF-β in the tumor progression, inhibiting TGF-β signaling emerges as a potential strategy in controlling tumor malignancy for cancer therapy (9, 14, 22, 23).

An oncolytic adenovirus armed with sTGFβRII (rAd-sTRII) was constructed by inserting the soluble 159-amino-acid residue domain of the TGFβRII into the dl01/07 adenoviral genome. As a control for viral replication, we also generated a replication-deficient adenovirus containing sTGFβRII (Ad-sTRII) and a replication-competent dl01/07 expressing herpes simplex virus thymidine kinase (rAd-TK). We investigated if the adenoviral vector–mediated expression of sTGFβRII in the extracellular environment can bind with TGF-β, resulting in the inhibition of TGF-β signaling in the target cells.

There was an initial concern that the adenoviral-mediated expression of sTGFβRII protein can potentially interfere with the viral replication. Therefore, one of our goals was to investigate the effect of sTGFβRII expression on the adenoviral replication in breast cancer cells. We examined the cytotoxicity and the replication potential of rAd-sTRII and Ad-sTRII in breast tumor cells. Our results suggest that whereas the infection of breast tumor cells with rAd-sTRII and Ad-sTRII produced the functional sTGFβRII protein, only the rAd-sTRII replicated in the tumor cells. Thus, it is possible to simultaneously achieve the adenoviral replication and the expression of the secreted form of functional sTGFβRII protein, indicating the potential use of rAd-sTRII for cancer therapy.

HEK-293 (ATCC CRL-1573), SK-BR-3, MDA-MB-468, MDA-MB-453, T47D, MCF-7, and MDA-MB-231 (kindly provided by Ruth Lupu, Evanston Northwestern Healthcare Research Institute) were cultured in DMEM (Mediatech, Inc., Herndon, VA) containing 10% fetal bovine serum (FBS; Mediatech) and 1% penicillin/streptomycin (Invitrogen, Carlsbad, CA).

For construction of replication-deficient adenovirus Ad-sTRII, ∼0.5-kb NotI-HindIII DNA fragment containing codon 1-159 of the TGFβRII gene from pBS-SK(−)/sTRII (24) was cloned in the NotI and HindIII region of pShuttle-CMV. The resulting shuttle vector pShuttle-CMV/sTRII was then recombined in Escherichia coli BJ5183 by homologous recombination with the E1- and E3-deleted pAdEasy-1 adenoviral backbone vector (Stratagene, La Jolla, CA) to generate a packagable adenoviral genome pAd-sTRII (25). Ad-sTRII vector was produced by transfecting PacI-digested pAd-sTRII into HEK-293 cells by LipofectAMINE 2000 (Invitrogen). For construction of replication-competent adenovirus rAd-sTRII, ∼550-bp XbaI fragment from pShuttle-CMV/sTRII containing codon 1-159 of the TGFβRII gene was cloned in XbaI-cut plasmid p309-CMV-poly(A) to produce a shuttle vector p309/sTRII. The 11-kb PacI-AscI fragment from p309/sTRII was recombined with BstBI- and SpeI-cut adenoviral backbone plasmid pTG07-4609 in E. coli BJ5183 to produce adenoviral genome plasmid pTG07-4609/sTRII. Adenovirus rAd-sTRII was generated by transfecting PacI-cut pTG07-4609/sTRII into HEK-293 cells. rAd-TK was constructed by similar procedures except that the herpes simplex virus thymidine kinase (HSV-TK) gene was inserted instead of the sTGFβRII gene. E1-deleted replication-deficient adenovirus devoid of any foreign cDNA (AdNull) was described previously (26). dl309 is a phenotypically wild-type adenovirus (27). Adenoviruses were amplified in HEK-293 cells and purified by cesium chloride gradient ultracentrifugation, and the titers were calculated using published methods (28, 29).

MDA-MB-231 cells (1 × 10⁶ per well in six-well plate) were plated in DMEM containing 10% FBS and incubated at 37°C overnight. The next morning, cells were infected with 100 plaque-forming units/cell (unless otherwise mentioned) of adenovirus for 3 hours. Cells were washed and incubated with DMEM without FBS for 24 hours. Media and cells were separately dissolved in SDS sample buffer and subjected to Western blot analysis as previously described (28, 30). Blots were probed with antibody reactive against TGFβRII (H-567; Santa Cruz Biotechnology, Santa Cruz, CA) or actin protein (I-19; Santa Cruz Biotechnology).

Sixty microliters of culture media from Ad-sTRII or rAd-sTRII infected MDA-MB-231 cells were denatured at 100°C for 10 minutes and treated with PNGase F (New England Biolabs, Beverly, MA) for 2 hours at 37°C according to manufacturer's instructions. The proteins were subjected to SDS-PAGE and analyzed by Western blot using rabbit anti-TGFβRII polyclonal antibody.

MDA-MB-231 cells (1 × 10⁶ per well in six-well plate) were uninfected or infected with different adenoviruses [100 multiplicities of infection (MOI)] for 3 hours in growth media. Cells were washed and incubated in 1.7 mL serum-free DMEM medium for 20 hours. The culture media were collected, and 200 μL of culture media were mixed with TGF-β1 (40 ng; Sigma, St. Louis, MO) for 1 hour at 4°C and 50 μL of wheat germ agglutinin-Sepharose agarose beads (Vector Laboratories, Burlingame, CA) were added and incubated for 1 hour at 4°C. The beads were washed six times with buffer [50 mmol/L NaCl, 10 mmol/L Tris-HCl, 5 mmol/L EDTA, 1% Triton X-100 (pH 7.4)] and subjected to SDS-PAGE (15%). The proteins were transferred onto Immun-Blot polyvinylidene difluoride membranes (Bio-Rad, Hercules, CA) and probed with rabbit anti-TGF-β1 polyclonal antibody (Promega, Madison, WI).

MDA-MB-231 cells grown in normal growth medium were serum starved overnight in DMEM without FBS, washed, and incubated fresh DMEM without FBS. TGF-β1 (5 ng/mL) was added to the cells and incubated for 0, 10, 20, 30, 60, 120, 180, and 240 minutes at 37°C. The total cells were subjected to Western blot using antibodies against phospho-p38 (sc-7975-R, Santa Cruz Biotechnology) or p38 (C-20, Santa Cruz Biotechnology).

MDA-MB-231 cells were incubated with 100 MOI of different adenoviruses for 3 hours in normal growth medium. Cells were washed and incubated in serum-free DMEM for 20 hours. The culture media were collected and centrifuged at 180,000 × g to remove contaminating adenovirus in the media. The 0.1 mL of this culture media was mixed with 0.7 mL DMEM without FBS and transferred to serum-starved MDA-MB-231 cells and incubated at 37°C for 1 hour. Cells were washed and dissolved in SDS sample buffer for Western blot analysis of p38 MAPK activation.

Cells were plated in triplicate in 96-well dishes (500 per well) and incubated for 24 hours at 37°C. Cells were exposed to varying concentrations of Ad-sTRII and rAd-sTRII and incubated for additional 7 days at 37°C. A colorimetric assay was done as described previously (28). Briefly, cells were fixed in 10% trichloroacetic acid for 1 hour, washed five times with water, and allowed to air dry. Cells were then stained for 10 minutes with 0.4% sulforhodamine B (Sigma), dissolved in 1% acetic acid, and rinsed five times with 1% acetic acid. Absorbance (A_{564 nm}) was obtained using Spectramax 250 (Molecular Devices, Sunnyvale, CA), which was used as a measure of cell number. The IC₅₀ (viral dose that caused 50% cytotoxicity) was calculated assuming the survival rate of uninfected cells to be 100%. The ratio of IC₅₀ was calculated by dividing the IC₅₀ of cells infected with Ad-sTRII by the IC₅₀ of cells infected with rAd-sTRII for each cell line.

To assay for viral replication, MDA-MB-231 cells were plated in six-well plates at about 70% confluence and then infected with Ad-sTRII, rAd-sTRII, rAd-TK, or dl309 for 3 hours at an MOI of 50, washed once with DMEM, and incubated in 1 mL DMEM for additional 1 hour at 37°C. At the end of the incubation, cells were washed and divided into two groups. In one group, cells were collected in 0.5 mL growth media and frozen at −70°C. In the second group, cells were maintained in growth media for additional 48 hours. Media and cells in both groups were collected, and cells were subjected to three cycles of freezing and thawing to release the viruses. The total viruses from media and cells were serially diluted and added to monolayers of 293 cells, respectively. After 3 hours of incubation at 37°C, the infected 293 cells were overlaid with 3 mL 1.25% SeaPlaque agarose (Cambrex, East Rutherford, NJ) in growth media. Plaques were counted following 7 to 10 days of incubation using published methods (29).

The recombinant adenoviruses Ad-sTRII and rAd-sTRII were constructed as described in Materials and Methods. To generate Ad-sTRII and rAd-sTRII, the cDNA for the complete extracellular domain of human TGFβRII (amino acid residues 1–159) under the control of cytomegalovirus promoter (CMV) was placed in their individual genomes. The schematic diagram of the structure of adenoviruses is shown in Fig. 1. It should be noted that the replication-deficient Ad-sTRII vector has an E1 deletion, whereas rAd-sTRII is a conditionally replicating adenovirus due to two short deletions in the E1A gene (dl01/07; Fig. 1; ref. 8).

We investigated whether the infection of human breast cancer cells with Ad-sTRII and rAd-sTRII can produce sTGFβRII protein. MDA-MB-231 breast cancer cells were exposed to AdNull, Ad-sTRII, or rAd-sTRII for 24 hours and subjected to Western blot analysis. As shown in Fig. 2A, there were no detectable protein bands reactive with antibody against TGFβRII in cells infected with AdNull. In contrast, strong protein bands appeared in both Ad-sTRII– and rAd-sTRII–infected cells. In cell lysates, there were protein bands with molecular weight ranging from 20 to 25 kDa, whereas in cell media, protein bands shifted to the higher position with molecular weight ranging from 20 to 40 kDa. Figure 2B and C shows the dose-dependent increase of sTGFβRII expression in both media and cell lysates. Infection of other breast tumor cells with these viruses also resulted in the overexpression of sTGFβRII (data not shown).

The predicted molecular mass of truncated TGFβRII (amino acid residues 1–159) is around 18 kDa. After cleavage of the hydrophobic leader sequence, the length of this truncated receptor is 136-amino-acid residues, and the predicted molecular mass is ∼15.5 kDa. However, as described above, in our experiments, the cells did not produce a distinct band, but instead, a smear of high molecular weight was observed. It was previously reported that the secreted soluble receptor contains complex N-linked oligosaccharides as well as additional sialic acid residues (31). We treated the secreted sTGFβRII receptor from both Ad-sTRII– and rAd-sTRII–infected cells with N-glycosidase F (PNGase F), an amidase that cleaves between the innermost GlcNAc and asparagine residues of high-mannose, hybrid, and complex oligosaccharides from N-linked glycoproteins (32). The protein smear was resolved into two major distinct bands (∼25 and ∼20 kDa), indicating that the sTGFβRII produced by MDA-MB-231 is a heterogeneously glycosylated protein (Fig. 3).

To test whether sTGFβRII could bind to TGF-β, cells were infected with adenoviruses (100 plaque-forming units/cell for 24 hours). sTGFβRII is known to bind with TGF-β1 with much higher affinity compared with TGF-β2 (33). The culture media from uninfected or infected cells were incubated with pure recombinant TGF-β1 and mixed with wheat germ agglutinin-Sepharose beads, which bind to glycosylated proteins, including soluble TGF-β receptor. Beads were washed and subjected to Western blot analysis and probed with anti–TGF-β1 antibody. TGF-β1 was clearly detectable in the precipitate from the medium of soluble TGF-β receptor expressing cells but not from the uninfected or AdNull-infected cells (Fig. 4A). These results indicate that the secreted soluble TGF-β receptor can bind with TGF-β.

Next, we wanted to investigate if the binding of soluble TGF-β receptor with TGF-β will abolish TGF-β signaling in breast cancer cells. Although several biochemical pathways are involved in TGF-β signaling, we chose to specifically examine p38 MAPK because the latter is known to be involved in TGF-β signaling in MDA-MB-231 cells (34). We examined the activation of p38 MAPK by TGF-β in Western blot assays. Antibodies specific for nonphosphorylated and phosphorylated p38 MAPK were used. As shown in Fig. 4B, the phosphorylation of p38 MAPK was increased in MDA-MB-231 cells after TGF-β1 addition to the media with maximal activation at 30 to 60 minutes. To test whether soluble TGF-β receptor produced by virus-infected cells can functionally inhibit TGF-β activities, we assessed its effect on p38 MAPK phosphorylation in MDA-MB-231 cells. MDA-MB-231 cells cultured in serum-free medium secrete multiple growth factors and cytokines, including TGF-β (35). Cells were infected with 100 MOI of either Ad-sTRII or rAd-sTRII for 24 hours. Culture media from the virally infected cells were collected and centrifuged at 180,000 × g. Under these conditions, adenoviruses are known to sediment at the bottom of the centrifuge tube (36). The overnight culture media from uninfected or virus-infected cells were used to treat new set of MDA-MB-231 cells for 1 hour. The cells treated with the culture media from both Ad-sTRII– and rAd-sTRII–infected cells exhibited decreased phosphorylation of p38 MAPK compared with cells treated with media from AdNull-infected or uninfected cells (Fig. 4C). These results indicate that the binding of TGF-β by sTGFβRII in the culture media prevented the maximal activation of p38 MAPK.

For cancer therapy purposes, it is important that rAd-sTRII–mediated production of soluble TGF-β RII does not compromise with the viral replication in the target cells. We therefore investigated the effect of adenoviral infections on the viral replication in two different assays: an indirect cytotoxicity assay and a direct method to evaluate the viral titers. To investigate the viral-mediated cytotoxicity, several breast tumor cell lines were exposed to varying doses of adenoviruses shown in Fig. 5A and B. The cytotoxicity assays were done as described in Material and Methods. In MDA-MB-231 cells, rAd-sTRII caused a dose-dependent increase in the cytotoxicity and markedly inhibited cell growth even at viral dosage levels <100 MOI. Under similar conditions, much higher doses of Ad-sTRII were required to induce comparable cytotoxicity. Similarly, rAd-sTRII was relatively more cytotoxic than Ad-sTRII in MCF-7 breast cancer cells. To investigate the contribution of sTGFβRII in cell killing, we compared the effect of rAd-sTRII with a control replicating adenovirus, rAd-TK. Both viruses exerted cytotoxic effect on the cells in a similar dose-dependent manner. In addition, we also compared the basal level toxicity of first-generation E1-deleted adenovirus expressing sTGFβRII with a control E1-deleted adenovirus devoid of any transgene. In this comparison, also nearly equal cytotoxicity to the tumor cells was observed. Thus, it seems that in our assay, sTGFβRII overexpression did not enhance the cytotoxicity of oncolytic or replication-deficient adenoviruses. Figure 5C shows the ratio of IC₅₀ caused by Ad-sTRII and rAd-sTRII in different breast tumor cell lines. These marked differences in cytotoxicity (5- to 500-fold) inflicted by rAd-sTRII were presumably the result of virus replication in these cancer cells.

To assess the replication ability of rAd-sTRII in a direct assay, we compared the vial production of rAd-sTRII with that of Ad-sTRII and two control adenoviruses rAd-TK and dl309 in MDA-MB-231 cells. The total viral particles in the culture medium and cell fraction were determined by performing plaque assay on 293 cells. After 48 hours of virus infection, the viral yield increased significantly (about 4 log differences compared with that of a 3-hour incubation) for rAd-sTRII, rAd-TK, and dl309 adenoviruses (Fig. 6). The titer of rAd-sTRII is only slightly lower than that of rAd-TK and was comparable with that of dl309. In contrast, the titer for replication-deficient Ad-sTRII did not increase but rather slightly decreased after a 48-hour incubation, indicating the inability of Ad-sTRII to replicate in MDA-MB-231 cells. These results suggest that the expression of sTGFβRII does not discernibly inhibit the replication of rAd-sTRII in MDA-MB-231 cells.

In recent years, replication-competent oncolytic adenoviral vectors as potential antitumor agents have been developed. To augment the anticancer effects of replicating adenoviruses, oncolytic adenoviruses can be armed with other genes, such as suicide genes, in a manner similar to the extensive development of the recombinant replication-deficient adenoviruses (1–4, 37). In this report, we have successfully placed the sTGFβRII gene in the genome of a replication-competent adenovirus and a control replication-deficient adenovirus and have conducted in vitro evaluation in breast tumor cells.

The sTGFβRII was overexpressed in breast cancer cells after infection with rAd-sTRII adenoviral vector. Vector-mediated expression of sTGFβRII was dependent on the viral dose. Western blot analysis of the infected cells indicated multiple size protein bands. However, the multiple protein bands were not due to the degradation product(s) of the sTGFβRII but due to the glycosylation of the sTGFβRII, as the treatment of the secreted proteins with N-glycosidase F converted the various heterogeneous bands into two distinct protein bands. More importantly, the secreted sTGFβRII protein was shown to bind with TGF-β1 and inhibited TGF-β–stimulated p38 MAPK in the target cells, suggesting that the sTGFβRII was fully functional. Similar levels of sTGFβRII functional proteins were produced by replication-deficient and replication-competent adenoviruses, suggesting that the viral replication had no adverse effect on the expression of sTGFβRII protein.

Overexpression of TGF-β ligands has been reported in many tumor types and elevated levels of TGF-β in tumor tissues correlate with markers of a more metastatic phenotype and/or poor patient outcome (38, 39). Based on our in vitro observations that rAd-sTRII–mediated expression and secretion of sTGFβRII into the extracellular environment can inhibit TGF-β signaling, we can perceive that the administration of rAd-sTRII in vivo will produce sTGFβRII that will be systemically released into the blood. This will inactivate the “overactive” TGF-β signaling associated with breast cancers and will result in the inhibition of tumor invasion and metastasis. However, in the absence of any experimental data, this remains an exciting hypothesis that needs to be tested in future.

In this study, we have shown that the rAd-sTRII is fully replication competent compared with a phenotypically wild type adenovirus dl309. It is an important observation given a tight interaction of the cellular machinery with the adenoviral replication heterologous protein can potentially interfere with the adenoviral replication, defeating the main advantage of replicating adenoviral vectors. Because the commonly used conditionally replicating viruses often exploit the differences in the cell cycle status, programmed cell death, and the cellular DNA synthesis between normal and tumor cells, the heterologous protein can potentially interact with these cellular pathways/machinery and interfere with adenoviral replication even in the tumor cells. Examples of such proteins are the regulators of cell cycle (p16^INK4A and p21^WAF1/Cip1), apoptosis (wild-type p53 and Bax), and DNA and protein synthesis (suicide gene plus the pro-drugs). Given the multiple pathways involved in the TGF-β–mediated signaling, there was a possibility that interfering with TGF-β pathways would the adenoviral replication. In this regard, it is a significant finding that overexpression of vector-mediated sTGFβRII does not compromise with the adenoviral replication in the limited number of breast tumor cells that we have tested thus far. However, it was surprising that sTGFβRII overexpression did not enhance the cytotoxic effect of the oncolytic virus. Although the exact reasons for this were not explored, in the future, we would further investigate this important observation.

Another point worth noting is the choice of the replicating adenoviral mutant to overexpress the transgene of interest. We chose to use dl01/07 adenovirus backbone, because dl01/07 expresses the mutant E1A gene product that is defective for binding both p300/CBP and pRb. pRb and p300 regulate the activity of E2F, which activates genes involved in transition from G₁ phase to the S phase of the cell cycle. All tumor cells exhibit uncontrolled cell growth due to deregulated G₁-S phase transition of the cell cycle. In cycling tumor cells, E2F is constitutively active because of disruption in the pRb/p16^INK4a/cyclin D pathway, including E2F-1 gene amplification. Therefore, dl01/07 can replicate in the tumor cells regardless of their genetic background (40), making it an attractive vector for treating a variety of cancers.

In summary, the rAd-sTRII replication in the infected cells and simultaneous production and release of sTGFβRII in the extracellular medium resulting in the inhibition of TGF-β signaling in the target cells offer us potentially a powerful tool to simultaneously treat the primary tumor and metastasis in breast cancer. In the future, we wish to take up this important endeavor to investigate the feasibility of using rAd-sTRII vector as an antitumor agent using the animal models and eventually in the clinical setting. Although in these studies, we have focused on the breast cancer cells as the target, it is likely that rAd-sTRII will find applications in targeting many cancers, especially those malignancies in which TGF-β overexpression seems to enhance tumorigenesis.

We thank Dr. Janardan D. Khandekar for his support of this research.