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Cancer Biol Ther. 2010 December 15; 10(12): 1290–1305.
Published online 2010 December 15. doi:  10.4161/cbt.10.12.13497
PMCID: PMC3047088

Enhancing mda-7/IL-24 therapy in renal carcinoma cells by inhibiting multiple protective signaling pathways using sorafenib and by Ad.5/3 gene delivery

Abstract

We have determined whether an adenovirus that comprises the tail and shaft domains of a serotype 5 virus and the knob domain of a serotype 3 virus expressing MDA-7/IL-24, Ad.5/3-mda-7, more effectively infects and kills renal carcinoma cells (RCCs) compared to a serotype 5 virus, Ad.5-mda-7. RCCs are a tumor cell type that generally does not express the receptor for the type 5 adenovirus; the coxsakie and adenovirus receptor (CAR). Ad.5/3-mda-7 infected RCCs to a much greater degree than Ad.5-mda-7. MDA-7/IL-24 protein secreted from Ad.5/3-mda-7-infected RCCs induced MDA-7/IL-24 expression and promoted apoptosis in uninfected “bystander” RCCs. MDA-7/IL-24 killed both infected and bystander RCCs via CD95 activation. Knockdown of intracellular MDA-7/IL-24 in uninfected RCCs blocked the lethal effects of conditioned media. Infection of RCC tumors in one flank, with Ad.5/3-mda-7, suppressed growth of infected tumors and reduced the growth rate of uninfected tumors implanted on the opposite flank. The toxicity of the serotype 5/3 recombinant adenovirus to express MDA-7/IL-24 was enhanced by combined molecular or small molecule inhibition of MEK1/2 and PI3K; inhibition of mTOR, PI3K and MEK1/2; or use of the multi-kinase inhibitor sorafenib. In RCCs, combined inhibition of cytoprotective cell signaling pathways enhanced the MDA-7/IL-24-induction of CD95 activation, with greater mitochondrial dysfunction due to loss of MCL-1 and BCL-XL expression and tumor cell death. Treatment of RCC tumors in vivo with sorafenib also enhanced Ad.5/3-mda-7 toxicity and prolonged animal survival. Future combinations of these approaches hold promise for developing a more effective therapy for kidney cancer.

Key words: ERK, JNK, PI3K, AKT, MDA-7/IL-24, sorafenib, PERK, MAPK, interleukin, RCC, kidney

Introduction

In the United States, renal cell carcinoma (RCC) is diagnosed in ~51,000 patients per annum. If the disease is detected at an early stage, in which a large portion or the entire kidney can be removed with the tumor, a high prolonged level of patient survival is noted (reviewed in ref. 1). However, if the disease has spread beyond the capsule of the kidney into the adrenal gland or surrounding fascia with nodal involvement the prognosis is poor with rapid nadir.1 This occurs even under ideal circumstances where the disease is still only locally advanced and essentially all of the tumor can be surgically removed and the patients are maximally treated with palliative radiation and chemotherapy. In part, this resistance is due to the fact that RCC is characterized as frequently being highly refractory to multiple established cytotoxic chemotherapy regimens.1 Clearly, there is a need to develop new approaches to treat this lethal disease.

The mda-7 gene (Interleukin 24, IL-24) was isolated from human melanoma cells induced to undergo terminal differentiation.2 Protein expression of MDA-7/IL-24 decreases with increasing staging in advanced melanomas, with almost undetectable levels of the protein in metastatic tumors.214 Enforced expression of MDA-7/IL-24 inhibits the growth, kills and also radio-/chemo-sensitizes a very broad spectrum of cancer cell types without exerting toxic effects in normal human epithelial or fibroblast cells.916 In kidney cancer cells we have previously shown that GST-MDA-7 kills these cells, but not normal kidney epithelial cells, in part through activation of the death receptor CD95.10 Mda-7/IL-24 has been evaluated in a Phase I clinical trial in advanced cancers that were refractory to further therapeutic intervention and this study indicated that an Ad.5-mda-7 (INGN-241) injected intra-tumorally was safe and with repeated injection a significant level of clinical activity was observed in up to ~44% of patients.10,11,13,14,17,18

The apoptotic pathways by which Ad.5-mda-7 induces cell death in tumor cells are complex and seem to subtly change based on the tumor cell type being examined.916,2028 In melanoma cells, MDA-7/IL-24 was shown to induce a decrease in BCL-2 and BCL-XL levels, concomitant with a modest upregulation of BAX and BAK expression; thus the ratio of protective to toxic BCL-2 family proteins in the tumor cell has been shifted towards a pro-apoptotic state.1013,19 The ability of Ad.5-mda-7 to induce apoptosis in DU145 prostate cancer cells, which do not produce the toxic BH3 domain protein BAX, demonstrates that MDA-7/IL-24 can mediate apoptosis in tumor cells through a BAX-independent pathway.914 More recently MDA-7/IL-24 toxicity has been strongly linked to alterations in endoplasmic reticulum (ER) stress signaling.2023 MDA-7/IL-24 physically associates with the ER-localized HSP70 family chaperone protein BiP/GRP78 that inactivates this protein resulting in the activation/dimerization of its chaperone: PKR-like endoplasmic reticulum kinase (PERK).1013,23,24 We have noted that high concentrations of GST-MDA-7 or infection of tumor cells with Ad.5-mda-7 kills tumor cells and does so in a PERK-dependent fashion that is dependent on mitochondrial dysfunction and in GBM cells, we went on to note that at least 5 toxic BH3 domain proteins facilitated MDA-7/IL-24 toxicity.2528 In a wide variety of cancer cells, overexpression of multiple protective BCL-2 family proteins protects cells from MDA-7/IL-24 toxicity.1013,20,22 In ovarian and renal carcinoma cells MDA-7/IL-24 was shown to initiate killing via the extrinsic apoptosis pathway that was dependent on ceramide generation and CD95 activation, and also upon ER stress-induced loss of MCL-1 expression.10,21,22,27

The ability of MDA-7/IL-24 to activate/inhibit the activities of many signaling pathways in transformed cells has been investigated by our laboratories and by other groups.1013,1532 Prior work, using bacterially synthesized GST-MDA-7, argues that in the 0.25–2.0 nM concentration range GST-MDA-7 causes growth arrest with little cell killing; effects that are due to elevated levels of ER stress signaling, whereas at ~20-fold greater concentrations, the cytokine causes profound ER stress signals, growth arrest and tumor cell death.1013,24,27,28 Our laboratories have demonstrated that Ad.5-mda-7 kills melanoma cells and established GBM cells in part by promoting p38 MAPK activation e.g., reviewed in reference 30. In primary human GBM cells however, MDA-7/IL-24-induced activation of p38 MAPK was shown to be a protective signal at what were considered to be “toxic” doses but that at “supra-toxic” doses p38 MAPK signaling reverted to being a toxic signal; JNK signaling was toxic in GBM cells.27,28 In RCCs we have noted that both MDA-7/IL-24-induced JNK1/2 and p38 MAPK signaling were toxic responses.10 Other groups have argued that inhibition of PI3K signaling, but not ERK1/2 signaling, modestly promotes Ad.5-mda-7 lethality in breast and lung cancer cells, whereas we have shown in GBM cells that inhibition of both PI3K and ERK1/2 signaling was required to potentiate the toxic effect of the cytokine.1013,21,27,28,31,32

The present studies were designed to define more effective ways of killing RCCs using mda-7/IL-24 delivered as a recombinant adenovirus. Previously we have used GST-MDA-7 as a tool to study MDA-7/IL-24 effects in RCCs; however GST-MDA-7 cannot be translated as a therapeutic for animals or for the clinic. In this manuscript we provide new data supporting the hypothesis that simultaneously inhibiting multiple cytoprotective pathways, including mTOR, PI3K and/or MEK1/2 signaling, is required to facilitate lethality of adenoviral mda-7/IL-24 delivery toward RCCs both in vitro and in vivo. In addition, we demonstrate in vitro and in vivo that the FDA approved RCC therapeutic multikinase inhibitor sorafenib, which inactivates signaling pathways and promotes ER stress signaling, enhances mda-7/IL-24 toxicity. Since delivery of therapeutic genes using Ad.5 has shown significant limitations in treating RCC, because of the need for Coxsackie adenovirus receptors (CAR) on tumor cells for virus entry, we evaluated the use of a tropism-modified adenovirus that uses a serotype 3 modification of the viral knob protein to facilitate delivery of mda-7/IL-24 in a CAR-independent manner.1013,21,29,33,34 This approach profoundly augmented therapeutic activity of MDA-7/IL-24 in RCCs, in vitro and in vivo, suggesting that combining both strategies, namely inhibition of cytoprotective pathways using sorafenib and virus tropism modification, may provide a means of developing an improved therapy to translate into the clinic for kidney cancer.1013,21,29

Results

RCCs are resistant to infection by serotype 5 adenoviruses due to lack of coxsakie and adenovirus receptor (CAR) expression.10,24,29,31 To circumvent this problem, we developed a chimeric serotype 5/serotype 3 modified knob adenovirus to deliver mda-7/IL-24 to RCCs and infect these cells in a CARi-ndependent fashion: Ad.5/3-mda-7.11,21,34,35 At low multiplicities of infection, Ad.5/3-mda-7 but not Ad.5-mda-7 infected RCCs and promoted expression of MDA-7/IL-24 (Fig. 1A). These findings were also reflected in colony formation assay data (Fig. 1B).

Figure 1A DFigure 1A D
Infection of RCCs with Ad.5/3-mda-7 causes MDA-7/IL-24 secretion into the growth media that has a potent toxic “bystander” effect via CD95 signaling. (A) A498 or UOK121LN cells were cultured for 24 h and then infected with increasing multiplicities ...

MDA-7/IL-24 is a novel cancer-specific cytotoxic cytokine that can be secreted from cells infected with a recombinant adenovirus containing the mda-7/IL-24 gene.912,37 We next determined whether direct infection of RCCs with Ad.5/3-mda-7 also permitted MDA-7/IL-24 to be secreted into the culture media and whether this also had a lethal “bystander” effect on uninfected RCCs. Infection of UOK121LN cells with Ad.5/3-mda-7, but not Ad.5-mda-7, increased expression of MDA-7/IL-24 in the culture media of cells (Fig. 1C, upper immunoblots). Transfer of the conditioned media containing MDA-7/IL-24, but not “spent” media from vector infected cells, onto uninfected UOK121LN cells increased expression of MDA-7/IL-24. It has been argued that MDA-7/IL-24 can set up a self-stimulatory paracrine loop in tumor cells and in agreement with data in other tumor types, knockdown of MDA-7/IL-24 expression in uninfected cells blocked the conditioned media containing the cytokine from inducing MDA-7/IL-24 expression (Fig. 1C, lower immunoblots).38 The lethal effects of Ad.5/3-mda-7, in infected cells were blocked by knockdown of CD95 expression (Fig. 1D). Transfer of media containing MDA-7/IL-24 onto uninfected RCCs promoted cell killing, an effect that was also blocked by knockdown of CD95 or knockdown of MDA-7/IL-24.

Infection of primary renal epithelial cells with Ad.5-mda-7 resulted in expression of MDA-7/IL-24 without causing significant toxicity to these non-transformed cells (Fig. 1E and data not shown). Forty-eight hour after infection, conditioned media from Ad.5-cmv, Ad.5-mda-7 and Ad.5-mda-7 SP- (lacking the signal peptide for cytokine secretion)-infected primary renal epithelial cells was removed and placed onto uninfected UOK121LN carcinoma cells. As a single agent, conditioned media containing from Ad.5-mda-7-infected cells, but not Ad.5-cmv or Ad.5-mda-7 SP-infected cells, suppressed the growth of RCCs as measured in an MTT assay by 38 ± 4% (Fig. 1E). In a dose-dependent fashion conditioned media containing MDA-7/IL-24 promoted cell killing in short-term true viability assays (Fig. 1F).

Previously we have noted that inactivation of ERK1/2 and AKT and activation of JNK1/2 and p38 MAPK were hallmarks of MDA-7/IL-24 toxicity in renal carcinoma cells.21 In A498 cells molecular inhibition of PI3K, mTOR or MEK1/2 suppressed basal levels of phospho-ERK1/2 and of phospho-(S473)-AKT and in cells infected with Ad.5/3-mda-7 the dephosphorylation of ERK1/2 and of AKT was enhanced wherein kinase activity was largely abolished (Fig. 2A). Knock down of mTOR reduced total and phospho-mTOR levels and MDA-7/IL-24 expression also reduced phospho-mTOR levels, and the combinations of Ad.5/3-mda-7 and molecular inhibition of PI3K, mTOR or MEK1/2 further reduced mTOR phosphorylation.

Figure 2
Ad.5/3-mda-7 lethality is enhanced by combined molecular inhibition of PI3K/MEK/mTOR pathways in A498 cells. (A) A498 cells were infected with empty vector control virus (Ad.5/3-cmv) or with viruses expressing MDA-7/IL-24 (Ad.5/3-mda-7); and in parallel ...

Expression of MDA-7/IL-24 enhanced JNK1/2 phosphorylation that was further increased by combined inhibition of signaling by: PI3K + MEK1/2; mTOR + MEK1/2; PI3K + mTOR; or PI3K + MEK1/2 + mTOR (Fig. 2A). This correlated with increased expression of the toxic BH3 domain protein BAX and decreased expression of MCL–1. Prior studies from our group demonstrated that MDA-7/IL-24-induced PKR-like endoplasmic reticulum kinase (PERK) signaling was essential for cytokine lethality.21,27 MDA-7/IL-24 enhanced PERK and eIF2α phosphorylation that was strongly enhanced by combined inhibition of PI3K + MEK1/2; mTOR + MEK1/2; PI3K + mTOR; or PI3K + MEK1/2 + mTOR. These findings correlated with decreased expression of the PERK chaperone BiP/GRP78.

We next determined the impact of inhibiting signaling pathways on Ad.5/3-mda-7 toxicity in A498 RCCs. Molecular inhibition of mTOR; PI3K; MEK1/2; PI3K + MEK1/2; mTOR + MEK1/2; PI3K + mTOR; or PI3K + MEK1/2 + mTOR caused modest increases in the basal level of apoptosis in Ad.5/3-cmv infected cells (Fig. 2B). Combined inhibition of PI3K + MEK1/2 or PI3K + mTOR modestly enhanced MDA-7/IL-24 toxicity whereas combined PI3K + MEK1/2 + mTOR inhibition strongly enhanced cell killing. The short-term viability data was also reflected in assays determining total cell numbers (Fig. 2C). However, in long-term colony formation assays only combined PI3K + MEK1/2 + mTOR inhibition strongly enhanced MDA-7/IL-24 toxicity (Fig. 2D). In UOK121LN RCCs only combined molecular inhibition of PI3K + MEK1/2 + mTOR signaling significantly enhanced MDA-7/IL-24 toxicity and suppression of growth in short term assays whereas molecular inhibition of any individual signaling pathway promoted MDA-7/IL-24 toxicity in long-term colony formation (Fig. 3A–C).

Figure 3
Ad.5/3-mda-7 lethality is enhanced by combined molecular inhibition of PI3K/MEK/mTOR pathways in UOK121LN cells. (A and B) UOK121LN cells were infected with empty vector control virus (Ad.5/3-cmv) or with viruses expressing MDA-7/IL-24 (Ad.5/3-mda-7); ...

Multiple drugs have been developed for clinical application that are designed to inhibit the PI3K/MEK1/2/mTOR signaling pathways and the multi-kinase inhibitor sorafenib which suppresses ERK1/2 pathway activity as well as inhibiting receptor tyrosine kinases is an FDA approved therapeutic for RCC. We next examined the impact of the PI3K inhibitor PX-866; the MEK1/2 inhibitor PD184352; and the mTOR inhibitors rapamycin and PI-103 on MDA-7/IL-24 lethality in RCCs. Use of kinase inhibitor drugs, as compared to transfecting plasmids to express dominant negative inhibitory proteins, resulted in a greater suppression of ERK1/2 and AKT signaling under basal conditions and promoted a greater level of PERK/eIF2α and JNK1/2 activation (Fig. 4A).37 Inhibition of MEK1/2 and PI3K significantly enhanced MDA-7/IL-24 lethality in RCCs, compared to inhibition of MEK1/2 and mTOR (Fig. 4B and C). Combined inhibition of mTOR, PI3K and MEK1/2 enhanced MDA-7/IL-24 toxicity to a greater extent than inhibition of MEK1/2 and PI3K (Fig. 4D). Sorafenib enhanced MDA-7/IL-24 toxicity (Fig. 4E).

Figure 4
Inhibition of mTOR, PI3K and MEK1/2 signaling using clinically relevant small molecule kinase inhibitors enhances Ad.5/3-mda-7 lethality. (A) A498 cells were infected with empty adenovirus vector (Ad.5/3-cmv) or an adenovirus vector expressing MDA-7/IL-24 ...

In general agreement with data in Figure 4B and C; Figure 4E, in colony formation assays combined inhibition of mTOR, PI3K and MEK1/2 or sorafenib treatment enhanced MDA-7/IL-24 toxicity to a greater extent than inhibition of MEK1/2 and PI3K or of mTOR (Fig. 4F). Using purified GST-MDA-7, we noted that sorafenib synergized in median dose effect colony formation assays with the cytokine to kill RCCs with combination index (CI) values of less than 1.00 (Table 1).

Table 1
GST-MDA-7 and sorafenib synergize to kill RCCs

Based on our data in Figures 24 we next determined the signaling and apoptosis pathways that were being modulated in RCCs. Expression of activated AKT and activated MEK1 significantly protected cells from inhibition of mTOR, PI3K and MEK1/2 to a similar extent as expression of activated AKT, activated MEK1 and activated mTOR (Fig. 5A, p < 0.05). Inhibition of JNK1/2 signaling blunted MDA-7/IL-24 lethality and it suppressed the elevation of MDA-7/IL-24 toxicity by combined inhibition of mTOR, PI3K and MEK1/2.37

Figure 5
Inhibition of signaling pathways enhances Ad.5-mda-7-induced CD95 activation and facilitates killing independently of CD95 activation. (A) A498 cells were infected with Ad.5/3-cmv or Ad.5/3-mda-7 and in parallel transfected with plasmids to express activated ...

Prior studies in RCCs have shown that MDA-7/IL-24 kills primarily via activation of CD95 (the extrinsic pathway), and our present studies also demonstrated that secreted MDA-7/IL-24 killed bystander RCCs in a CD95 dependent fashion (Fig. 1D). The ability of MEK1/2 and PI3K inhibition to enhance MDA-7/IL-24 toxicity was suppressed by ~50% when extrinsic apoptosis pathway signaling was blocked (Fig. 5B). However, when signaling via the intrinsic pathway was blocked, MDA-7/IL-24 toxicity was abolished. As MDA-7/IL-24 activates CD95 in RCCs we determined whether modulation of signaling pathway functions altered the levels of MDA-7/IL-24-induced CD95 activation. Use of molecular tools modestly enhanced MDA-7/IL-24-induced CD95 surface localization and DISC formation whereas use of small molecule kinase inhibitors, that inhibited the signaling pathways to a greater extent, caused a much larger enhancement of CD95 surface localization, that in the case of PX-866 + PD184352 treatment or sorafenib treatment, was greater than additive (Fig. 5C). These findings using kinase inhibitors correlated with increased association of pro-caspase 8 and FADD with CD95 (Fig. 5C, upper blots).

Based on the data in Figure 1, we wished to determine whether the in vitro toxic “bystander” effect of secreted MDA-7/IL-24 on uninfected tumor cells could be translated into an in vivo model system. A498 RCC tumors were established on two flanks of an athymic mouse. One tumor was injected every second day with Ad.5/3-cmv or with Ad.5/3-mda-7 (total two injections) and the volumes of the tumors on both sides of the animal measured using calipers. Infection of tumors with Ad.5/3-mda-7, but not Ad.5/3-cmv, caused a rapid suppression of the tumor growth rate and significantly increased animal survival (Fig. 6A and B (p < 0.05)). Infection of animals with Ad.5/3-mda-7, but not Ad.5/3-cmv, also suppressed the growth rate of uninfected tumors growing on the opposite flank, as previously observed in the context of prostate tumors implanted on opposite flanks of nude mice.34 This correlated with caspase 3 cleavage; MDA-7/IL-24 expression and increased apoptosis in both the Ad.5/3-mda-7-infected and uninfected “bystander” RCC tumors (Fig. 6C and D). Of particular note, Ad.5/3-cmv virus infection promoted recruitment of CD11c and CD11b positive immune cells (dendritic and natural killer cells) into tumors, as judged by CD11c and CD11b staining in Ad.5/3-cmv infected tumors but not in the contra-lateral uninfected “bystander” tumor. In contrast to empty vector virus, Ad.5/3-mda-7 infection resulted in CD11c and CD11b staining in both the infected tumor and in the contra-lateral uninfected bystander tumor. Together with our in vitro data, these findings argue that MDA-7/IL-24 secreted from a renal carcinoma tumor in vivo has antitumor activity on an uninfected distant RCC tumor.

Figure 6Figure 6
Ad.5/3-mda-7 infection of RCC tumors in vivo exhibits a toxic bystander effect on uninfected RCC tumors. (A) A498 cells were injected into the rear right and left flanks of athymic mice. Tumors of ~150 mm3 grew over the following 29 days. Animals ...

Finally, based on our in vitro studies we determined whether the in vivo RCC antitumor effects of MDA-7/IL-24 could be enhanced by the clinically relevant RCC therapeutic sorafenib.38,39 Pre-formed A498 tumors (~150 mm3) were injected with either Ad.5/3-cmv or Ad.5/3-mda-7. A second adenoviral infusion was performed 48 h after the first infection. Twenty-four hour after the first viral infection animals were treated with vehicle or sorafenib (20 mg/kg) for the following 4 days. Infection with Ad.5/3-mda-7 or treatment with sorafenib reduced the rate of tumor growth (Fig. 7A). However, combined exposure to Ad.5/3-mda-7 and sorafenib promoted a significantly greater suppression of tumor growth than either agent individually. Based on animal welfare guidelines, animals carrying tumors > 2.0 cm3 in volume are humanely sacrificed; treatment of animals with empty vector virus and sorafenib modestly increased animal survival whereas treatment with Ad.5/3-mda-7 significantly increased survival (Fig. 7B). Combined treatment of animals with sorafenib and Ad.5/3-mda-7 enhanced animal survival beyond that caused by administration of Ad.5/3-mda-7 alone (p < 0.05). In isolated tumors it was noted that cleavage of pro-caspase 3 and TUNEL positivity was increased by Ad.5/3-mda-7 + sorafenib treatment and decreased Ki67 reactivity (Fig. 7C and D). Infection with Ad.5/3-mda-7 also promoted recruitment of CD11b and CD11c positive immune cells into the tumor; immune cells including monocytes, macrophages/dendritic cells, neutrophils, natural killer cells and B cells. In addition we found that: (a) Ad.5/3-mda-7 and sorafenib did not interact to kill primary human renal epithelial cells in vitro and; (b) no obvious normal tissue toxicity by H&E staining was noted in to be induced in either the kidneys or livers of animals exposed to Ad.5/3-MDA-7 and sorafenib (data not shown). Collectively, our data argue that MDA-7/IL-24 augments the antitumor effects of sorafenib against established renal carcinoma tumors.

Figure 7Figure 7
Sorafenib enhances the lethal effects of Ad.5/3-mda-7 in vivo. (A) A498 tumors formed over 29 days in the flank of athymic mice (~150 mm3). Animals were separated into tumor volumes of approximate equivalent mean tumor size and standard error. ...

Discussion

The present studies have focused on developing enhanced therapies for renal carcinoma, a tumor that frequently displays a rapid progression beyond the kidney and with no treatment providing long-term clinical benefit for metastatic disease. To achieve this objective we utilized mda-7/IL-24, which has demonstrated tumor cell-specific killing in a wide variety of tumor cell types.1013,14 Based on previous observations that specific signaling pathways might provide protection from mda-7/IL-24-induced toxicity, we have now explored combinatorial approaches targeting multiple specific cytoprotective signaling pathways in combination with mda-7/Il-24 on human RCCs both in vitro and in vivo. The studies in this manuscript highlight combinations of clinically relevant protein and lipid kinase inhibitors that increase MDA-7/IL-24 toxicity and we also identify a means of enhancing the therapeutic delivery of MDA-7/IL-24 for RCC using a tropism-modified Ad.5/3.

Serotype 5 human adenoviruses bind to and infect, human cells through the Coxsackie and adenovirus receptor (CAR); a protein whose expression has been shown in several studies to be significantly reduced in primary human RCCs in culture and in situ (reviewed in ref. 1013, 21 and 22). Thus, delivery of therapeutic genes by this method to RCCs was hampered by the relative inefficiency of this gene delivery method. Multiple laboratories are attempting to solve the relative inefficiency of serotype 5 adenovirus infections in RCCs and other malignancies by modifying specific sequences within virus capsid proteins that directly associate with CAR, i.e., they are applying targeting strategies to enhance viral infectivity via CAR-independent pathways. Initial studies from several groups modified the infective viral capsid “knob” to bind instead to surface integrin proteins whose expression is enhanced upon transformation (RGD modification) or by insertion into the knob of multiple lysine residues (pK7) which will increase viral interaction with cells by electrostatic effects on the cell's surface.

Subsequently, more subtle modifications to the proteins expressed as part of the viral capsid knob have been made with the development of viruses expressing chimeric knobs containing components of different serotype viruses. For example, in some of these viruses, the infective capsid knobs from serotype 3 adenoviruses were incorporated into the adenovirus type 5 knob and our present studies have demonstrated that modified serotype 5/3 knob adenoviruses are able to achieve enhanced gene transduction into low-CAR containing RCCs in vitro and in vivo.33,34

Infection of RCCs with a dose of Ad.5/3-mda-7 virus particles that caused modest levels of toxicity ~48 h after exposure correlated with activation of the JNK1/2 and p38 MAPK pathways as well as PERK. This treatment, in parallel, suppressed ERK1/2, AKT, mTOR and p70 S6K signaling. Multiple studies using a number of toxic stimuli document that prolonged JNK1-3 and/or p38 MAPK activation in a wide variety of cell types can trigger cell death.3537 It is also well established that the balance between the readouts of ERK1/2 and JNK1-3 signaling can represent a general key homeostatic mechanism that regulates cell survival versus cell death processes.3537 Prior in vitro studies using RCCs treated with GST-MDA-7 have demonstrated that JNK1-3 and p38 MAPK signaling represent key pro-apoptotic signals generated by mda-7/IL-24 exposure. Inhibition of MEK1/2 and PI3K enhanced MDA-7/IL-24-induced JNK1-3 activation that was causal in promoting cell killing, but combined inhibition of MEK1/2 and PI3K and mTOR did not lead to a further activation of the JNK pathway yet caused enhanced tumor cell killing. Inhibition of the JNK pathway in vitro, however, suppressed the toxicity of MEK1/2 and PI3K and mTOR inhibition when combined with Ad.5/3-mda-7.

As previously noted using bacterially synthesized GST-MDA-7 protein, infection of RCCs with Ad.5/3-mda-7 caused activation of the CD95 death receptor that played a key role in MDA-7/IL-24 lethality. Inhibition of PI3K + MEK1/2 or use of the multi-kinase inhibitor sorafenib enhanced Ad.5/3-mda-7 lethality that correlated with increased activation of CD95 and decreased levels of MCL-1 and c-FLIP-s. Overexpression of c-FLIP-s reduced Ad.5/3-mda-7 lethality by >80% but only suppressed the enhanced killing of Ad.5/3-mda-7 cells treated with PI3K + MEK1/2 inhibitors by ~40%. Overexpression of BCL-XL or dominant negative caspase 9 blocked not only Ad.5/3-mda-7 toxicity but also the potentiation of Ad.5/3-mda-7 toxicity by PI3K + MEK1/2 inhibitors. Thus, although PI3K + MEK1/2 inhibitors did increase MDA-7/IL-24-induced CD95 activation our data argues that the primary site of PI3K + MEK1/2 inhibitor action is by promoting further mitochondrial dysfunction due to the loss of MCL-1 and BCL-XL expression.

Activation of PERK by MDA-7/IL-24 has been linked to increased tumor cell killing and in RCCs we noted that the majority of PERK activation caused by GST-MDA-7 treatment was dependent on CD95-induced activation of PERK rather than GST-MDA-7-induced inactivation of BiP/GRP78 with the subsequent activation of the BiP/GRP78 chaperone PERK.21 Expression of MDA-7/IL-24 increased BiP/GRP78 levels, an effect that was suppressed by inhibition of multiple protective signaling pathways and the loss of BiP/GRP78 induction correlated with increased activation of PERK and eIF2α and with enhanced tumor cell killing. Overexpression of BiP/GRP78 has been linked in several systems to chemotherapy resistance and to tumor promotion.

Sorafenib (Bay 43-9006, Nexavar®; a Raf family kinase inhibitor) is a multi-kinase inhibitor that was originally developed as an inhibitor of Raf-1, a component of the ERK1/2-MEK1/2 pathway, but which was subsequently shown to inhibit multiple other kinases, including class III tyrosine kinase receptors such as platelet-derived growth factor, vascular endothelial growth factor receptors 1 and 2, c-Kit and Flt3.38,39 Antitumor effects of sorafenib in RCC have been ascribed to anti-angiogenic actions of this agent through inhibition of pro-angiogenic growth factor receptors.40,41 Several groups, including ours, have shown in vitro that sorafenib kills human tumor cells at concentrations well below the maximum achievable dose (Cmax) of 15–20 µM, through a mechanism involving downregulation of the antiapoptotic BCL-2 family member MCL-1; MCL-1 has a short half-life of ~30 min. In these studies sorafenib-mediated MCL-1 downregulation occurred through a translational rather than a transcriptional or post-translational process that was mediated by endoplasmic reticulum (ER) stress signaling.36 This effect is almost identical to the mechanism of action by which MDA-7/IL-24 suppresses MCL-1 levels.42 Sorafenib enhanced Ad.5/3-mda-7 lethality and enhanced CD95 activation. Sorafenib and expression of MDA-7/IL-24 combined to abolish MCL-1 and BCL-XL expression. As sorafenib is an FDA-approved therapeutic modality for renal carcinoma patients we determined whether the drug could promote MDA-7/IL-24-induced antitumor effects in vivo. Treatment of established RCC tumors with either sorafenib or Ad.5/3-mda-7 suppressed RCC tumor growth in vivo. Combined exposure to MDA-7/IL-24 and sorafenib caused a prolonged suppression of RCC tumor growth and promoted recruitment of immune cells into the tumor.

In conclusion, the present studies demonstrate that clinically relevant protein and lipid kinase inhibitors enhance the toxicity of MDA-7/IL-24 in vitro and in vivo. Further studies will be required to determine whether MDA-7/IL-24 (Ad.5/3-mda-7) is able to enhance the tumor controlling effects of sorafenib in patients with metastatic renal carcinoma. Based on the poor prognosis of patients with advanced renal cancer, defining novel and effective therapeutic approaches is clearly a priority. Considering the safety of sorafenib and mda-7/IL-24 as single agents, future studies of this combination could prove of immense value in treating RCC patients whose disease has progressed beyond the renal capsule.11,17,18,43,44

Materials and Methods

Materials.

Phospho-/total-ERK1/2, Phospho-/total-JNK1-3, Phospho (S473)-/total-AKT, Phospho-/total-p38 MAPK, Phospho-/total mTOR, Phospho-/total p70 S6K and P-PERK antibodies were purchased from both Cell Signaling Technologies (Worcester, MA) and from Santa Cruz Biotechnology (Santa Cruz, CA). Sorafenib tosylate was purchased from Eton Bioscience Inc., (San Diego, CA). The plasmid to express mTOR was from Addgene. The JNK inhibitor peptide (JNK IP), PI-103 and PX-866 were supplied by Calbiochem (San Diego, CA) as a powder, dissolved in sterile DMSO and stored frozen under light-protected conditions at −80°C. A498 cells were from the ATCC (Bethesda, MD). UOK121LN cells were supplied by Dr. W.M. Lineham (NIH, Bethesda, MD). A498 cells are re-purchased every 6–9 months. No other cell line authentication was performed by the authors. Trypsin-EDTA, DMEM and RPMI medium and penicillin-streptomycin were purchased from GIBCOBRL (GIBCOBRL Life Technologies, Grand Island, NY). Other reagents were of the highest quality commercially available.1013,21,27,28,35,36

Methods.

Generation of Ad.5-mda-7 or Ad.5/3-mda-7. Recombinant type 5 and 5/3 adenoviruses to express MDA-7 (Ad.5-mda-7 and Ad.5/3-mda-7), control (Ad.cmv empty vector) were generated as described in reviewed in ref. 1013, 16, 27, 28 and 3336.

Cell culture and in vitro infection/exposure of cells to Ad.mda-7 and drugs. All cell lines were cultured at 37°C (5% (v/v CO2)) in vitro using RPMI supplemented with 5% (v/v) fetal calf serum and 10% (v/v) Non-essential amino acids. For short-term cell killing assays and immunoblotting, cells were plated at a density of 3 × 103 per cm2 and 36 h after plating were treated with MDA-7/IL-24 and/or various drugs, as indicated. In vitro small molecule inhibitor treatments were from a 100 mM stock solution of each drug and the maximal concentration of Vehicle (DMSO) in media was 0.02% (v/v). For Ad infection, cells were infected 12 h after plating and the expression of the recombinant viral transgene allowed to occur for 24 h prior to any additional experimental procedure. Cells were not cultured in reduced serum media during any study.1013,27,28

Cell treatments, SDS-PAGE and western blot analysis. Cells were treated with various drug concentrations and viral multiplicities of infection, as indicated in the Figure legends. For SDS PAGE and immunoblotting, cells were lysed in either a nondenaturing lysis buffer and prepared for immunoprecipitation as described in reference 36 or in whole-cell lysis buffer (0.5 M Tris- HCl, pH 6.8, 2% SDS, 10% glycerol, 1% β-mercaptoethanol, 0.02% bromophenol blue), and the samples were boiled for 30 min. After immunoprecipitation, samples were boiled in whole cell lysis buffer. The boiled samples were loaded onto 10–14% SDS-PAGE and electrophoresis was run overnight. Proteins were electrophoretically transferred onto 0.22 µm nitrocellulose and immunoblotted with indicated primary antibodies against the different proteins.1013,27,28

Recombinant adenoviral vectors; infection in vitro. We generated and purchased previously noted recombinant adenoviruses to express constitutively activated and dominant negative (dn) AKT and MEK1 proteins, dn caspase 9, XIAP, c-FLIP-s, CRM A and BCL-XL (Vector Biolabs, Philadelphia, PA). Note: UOK121LN cells were infected with these adenoviruses at an approximate m.o.i. of 400. A498 cells, even at 400 m.o.i. were not infected by serotype 5 adenovirus. Cells were incubated for 24 h to ensure adequate expression of transduced gene products prior to drug exposures.1013,27,28

Detection of cell death by trypan blue, hoechst, TUNEL and flow cytometric assays. Cell death assays were carried out as described in references 1013, 21 and 2536.

Plasmid transfection. Plasmid DNA (0.5 µg/total plasmid transfected) was diluted into 50 µl of RPMI growth media that lacked supplementation with FBS or with penicillin-streptomycin. Lipofectamine 2000 reagent (1 µl) (Invitrogen, Carlsbad, CA) was diluted into 50 µl growth media that lacked supplementation with FBS or with penicillin-streptomycin. The two solutions were then mixed together and incubated at room temperature for 30 min. The total mix was added to each well (4-well glass slide or 12-well plate) containing 200 µl growth media that lacked supplementation with FBS or with penicillinstreptomycin. The cells were incubated for 4 h at 37°C, after which time the media was replaced with RPMI growth media containing 5% (v/v) FBS and 1X pen-strep.

Flank inoculation of A498 cells.

Athymic female NCr-nu/nu mice (NCI-Fredrick) weighing ~20 g, were used for this study. Mice were maintained under pathogen-free conditions in facilities approved by the American Association for Accreditation of Laboratory Animal Care and in accordance with current regulations and standards of the US Department of Agriculture, Washington, DC, the US Department of Health and Human Services, Washington, DC, and the National Institutes of Health, Bethesda, MD. A498 cells were cultured in RPMI supplemented with 5% (v/v) fetal calf serum and 100 µg/ml (1% v/v) penicillinstreptomycin. Cells were incubated in a humidified atmosphere of 5% (v/v) CO2 at 37°C. Rear flank injection of 5 × 106 A498 cells 10 µl of medium was performed over 10 min. Tumors (~150 mm3) were permitted to form over the following month. Adenoviral vectors, Ad.5/3-mda-7 or Ad.5/3-cmv were administered ~1 month after tumor cell implantation into animals. Viral vectors (Ad.5/3-mda-7 or Ad.cmv, 1 × 109 p.f.u.) suspended in 2 µl of PBS were delivered by slow infusion over a 6 min period.

Immunohistochemistry and staining of fixed tumor sections. Postsacrifice, tumors were fixed in OCT compound (Tissue Tek); cryostat sectioned (Leica) as 12 µm sections. Nonspecific binding was blocked with a 2% (v/v) Rat Sera, 1% (v/v)m Bovine Sera, 0.1% (v/v) Triton X100, 0.05% (v/v) Tween-20 solution then sections were stained for cell signaling pathway markers: Animals that received recombinant adenoviruses were monitored twice weekly for survival or signs of disease. Other animals were sacrificed 14–140 days as indicated after adenovirus injection. Post-sacrifice, mouse tumors were fixed in OCT compound (Tissue Tek); cryostat sectioned (Leica) as 12 µm sagittal sections. Nonspecific binding was blocked with a 2% (v/v) Rat Sera, 1% (v/v) Bovine Sera, 0.1% (v/v) Triton X100, 0.05% (v/v) Tween-20 solution then sections were stained for cell signaling pathway markers: MDA-7/IL-24; Ki67; CD11b; CD11c; cleaved caspase 3. Staining was performed as described in references 1013, 21, 26 and 36.

Data analysis. Comparison of the effects of various treatments was performed using one-way analysis of variance and a two-tailed Student's t-test. Differences with a p value of <0.05 were considered statistically significant. Statistical examination of in vivo animal survival data utilized log rank statistical analyses between the different treatment groups. Experiments shown are the means of multiple individual points from multiple experiments (±SEM).

Acknowledgements

Support for the present study was provided: to P.D. from PHS grants (P01-CA104177, R01-CA108520, R01-DK52825; R01-CA141703; R01-CA150214), The Jim Valvano “V” foundation and Department of Defense Award (W81XWH-10-1-0009); to P.B.F. from PHS grants (P01-CA104177, R01-CA097318; R01-CA127641; R01-CA134721; R01-CA108520), the Samuel Waxman Cancer Research Foundation (SWCRF) and the National Foundation for Cancer Research (NFCR); to D.T.C. from PHS grant (P01-CA104177). P.D. is The Universal Inc., Professor in Signal Transduction Research. PBF holds the Thelma Newmeyer Corman Chair in Cancer Research at the VCU Massey Cancer Center, VCU, School of Medicine and is a SWCRF Investigator.

Abbreviations

ERK
extracellular regulated kinase
MEK
mitogen activated extracellular regulated kinase
JNK
c-Jun NH2-terminal kinase
PI3K
phosphatidyl inositol 3 kinase
MDA-7/IL-24
melanoma differentiation associated gene-7/interleukin-24
PERK
protein kinase R-like endoplasmic reticulum kinase
MAPK
mitogen activated protein kinase
ca
constitutively active
dn
dominant negative
EGFR
epidermal growth factor receptor
IL
interleukin
PTEN
phosphatase and tensin homolog on chromosome ten
RCC
renal cell carcinoma

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