Scholarly article on topic '/IL-24: Exploiting Cancer's Achilles' Heel'

/IL-24: Exploiting Cancer's Achilles' Heel Academic research paper on "Biological sciences"

CC BY-NC-ND
0
0
Share paper
Academic journal
Mol Ther
OECD Field of science
Keywords
{""}

Academic research paper on topic "/IL-24: Exploiting Cancer's Achilles' Heel"

mda-7/IL-24: Exploiting Cancer's Achilles' Heel

Irina V. Lebedeva,1 Moira Sauane,1 Rahul V. Gopalkrishnan,1 Devanand Sarkar,1 Zhao-zhong Su,1 PankajGupta,1 John Nemunaitis,2 Casey Cunningham,2 Adly Yacoub,3 Paul Dent,3 Paul B. Fisher,1'4'5'*

1Department of Pathology, 4Department of Urology, and 5Department of Neurosurgery, Herbert Irving Comprehensive Cancer Center, College of Physicians and Surgeons, Columbia University Medical Center, 630 West 168th Street, New York, NY 10032, USA 2US Oncology, Inc., Mary Crowley Medical Research Center, Dallas, TX 75246, USA 3Department of Radiation Oncology, Virginia Commonwealth University, 401 College Street, Richmond, VA 23298, USA

*To whom correspondence and reprint requests should be addressed. Department of Pathology, Herbert Irving Comprehensive Cancer Center, College of Physicians and Surgeons, Columbia University Medical Center, 630 West 168th Street, New York, NY 10032.

Fax: (212) 305 8177. E-mail: pbf1@columbia.edu.

Available online 28 October 2004

The mda-7/IL-24 cDNA was isolated almost a decade ago in a screen for genes differentially upregulated following growth arrest and terminal differentiation of a human melanoma cell line employed as an in vitro cell differentiation model. The underlying rationale for the screen was that oncogenesis arises from a cellular dedifferentiation process culminating in uncontrolled proliferation and acquisition of invasive and metastatic potential. Identification of genes upregulated during the process of reactivation of faulty or inoperational differentiation maintenance programs was postulated to have cancer gene therapeutic potential. In this context, it is heartening to note that mda-7/IL-24 has made a methodical and progressive journey, from an unidentified novel sequence with little homology to known genes at its time of isolation to currently having the status of a molecule belonging to the IL-10-related family of cytokines, with considerable cancer gene therapeutic potential. Extensive in vitro and in vivo human tumor xenograft studies have established its transformed cell apoptosis-inducing capacity in various model systems. It has recently taken an important step for a candidate cancer gene therapeutic molecule, in the ultimate goal of benchtop to clinic, by being currently utilized in human Phase I/II clinical trials. This review provides a current perspective of our understanding of mda-7/IL-24, including established and more recent information about the molecular properties, specificity of anti-tumor-cell apoptosis-inducing activity, and underlying mechanisms of this action relative to its cancer gene therapeutic potential.

Contents

Introduction........................................................................................................................4

Cloning and Characterization....................................................................................................5

Molecular Pathways Involved in mda-7/IL-24 Activity......................................................................6

Diverse Mechanisms Underlying Apoptosis-Inducing Properties of the mda-7/IL24 Gene..............................8

Radiosensitizing Properties of mda-7/IL-24: Implications in Cancer Gene Therapy....................................10

Newer Information on Cancer Cell-Specific Apoptosis Induction, Including an Intracellular Role

of Action for mda-7/IL-24..................................................................................................11

Phase I Clinical Studies Utilizing Ad.mda-7 (INGN-241)..................................................................14

Concluding Perspective on mda-7/IL-24: a Gene with a Potentially Bright Future....................................16

Acknowledgments................................................................................................................16

References........................................................................................................................16

Introduction

required to trigger JAK/STAT signaling after cognate Recent papers by Sauane et al. [1] and Sieger et al. [2] receptor binding is not an obligatory event in its ability indicate that extracellular secretion of mda-7/IL-24 to induce apoptosis in transformed cells, but rather,

intracellular localization to the endoplasmic reticulum (ER) is necessary and sufficient. Earlier work had provided important lead evidence, indicating that this apoptosis-inducing activity occurred even in contexts in which JAK/STAT signaling had been blocked by pharmacological means or by mutation or in situations in which functional cognate receptors were not expressed by susceptible cells [3]. This unprecedented mode of activity of mda-7/IL-24, in the context of its belonging to and displaying all the characteristics of a secreted cytokine, serves to highlight the fact that despite concerted efforts of several independent groups, the mechanistic basis of its anti-tumor cell activity approximates an enigma wrapped in a mystery.

This is not to diminish credit from a large number of published and ongoing studies that are steadily chipping away and have peeled back some layers of the complexity surrounding mda-7/IL-24's novel and therapeutically relevant activities (reviewed in [4-9]). What is now evident is that a diverse array of proapoptotic signals and pathways are triggered in cell types that may be functionally defined as "transformed" or "oncogenic," while "immortalized-normal" counterparts or "normal" low-passage primary cells remain unaffected after exposure to the mda-7/IL24 gene product (reviewed in [5-9]). These studies were extended considerably with the successful completion of a Phase I clinical trial [6,1012] and positive results generated so far in an ongoing Phase I/II clinical trial, indicating safety and potential anti-tumor efficacy of this gene when delivered via a nonreplicating adenovirus vector [13]. What has displayed a remarkable pattern of consistency is that mda-7/IL-24 is able to induce transformed cell-specific apop-tosis in almost every type of cancer cell line so far tested (reviewed in [5,6,8]). There has been only one reported instance of a cancer cell-related mutation that confers resistance to mda-7/IL-24-induced killing and that too in only one specific cancer type, pancreatic carcinoma [14]. Tumor suppressor mutation status such as that of p53 and Rb has no influence on the proapoptotic outcome following mda-7/IL-24 treatment of cells, it being equally active in either mutant or wild-type contexts [15-18]. Combined with a potent bystander effect [1,14] and ability to radiosensitize cells [18-22], tumor suppressor-status independence gives mda-7/IL-24 a potent edge over existing gene-based anti-cancer therapies. This multiplicity of features is a mixed blessing when it comes to elucidating the mechanistic basis of tumor cell specificity, since this endeavor becomes vastly more complicated due to lack of defining boundaries.

Subsets of genes triggered during mda-7/IL-24-induced apoptosis or, conversely, a specific gene or pharmacological agent that offers protection from killing varies from cancer to cancer as well as between cell lines derived from the same type of cancer [2,3,17,23-25]. Therefore, a common mode of action or cancer cell-specific apoptosis

trigger or some universal principle underlying transformed cell apoptosis-inducing specificity of mda-7/IL-24 has yet to emerge. Moreover, it is now readily apparent from the accumulating body of information cataloging activity is an awareness of an increasingly diverse set of mechanisms involved in mda-7/IL-24's mode of action (reviewed in [5,6,8,9,26]). What can be said with some certainty is that each study has led to increasing confidence of the feasibility of utilizing mda-7/IL-24 as a cancer gene therapeutic. We have attempted here to assess the many divergent aspects of this molecule in the cancer gene therapy context.

Cloning and Characterization

The isolation of mda-7/IL24 and its steady development to the level of a viable cancer gene therapeutic molecule can be viewed as a practical validation of the concept of "differentiation therapy," which forms the theoretical basis of the original screen [27-29]. HO-1 human metastatic melanoma cells [30,31] were induced to differentiate in vitro, resulting in growth arrest, increased melanogenesis, and morphological changes accompanied by induction and differential expression of numerous genes likely related to this phenomenon, a full list of which has not yet been compiled since several rounds of differential screening have been performed using this model [27-29,32]. Many of the isolated genes had known or potential growth regulatory properties [27,29,33-35]. However, some cDNAs, including the cDNA corresponding to mda-7/IL24, appeared to be novel and possessed no predictable or apparent functional attributes at the time of isolation [15,27,28,36]. In hindsight, differential screening of the HO-1 human melanoma system [29,32,35] turned out to be a serendipitous choice in the isolation of mda-7/IL24, since to our knowledge this gene has not turned up with notable frequency in any other screen performed in human systems. The currently known structural attributes of mda-7/IL-24 have been summarized in Fig. 1.

When overexpression studies were performed, they rapidly established mda-7/IL-24's capacity to inhibit cell growth when overexpressed by a plasmid expression vector [15,28]. Tests were subsequently performed to determine if overexpression of mda-7/IL-24 displayed transformed cell growth inhibitory specificity. Comprehensive initial documentation of growth inhibition in a range of human and rat transformed cells with minimal effects on primary untransformed counterparts was made by Jiang et aZ. [15,28]. While this report did not address the underlying mechanistic basis of tumor cell specificity, it noted the lack of dependence of mda-7/IL-24 on p53 and pRb status of susceptible cells [15]. Successful mouse-human tumor xenograft studies, induction of apoptosis, and involvement of the proapoptotic molecules (Bax) in mda-7/IL-24-induced transformed cell death were first

fig. 1. Schematic representation of the MDA-7/IL-24 polypeptide showing various predicted and established protein motifs.

reported by Su et al. [16] in a human breast cancer context. These and other initial studies performed in a concurrent time frame [27,33,37] established the experimental foundation of the therapeutic potential of this (at the time) novel sequence, thereby validating the underpinning concept of the experimental strategy of differentiation therapy.

At the present time, it is possible that an exhaustive and detailed study cataloging all possible cell types and tissues naturally expressing mda-7/IL-24 has not yet been performed. However, it is quite likely that most or all tissues and cell types expressing this gene may have been recorded in discrete reports [38-42]. In general, the published literature indicates that this gene has a tissue-specific expression pattern, being restricted to melano-cytes and certain lymphocyte and leukocyte subtypes [38,39,42]. The possible IL-10-related cytokine nature of mda-7/IL-24 was first noted by Jiang et al. [28], who reported the presence of an IL-10 homology domain in a 42-aa region of the putative open reading frame of the sequence. A highly hydrophobic N-terminal region was also recognized in the sequence in this report but due to poorly developed computer algorithms at that time and the atypically long nature of the human mda-7/IL-24 signal peptide [43,44] this feature was reanalyzed and confirmed only at a later time [23,38,41,45,46]. Several reports appearing in 2001 [38,45] determined by either bioinformatic and/or experimental data that the sequence encoded by mda-7/IL-24 (called mda-7 up to that point) was indeed an IL-10-related secreted cytokine (Fig. 1). Blumberg et al. [47] first reported the genomic

cluster of IL-10-related genes residing on human chromosome 1q32, including IL-10, IL-19, IL-20, and mda-7/ IL-24. Su et al. [14] noted functional secretion of mda-7/ IL-24, resulting in a potent transformed cell killing bystander effect. These studies culminated in the identification of the receptor complexes for this cytokine by Dumoutier et al. [45], which was later reconfirmed by Wang et al. [46]. A comprehensive study of genomic structure, chromosomal localization, and expression profile was also published in 2001 by Huang et al. [38]. In the following year (2002) several additional studies documented secretion from melanoma cells and mela-nocytes [23,41] and documented immunostimulatory activity [39]. With all of the studies highlighted above characterization of mda-7/IL-24 as a secreted IL-10-related cytokine possessing potent antitumoral activity was achieved.

Molecular Pathways Involved in mda-7/IL-24 ACTIVITY

Expanding studies in diverse systems reveal that the signal transduction pathways mediating Ad.mda-7-induced apoptosis are versatile and different signaling pathways are activated in different cell lines (summarized in Fig. 2). Experiments with inhibitors of various signaling pathways revealed that SB203580, a specific inhibitor of the p38 MAPK pathway, could effectively protect against Ad.mda-7-induced apoptosis in melanoma cells [24]. Ad.mda-7 infection resulted in phos-phorylation of p38 MAPK and induction of the growth-

fig. 2. Overview of the signaling pathways associated with Ad.mda-7 and MDA-7/IL-24 activity in cancer cells and in the immune system. Abbreviations: P, phosphorylation; PHA, phytohemagglutinin; LPS, lipopolysaccharide; IL, interleukin; TNF-a, tumor necrosis factor-a; IFN-g, interferon-g; GM-CSF, granulocyte macrophage-colony stimulating factor; VEGF; vascular endothelial growth factor; TGF-ß, transforming growth factor-ß; PI3K/PKB, phosphatidylinositol 3-kinase/ protein kinase B; FAK, focal adhesion kinase; MMP, matrix metalloproteinase; PKR, double-stranded RNA-dependent protein kinase R; MAPK, mitogen-activated protein kinase; eIF2a, eukaryotic translation initiation factor-2a; Tyk2, tyrosine kinase-2; STAT, signal transducer and activator of transcription; GADD, growth-arrest and DNA-damage inducible; Hsp, heat shock protein; Pp2A, protein phosphatase-2A; iNOS, inducible nitric oxide synthase. (Modified from Sarkar et al. [5]. Used by permission of the publisher.)

arrest and DNA-damage-inducible (GADD) family of genes in melanoma cells, but not in normal melanocytes [24]. The GADD gene family consists of GADD34, GADD45a, GADD45h, GADD45g, and GADD153, which encode highly acidic proteins [48,49]. Overexpression of each GADD gene causes growth inhibition and/or apoptosis and combined overexpression of the GADD genes leads to synergistic or cooperative antiproliferative effects [49]. Ad.mda-7 infection resulted in marked induction of GADD153, GADD45a, , and GADD34 and a modest induction of GADD45g [24]. Inhibition of the p38 MAPK pathway, either with SB203580 or by an adenovirus expressing a dominant negative p38 MAPK, abrogated the induction of the GADD family of genes by Ad.mda-7 and also protected

melanoma cells from apoptosis. In addition, inhibition of the GADD family of genes by an antisense approach also rescued cells from Ad.mda-7-mediated cell death. GADD153 functions by downregulating the bcl-2 promoter [50] and treatment with SB203580 prevented Ad.mda-7-mediated downregulation of bcl-2. These findings indicate that activation of the p38 MAPK pathway followed by induction of the GADD family of genes plays a crucial role in the cancer-selective apoptosis-inducing effect of Ad.mda-7. The induction of the GADD family of genes following Ad.mda-7 infection is also observed in Ad.mda-7-infected glioblastoma multiforme [18], prostate cancer, and breast cancer cells and in pancreatic cancer cells infected with Ad.mda-7 in combination with antisense K-ras (unpublished data).

Ad.mda-7 induces apoptosis of lung cancer cells via upregulation of double-stranded RNA-dependent protein kinase (PKR) [51]. Once activated, PKR is able to phos-phorylate a variety of substrate targets, the most well characterized being eIF2a, which can lead to the inhibition of protein synthesis, growth suppression, and induction of apoptosis [52]. Infection with Ad.mda-7 resulted in phosphorylation of PKR and also its downstream targets eIF2a, Tyk2, Stat1, Stat3, and p38 MAPK [51]. Ad.mda-7 infection also produced apoptosis that was associated with activation of caspases 3, 8, and 9 and cleavage of Bid and PARP. The activation of PKR appeared to be upstream of caspase activation because pretreatment with caspase inhibitors failed to block PKR phosphorylation. However, treatment with a specific serine/threonine kinase inhibitor 2-amino purine blocked Ad.mda-7-mediated apoptosis-induction and also activation of PKR and eIF2a [51]. An involvement of PKR in Ad.mda-7-mediated apoptosis was demonstrated in PKR-null (—/—) mouse embryonic fibroblasts (MEF). Infection with Ad.mda-7 was reported to induce apoptosis in PKR wild-type MEFs, but not in PKR-null (—/—) MEFs. However, considering the preponderance of studies demonstrating that Ad.mda-7 does not induce apoptosis in normal cells, the significance of this enigmatic finding is questionable and requires further independent confirmation. The common findings between these studies is activation of p38 MAPK following Ad.mda-7 infection [24,51]. It is possible that in melanoma cells p38 MAPK activation is downstream of PKR activation, although in melanoma cells the post-p38 signal transduction changes appear to be more important in Ad.mda-7-induced apoptosis. It has been documented that eIF2a phosphorylation activates the transcription factor ATF4, which activates GADD153 [53]. Thus, there is a significant level of cross talk between the PKR and the p38 MAPK signal transduction pathways. Further studies are required to identify the upstream molecules activating PKR and p38 MAPK pathways to delineate the relevant signal transduction pathways involved in Ad.mda-7 -induced apoptosis.

Another pathway important in Ad.mda-7-induced apoptosis is the c-Jun NH2-terminal kinase (JNK) pathway. Infection with Ad.mda-7 radiosensitizes glioma cells [18,20]. JNK was activated in Ad.mda-7-infected, irradiated cells and treatment with a specific JNK inhibitor, SP600125, protected glioma cells from the synergistic killing effect of radiation and Ad.mda-7 [20]. In a separate study it was shown that curcumin, a dietary pigment that inhibits JNK activation, inhibits phosphorylation of c-jun and radiosensitization by Ad.mda-7 in non-small-cell lung cancer cells [19].

In addition to the positive regulation of p38 MAPK, PKR, and JNK, Ad.mda-7 has been shown to regulate the h-catenin and phosphatidylinositol 3-kinase (PI3K) pathways negatively [25]. In breast and lung cancer cells but not in normal human endothelial cells

(HUVEC), Ad.mda-7 infection resulted in changes in localization of h-catenin from the nucleus to the plasma membrane that lead to inhibition of h-catenin-mediated transcriptional activation. APC and GSK-3h, negative regulators of h-catenin, were upregulated by Ad.mda-7 infection. Proto-oncogenes associated with the PI3K pathway, such as PI3K (p85), FAK, ILK-1, and PLC-g, were downregulated and PTEN, a negative regulator of PI3K, was upregulated by Ad.mda-7 infection. However, the significance of the downregulation of these pathways in the context of Ad.mda-7-mediated apoptosis was not clearly elucidated. It was not tested if overexpression of h-catenin or activation of the PI3K pathway might protect these cells from Ad.mda-7-induced apoptosis.

Diverse Mechanisms Underlying Apoptosis-Inducing Properties of the mda-7/IL24 Gene

The selective growth inhibition and targeted killing of cancer cells by Ad.mda-7 places this tumor growth suppressor gene in a unique position for potential gene therapy applications and distinguishes it from the majority of currently identified tumor suppressor genes [6,10,15,16].

Studies of mda-7 mRNA and protein expression in normal melanocytes and in melanoma cells revealed a correlation between mda-7 expression and reversion in the cancer phenotype of melanoma cells (including induction of terminal differentiation and terminal growth arrest) and support the concept that mda-7 is a negative regulator of cell growth and progression in melanoma cells [4,23,28,33,38,40,41,54]. Transfection of different cancer cell types with an mda-7/IL-24 expression vector documented the wide-range tumor-suppressing properties of mda-7/IL-24 [4,15]. Ectopic expression of mda-7/IL-24 administered by adenovirus (Ad.mda-7) results in suppression of cellular proliferation, G2/M cell cycle arrest, and activation of the apoptotic cascade in diverse cancer cells (including osteosarcoma, melanoma, glioblastoma, and carcinomas of the breast, lung, cervix, colon, nasopharynx, ovary, and prostate) without any effects in their normal counterparts [15,16,18,23,55,56]. These results firmly established that mda-7/IL-24 functions as a growth suppressor specifically in the context of cancer cells. Interestingly, mda-7/IL-24 inhibits cancer cell growth irrespective of the status of other tumor suppressor genes (p53, Rb, orp16ink4) in these cancer cells [4,15,16,23,57].

The apoptotic nature of growth suppression in cancer cells prompted by Ad.mda-7 was confirmed by multiple assays (DNA fragmentation analysis, annexin V and PI staining, TUNEL assay) [14,16,18,23,57,58]. Treatment with a pancaspase inhibitor, z-VAD.fmk, significantly inhibits apoptosis induced by Ad.mda-7 in a variety of

cancer cell types [17,57]. Studies using cell lines from different tumor types have emphasized a variety of diverse cellular mediators of the apoptotic response of tumor cells to Ad.mda-7. Ectopic expression of mda-7 can increase expression of p53, Bax, and Bak and increase mitochondrial cytochrome c release in lung, mesothelioma, and breast cancer cell lines [14,16-18,23,59]. Apoptosis induction associates with activation of the caspase cascade in specific tumor systems, including activation of caspase-9 and caspase-3 and cleavage of PARP, a caspase substrate [20,21,51,55,57]. In lung cancer cells, Ad.mda-7 also induces apoptosis that was associated with activation of caspases 3, 8, and 9 and cleavage of Bid and PARP [51]. In this context, a combination of intrinsic (mitochondria-mediated) and extrinsic (death receptor-mediated) pathways is likely to be active in mda-7/IL-24-induced apoptosis and the contribution of each probably varies depending on the genetic context of the transformed cancer cell.

Infection with Ad.mda-7 resulted in an increase in the ratio of proapoptotic (Bax and Bak) to antiapoptotic (Bcl-2 and Bcl-xL) proteins [14,16-18,21,23,55]. A potential role of mitochondrial-mediated events in apoptosis was suggested by the fact that overexpression of Bcl-2, Bcl-xL, and adenoviral E1B protein protects cancer cells from Ad.mda-7-induced apoptosis [16,17,20,21,59]. Bcl-2 and Bcl-xL appear to preserve, directly or indirectly, the integrity of the outer mitochondrial membrane thus preventing release of proapoptotic factors from mitochondria to cytosol. A recent study presents definitive

evidence that changes in mitochondrial function and ROS production are key components associated with selective killing of prostate cancer cells by mda-7/IL-24. In this study, Lebedeva et al. [58] demonstrated that Ad.mda-7 selectively induces apoptosis in prostate cancer cells by promoting mitochondrial dysfunction and reactive oxygen species (ROS) production. Antioxidants (N-acetyl-L-cysteine and Tiron) and inhibitors of mitochondrial permeability transition (MPT) (cyclosporin A and bongkrekic acid) inhibit Ad.mda-7-induced apoptosis. Conversely, agents augmenting ROS production (arsenic trioxide, dithiophene NSC656240) and mitochondrial pore opening (PK11195) facilitate Ad.mda-7-induced apoptosis. The fact that ectopic expression of Bcl-2 and Bcl-xL inhibits mitochondrial changes, ROS production, and apoptosis provides additional support for an association between mitochondrial dysfunction and Ad.mda-7-mediated apoptosis. This proposed model for Ad.mda-7-induced apoptosis in prostate cancer cells (Fig. 3) implies that after Ad.mda-7 infection, MDA-7 protein affects mitochondria directly or indirectly, causing alterations in mitochondrial function (decrease in mitochondrial membrane potential and MPT) and ROS production. These mitochondrial changes precede early apoptotic changes and are caspase-independent since they are not inhibited by the general caspase inhibitor z-VAD.fmk. Moreover, mitochondrial changes can be blocked by inhibitors of MPT, such as cyclosporin A and bongkrekic acid, and can be promoted by activators of MPT, such as PK11195. ROS inhibitors (N-acetyl-L-cysteine and Tiron)

fig. 3. Proposed model for Ad.mda-7-induced apoptosis in prostate cancer cell lines. Following Ad.mda-7 infection, MDA-7 protein affects mitochondria directly or indirectly, causing alterations in mitochondrial function (decrease in and MPT) and ROS production. The reductions in and MPT are caspase-independent, since they are not inhibited by the general caspase inhibitor z-VAD.fmk. Moreover, MPT can be blocked by inhibitors of MPT, such as CsA and BA, and can be promoted by activators of MPT, such as PK11195, a PBzR agonist. ROS inhibitors (NAC and Tiron) block Ad.mda-7-induced apoptosis, while ROS producers (As2O3 and NSC656240) enhance apoptosis only in the context of prostate cancer cells. Abbreviations: mitochondrial trans-

membrane potential; MPT, mitochondrial permeability transition; ROS, reactive oxygen species; z-VAD.fmk, N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone; CsA, cyclosporin A; BA, bongkrekic acid; PK11195, 1-(2-chlorophenyl)-N-methyl-N-(1-methyl-propyl)-3-isoquinolinecarboxamide; PBzR, peripheral benzodiazepine receptors; As2O3, arsenic trioxide; NSC656240, dithiophene. (Reproduced, by permission of the publisher, from Lebedeva et al. [58].)

block Ad.mda-7-induced apoptosis, whereas ROS producers (As2Ü3 and NSC656240) enhance apoptosis. All of these changes occur upon Ad.mda-7 infection only in the context of prostate cancer and not in immortalized normal prostate epithelial cells. However, in cancer cells of different origin, the mechanism of Ad.mda-7-induced apoptosis could be unrelated to early mitochondrial changes and MPT. In a recent study using two lung cancer cell lines, Ad.mda-7 induced changes in mito-chondrial potential in one line, and cyclosporin did not

prevent cell death in either of these cell types [60]. The reason for these differences among prostate and lung carcinoma cell lines is not currently understood but could reflect inherent differences in the mode of action of mda-7/IL-24 in these two tumor cell types or they could represent differences in experimental protocols (timing of treatment, concentration of inhibitors, etc.). An involvement of ROS production in mda-7/IL-24-modulated apoptosis was confirmed for glioblastoma and renal cancer cell lines [21,61].

Ad.SP-mda-7

pellet

supernatant

P69 DU-145 LNCaP PC-3

Ad.mda-7

Ad.mda-7 (P69)

DU 145

untreated PD98059 (10uM)

control Ad. vector Ad.SP-mda-7 Ad.mda-7

P69 6.09 7.07 7.53 4.99

DU-145 6.54 8.98 22.4 17.1

Щ pERKl/2 total ERK

Pancreatic cancer represents a notable exception to the apparently ubiquitous cancer-specific apoptosis-inducing properties of Ad.mda-7 [14]. A frequent genetic alteration in pancreatic cancer involves activation of the K-ras oncogene (85-95%) [62,63]. Infection with Ad.mda-7 alone does not kill pancreatic cancer cells irrespective of their K-ras status. As a result of Ad.mda-7 infection, robust expression of mda-7 mRNA is observed, but MDA-7 protein translation is impaired. However, downregula-tion of K-ras expression by means of antisense phosphor-othioate oligonucleotides in combination with Ad.mda-7 treatment results in MDA-7 protein production followed by significant growth inhibition and apoptosis only in mut K-ras pancreatic cancer cells. This observation provides a basis for potentially developing rational approaches for selectively killing pancreatic cancer cells with clear potential for increasing efficacy of mda-7/IL-24 in additional cancers (see [14,64]). A recent study demonstrated that ROS production in combination with Ad.mda-7 treatment is also able to remove the MDA-7 protein translational block and result in apoptosis and cell death of pancreatic cancer cells irrespective of their K-ras status [65].

Data regarding MDA-7 protein action on cancer cells are contradictory. In vitro, 293 cell-derived secreted sMDA-7/IL-24 did not demonstrate significant antiproli-ferative activity against HUPEC, HUVEC, or lung tumor cells even at high (about 2 nM) concentrations [66]. Another group has found inhibition of ovarian carcinoma cell proliferation by a high concentration (about 30 nM) of glycosylated mammalian sMDA-7/IL-24 [67]. In contrast, studies with recombinant GST-MDA-7 protein in diverse cancer cell lines further confirmed its potent tumor-suppressing and apoptosis-inducing properties [20,21,61,68]. Recent data demonstrated that it was the intracellular and not the secreted form of MDA-7 that was primarily responsible for eliciting cell death in prostate and melanoma cells [1] and in lung cancer cells [2]. However, in the case of lung cancer cells, the killing by MDA-7 protein was not observed [2].

Experiments in vivo document that Ad.mda-7 inhibits human tumor formation and progression in human

xenograft models of breast [16], cervical [4], pancreatic [14], glioblastoma [20,21] and lung cancer [69,70]. Tumors from mice treated with Ad.mda-7 demonstrated extensive apoptosis by TUNEL staining (17%), whereas tumors from animals treated with Ad.luc showed minimal apoptotic cell death (3%) [69]. Studies in tumor lung xenografts in nude mice also demonstrated antiangio-genic activity of Ad.mda-7 [66,69], and this effect was enhanced by radiation [70]. The recent study by Ramesh and colleagues [71] proved that adenoviral overexpression of mda-7/IL-24 inhibits invasion and migration of lung cancer cells in vitro and in vivo by downregulating proteins associated with these processes (such as p85, PI3K, and FAK), resulting in reduced metastasis.

Radiosensitizing Properties of mda-7/IL-24: Implications in Cancer Gene Therapy

The possibility that MDA-7 could interact with ionizing radiation to radiosensitize lung tumor cells was first reported at the 2001 American Association for Cancer Research Meeting. Subsequent peer-reviewed studies by several laboratories have now demonstrated that MDA-7/ IL-24, either as a purified protein or generated via a recombinant adenovirus (Ad.mda-7) can radiosensitize human and rodent tumor cells from multiple tissue origins, without causing cytotoxicity or radiosensitiza-tion to nontransformed cells of the same organ [1821,72]. Su etal. [18] have demonstrated greatly enhanced inhibition of growth and apoptosis induction in malignant human glioma-derived cell lines exposed to radiation, irrespective of p53 status and concurrent with upregulation of the GADD genes. Heterogeneity of p53 status is common in the clinical settings of malignant glioma, where tumor cells with a wild-type and a mutant p53 genotype can be found within the same cancer, making this observation particularly relevant from a gene therapy viewpoint. Work by Yacoub et al. [20,21] has extended these findings to show a greater than additive efficacy of combined Ad.mda-7 and radiotherapy in treatment of rodent and human malignant glioma cell

fig. 4. Comparative growth inhibition, apoptosis induction, and MDA-7 expression in cells infected with Ad.vec, Ad.mda-7, or Ad.SP~mda-7. (A) Growth inhibition in prostate cancer cell lines. Cells were infected with 100 pfu/cell of Ad.vec, Ad.mda-7, or Ad.SP~mda-7, and cell viability as a ratio of specific treatment versus untreated cells was determined by MTT proliferation assay 5 days after infection. An average of three independent experiments is shown +SD. (B) Apoptosis induction in prostate cancer cell lines. DU-145 and P69 cells were treated as described for (A), collected 24 h later, fixed in 70% ethanol, and stained with propidium iodide. Percentage of the cells in A0 fraction was quantitated by flow cytometry using the CellQuest software. (C) MDA-7/IL-24 expression in DU-145 cells. Protein lysates were collected from uninfected DU-45 cells and DU-145 cells infected with Ad.vec, Ad.mda-7, or Ad.SP~mda-7 as described for (A). Samples were analyzed by Western blotting using polyclonal rabbit anti-mda-7/IL-24 antibody. (D) Effects of ERK1/2 inhibitor on mda-7/IL-24-induced killing in prostate cancer cell lines (top). Cells were incubated in the presence or absence of PD98059 (10 aM) after infection with Ad.vec, Ad.mda-7, or Ad.SP~mda-7. Cell viability was determined by MTT assay 6 days after infection and is presented as a ratio of treated cells to untreated +SD. ERK1/2 induction following mda-7 expression in prostate cancer cell lines (bottom). Activation of the ERK1 /2 pathway was determined 24 h postinfection as described for (C) by Western blotting using phosphospecific ERK1/2 antibody. Abbreviations: Ad.vec, replication-incompetent adenovirus lacking a gene insert; Ad.mda-7, replication-incompetent adenovirus containing the full-length mda-7 gene; Ad.SP~mda-7, replication-incompetent adenovirus containing the mda-7 gene lacking the 49-aa secretory motif; MTT, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide thiazolyl blue; ERK1/2, extracellular signal-regulated kinases 1 and 2. (Modified from Sauane et a/. [1]. Used by permission of the publisher.)

lines. Recent studies have extended this observation to primary nude mouse tumor-maintained human glioblas-toma multiforme cells [22]. As described below, the JNK pathway appears to be integral to apoptosis induction in this context. The cell cycle profile of combination treatments showed accumulation of cells in both G1/G0 and G2/M phases, indicating that mda-7/IL-24 causes radio-sensitization in a cell-cycle-independent manner. In addition, there appeared to be some protective effects of the ERK1/2 and PI3K signaling pathways [73-76] since inhibition of either alone did not, but a combined inhibition of both did, enhance killing [20]. A recent report by Lebedeva et al. [58] in prostate cancer cells has shown that apoptosis induction by mda-7/IL-24 occurs via induction of mitochondrial dysfunction and generation of reactive oxygen species. Overexpression of Bcl-2 and Bcl-xL proteins in this context served a cytoprotective function with respect to apoptosis induction. These data apparently tie in with the ability of mda-7/IL-24 to radiosensitize cells. Recent studies by Nishikawa [70] demonstrated that radiation enhanced the anti-tumor activity of Ad.mda-7 in lung cancer cells, an effect mediated by suppression of angiogenesis. However, the mechanism(s) by which MDA-7 causes radiosensitization is not fully understood.

One key event, which has been closely linked to enhanced cell killing by mda-7/IL-24, is the generation of reactive oxygen/nitrogen species and activation of the JNK pathway. Ionizing radiation, in a variety of cell types, has been shown to activate JNK1/2/3 signaling, potentially via the generation of ceramide;however, activation of many signaling pathways using modest radiation doses (1-4 Gy) causes only a transient or low sustained activation of MAP kinase signaling pathways [75,76]. In some cell types, e.g., lung carcinoma cells, Ad.mda-7 as a single agent has been shown to cause intense JNK1/2 activation [19,25,71], whereas in other tumor cell types, e.g., glioma, breast, and prostate carcinoma, Ad.mda-7 and purified MDA-7 protein have had little or no discernable effect on the activity status of this pathway [3,20,61,72]. In glioma, breast, and prostate carcinoma cells, however, radiation and Ad.mda-7 interact to cause prolonged intense activation of the JNK1/2/3 pathway [18-22,72]. Inhibition of JNK1/2/3 signaling using the relatively specific small molecule inhibitor SP600125 abolished the radiosensi-tizing properties of Ad.mda-7 and GST-MDA-7 in these cell types. The precise mechanism(s) by which MDA-7 causes prolonged JNK pathway activation remains to be defined.

The ability of radiation to enhance the capacity of mda-7/IL-24 to induce growth suppression and apoptosis in a tumor-specific manner represents a significant means of increasing the therapeutic index of this novel cancer-selective apoptosis-inducing gene. Recent studies indicate that radiation can be combined with Ad.mda-7 to induce

growth inhibition and apoptosis in both parental prostate tumor cells and prostate tumor clones displaying inherent resistance to mda-7/IL-24 because of overexpression of the antiapoptotic genes bcl-2 or bcl-xL [72]. These findings provide further impetus for the combinatorial use of radiation and mda-7/IL-24 to target cancer cells for death, including those tumors that may display resistance to either agent used alone. In this context, the window of therapeutic applications of mda-7/IL-24 can be further amplified by using ionizing radiation, which could provide a means of circumventing resistance to single modality treatments.

Newer Information on Cancer Cell-Specific Apoptosis Induction, Including an Intracellular role of action FOR mda-7/IL-24

Based on a progressive series of advancements, the likelihood of mda-7/IL24 becoming a mainstream cancer gene therapeutic appears highly probable [6,9,10]. Consequently, considerable interest now exists in elucidating the mechanism by which mda-7/IL-24 distinguishes between normal and transformed cells. Experimental evidence has confirmed that ectopically expressed (or endogenous) MDA-7/IL-24 protein is processed via classical secretory pathways and is found extracellularly as a multiply glycosylated protein [6,8,57]. Further, this molecule has been shown to bind specific cytokine receptor complexes (IL-20R and IL-22R receptors) and consequently activate the JAK/STAT signal transduction pathway, specifically STAT3 and STAT1 [45,66]. Currently, the downstream targets activated subsequent to receptor binding and STAT activation are unknown [77,78]. On the other hand, when MDA-7/IL-24 is expressed at supraphysiological levels by adenovirus administration, the molecule can function independent of JAK/STAT signal transduction pathways that are classically involved in cytokine-mediated activities [3]. We have additionally demonstrated by sensitive RT-PCR methodology that apoptosis can be induced in tumor cells not expressing detectable levels of IL-20/IL-22 receptors [3]. These results raise the possibility of the existence of an alternative receptor complex mediating Ad.mda-7 killing that might be only partially dependent on and/or completely independent of JAK/STAT-trans-duced mechanisms or that transformed cell killing operates through a receptor-independent pathway. A further indirect confirmation of our findings is that Ad.mda-7 infection of different cell lines is documented to trigger several independent pathways [6,7]. Infection with Ad. mda-7 activates the PKR pathway, GADD genes, and components of the MAPK pathway, including ERK and p38 MAPK [18,20,21,24,25,51]. Infection with an mda-7/IL-24-expressing virus or with purified MDA-7/IL-

24 protein also inhibits angiogenesis [66,69,70]. These diverse activities appear to be inconsistent with or attributable entirely to potential cytokine-related properties of mda-7/IL-24.

The next logical step in pursuing our findings pertaining to JAK/STAT independence was to determine if the apoptotic effect could be triggered by intracellular fractions (possibly by receptor-independent mechanisms) or if extracellular MDA-7/IL-24 protein (receptor mediated) was mandatory for activity. To achieve this objective, an adenovirus vector was constructed that expressed a nonsecreted version of MDA-7/IL-24 protein by deleting the signal peptide (Ad.SP~mda-7) [1]. When the extent and modality of killing were compared between the full-length mda-7/IL-24-expressing virus (Ad.mda-7) and Ad.SP~mda-7, the results indicated that the effects of Ad.SP~mda-7 and Ad.mda-7 infection were similar with respect to transformed cell apoptosis induction [1] (Figs. 4A and 4B). MDA-7/IL-24 protein was shown to localize to the ER/Golgi compartments by two independent studies, one utilizing mutant Ad.SP~mda-7 adenovirus [1] and the other utilizing plasmid-based analyses [2]. Localization of MDA-7/IL-24 protein was similar irrespective of the presence or absence of signal peptide as well as in both normal and transformed cells and therefore the differences in cellular localization of this protein can be excluded as a direct mechanism underlying the differential apoptosis-inducing activity of MDA-7/IL-24 toward cancer cells (Fig. 6). Western blot analyses performed on protein-derived cytosolic and extracellular fractions of cells infected with both full-length mda-7/IL-24 (Ad.mda-7) and Ad.SP~mda-7 viruses indicated that only full-length MDA-7/IL-24 was processed and secreted [1] (Fig. 4C).

Based on its ER localization, one hypothesis is that MDA-7/IL-24 protein induces the phenomenon of "ER stress." ER stress is caused by different conditions that perturb ER function, including the accumulation of misfolded proteins. In this particular case, to eliminate ER stress caused by misfolded protein accumulation, a highly conserved unfolded protein response (UPR) signal transduction pathway is activated [79]. The UPR is characterized by the coordinated activation of multiple signal transduction pathways that lead to the suppression of the initiation step of protein synthesis and trigger the expression of genes encoding ER chaperones, enzymes, and structural components of the ER. Prolonged activation of this pathway leads ultimately to apoptosis. ER stress or UPR induction by mda-7/IL-24 in turn induces proapoptotic events [1]. Earlier findings from our group support this hypothesis since induction of the GADD genes is classically associated with the stress response including ER-stress pathways [80,81]. Induction of GADD genes and further upstream events such as activation of p38 MAPK was shown to be mda-7/IL-24 induced in a transformed cell-

specific manner after Ad.mda-7 or Ad.SP~mda-7 infection [1,24]. This treatment also specifically activated the p44/42 MAPK pathway [1,20] (Fig. 4D). Furthermore, Ad.mda-7 infection produced an upregulation in inositol 1,4,5-trisphosphate receptor (IP3R) in H1299 cells [25]. IP3R is an intracellular calcium-release channel implicated in apoptosis and localized in the ER. Our observation that mda-7/IL-24 induces ER-stress response is consistent with another microarray-based study that has been published recently, showing that mda-7/IL-24 is able to induce the expression of ER-stress response genes such as BiP, PP2A, HSJ1, and TRA1 in H1299 cells [2].

The major issue that arises is why mda-7/IL-24 is able to induce killing differentially in transformed compared to normal cells when interpreted through the newer information described above. There are two possibilities based on the new ER-stress induction mechanism that we have recently uncovered. One is that enhanced sensitivity may be due to the "activated" or "destabilized" nature of tumor cells, which enhances cell death after the ER-stress response is triggered, compared to normal cells. The other possibility is that mda-7/IL-24 not only induces the classical ER-stress response that favors apoptosis but it also induces additional specific pathways that cause apoptosis only in transformed cell lines. Our data do not permit a clear distinction as to whether mda-7/IL-24 specifically induces killing in transformed cells only by induction of ER stress and the selectivity is caused by the nature of the cancer cells or whether mda-7/IL-24 triggers tumor cell-specific proapoptotic signals in coordination with induction of ER stress. Our studies provide some support for both hypotheses since we have determined that mda-7/IL-24 localizes to the ER compartment in normal cells as well as cancer cells and is therefore in a position to induce this pathway irrespective of transformation status of the cell [1,2] (Fig. 5). When micro-array studies were performed to determine gene expression patterns following Ad.mda-7 infection, no comparative data appear to have been generated regarding comparisons between normal and cancer cells [2], which would have greatly clarified the question of ER-stress induction specificity. Such studies will clearly need to be performed to address this critical issue. Our own studies have shown that GADD family gene induction as well as p38 MAPK activation is induced only in transformed cells and not in normal cells [24], indicating that the differential response might be due to differential activation, either in strength or in duration of the ER-stress response. However, this does not preclude the additional activation of other pathways specifically in cancer cells. While further investigations are clearly needed to determine the mechanism of specificity of mda-7/IL-24-triggered tumor cell apoptosis, our recent findings indicate that ER stress is likely to play some role

Ad.mda-7

fig. 5. Localization of the MDA-7 protein after infection with Ad.SP mda-7 or Ad.mda-7. DU-145 cells were infected with 100 pfu/cell of Ad.vec, Ad.mda-7, or Ad.SP—mda-7. After 48 h, cells were fixed and MDA-7/IL-24 protein was detected by indirect immunofluorescence using rabbit anti-mda-7/IL-24 antibody. Images of Golgi, ER, and mitochondria were obtained using anti-G130, anti-calreticulin, and MitoTracker, respectively. Images of the different compartments and MDA-7/IL-24 were merged. Similar localization of MDA-7/IL-24 protein was observed following infection with the different viruses in P69 cells (data not shown). (Reproduced, by permission of the publisher, from Sauane et al. [1].)

in this phenomenon. Overall, these findings uncover a new intracellular locus and mechanism for the activity of mda-7/IL-24 in inducing transformed cell-specific apop-tosis that may prove amenable for potential cancer therapy.

Phase I Clinical Studies Utilizing Ad.mda-7 (INGN-241)

Based on the extensive preclinical data described above, a Phase I trial was initiated utilizing a nonreplicating adenoviral vector expressing mda-7/IL-24, designated INGN-241, which was essentially identical to the Ad.mda-7 vector utilized in preclinical and other mechanistic investigations [11-13,82,83].

Sequential cohorts of patients received escalating doses of Ad.mda-7 from 2 x 1010 to 2 x 1012 viral particles. Ad.mda-7 was injected into the center of the accessible target tumor lesion. In the first five cohorts, injected lesions were resected at 24-96 h postinjection (Fig. 6A) and then analyzed to determine distribution and effect (mRNA and protein expression, other biologic parameters) on the tumor cells. In the next three cohorts, injected lesions were not removed early and biopsies were performed at baseline and 30 days posttreatment. Additionally, the final cohort of patients received repeat injections (2 x 1012 viral particles injected twice weekly for 3 weeks of a 28-day cycle). Twenty-eight patients were treated in all. After the first 11 patients (cohorts 1-6) were evaluated an excellent safety profile was determined at doses up to 2 x 1012 viral particles per injection. Patients entered into cohort 7 (n = 7) received a single injection of 2 x 1012 viral particles. Excisional biopsy was performed 30 days after treatment in 5 of these patients. Cohort 8 patients (n = 8) received 2 x 1012 viral particles two times a week for 3 weeks. Five patients completed treatment and 3 underwent excisional biopsy 30 days after their last injection.

Ad.mda-7 vector DNA and mRNA copies were detected in all injected lesions. Expression was highest near the injection site and decreased toward the perimeter of the tumor. Protein expression of MDA-7/ IL-24 determined by immunostaining paralleled DNA expression [12] (Fig. 6B). Apoptosis staining by TUNEL reactivity also closely correlated with the expression pattern of the MDA-7 protein (Figs. 6C and 6D). These results convincingly demonstrate correlation of delivered DNA with expressed RNA/protein and the intended functional effect (apoptosis).

Common toxicities related to Ad.mda-7 included injection site pain and fever (¿grade 2). Fever generally occurred within 24 h of tumor injection and was observed more frequently at higher doses. All fevers resolved within 48 h following injection. No other significant short-term or long-term toxic effects have been identified.

fig. 6. Spread of mda-7/IL-24 RNA, DNA, and protein and biological effects (apoptosis) 24 h after intratumoral injection. (A) Schematic representation of serial sections of tumor. (B) Decay of INGN 241 (Ad.mda-7) vector at the injection site. Immunohistochemical staining of different tumor sections and the median numbers of DNA and RNA copies determined by PCR and RT-PCR, respectively, are shown for each section. (C) Spread of MDA-7/IL-24 protein and biological effect (apoptosis) at the injection site. Protein expression correlates with apoptosis. Serial sections from each tumor were evaluated for MDA-7 expression and TUNEL reactivity using immunohistochemistry. (D) Data from TUNEL assay and immunohistochemistry are plotted to indicate signals compared to distance from injection site.

Response was difficult to evaluate since in the first six cohorts the injected lesions were excised between 24 and 96 h after injection. No patients in cohort 7 (injected tumor excised on day 30) demonstrated evidence of response. However, of the patients receiving multiple injections (cohort 8), two of seven patients achieved clinically significant responses (greater than 50% shrinkage of injected lesion) to Ad.mda-7 [12]. One patient with refractory metastatic melanoma had a 2 x 2-cm supraclavicular node injected. Within the first week a partial response was achieved. Local regional erythema and warmth developed within 24 h of injection, but regression was not observed until day 5. As the erythema resolved regression continued to the point at which no palpable disease was identified. However, evidence of systemic activity was not observed. After the first week a second lesion was injected on the same patient. The lesion was on the dorsum of the right hand. Maximum diameter of the second lesion was also 2 cm and within the first week again complete regression was demonstra-

ted. This time the site of injection was resected and microscopically a marked inflammatory lymphoplasma-cytic infiltrate was observed throughout the residual nodule. There was extensive necrosis and no viable tumor cells. We then injected a third lesion located in the anterior right thigh in the same patient. However, the third lesion despite undergoing a minor response did not fulfill criteria for a partial response. This patient remains alive today with continued detectable disease at unin-jected sites 491 days after initial injection. No further treatments were administered. A less dramatic response was seen in another patient with squamous cell carcinoma of the penis in which one of several skin nodules were injected. The injected nodule was a maximum diameter of 3 cm. It underwent a significant central necrotic response satisfying criteria for a partial response. However, other regionally located nodules did not show evidence of response and within several months following the initial course the perimeter of the injected lesion and several uninjected lesions progressed.

Clinical investigation in this first trial demonstrated that MDA-7/IL-24 protein could be detected at the periphery of injected lesions and correlation of DNA, RNA, and protein expression was made with apoptosis. Interestingly, apoptosis demonstrated in patients receiving MDA-7/IL-24 was significantly greater than what was observed following treatment with Ad p53 in non-small-cell lung cancer [84,85]. However, it is unclear if the observed apoptotic effect contributed to the antitumor activity in the two responding patients.

MDA-7/IL-24 also has immune stimulatory activity. It activates IL-6, TNF-a, and interferon-g production [39] and has been shown to downregulate significantly TGF-h (potent immune suppressor of anti-cancer activity) [69]. The potential for enhanced dendritic priming and maturation at the local injection site is possible although it is unlikely that the initial response, which occurred within 5 days, was associated with a dendritic priming affect. Our clinical data indicate transient increases in circulating cytokines such as IL-6, IL-10, and TNF-a in response to MDA-7/IL-24 treatment. Significantly higher elevations of IL-6 and TNF-a were observed in patients receiving repeat treatment who demonstrated evidence of activity related to Ad.mda-7 [13]. The majority of patients also showed a marked increase in CD3+ and CD8+ T cells at day 15 following injection, suggesting that Ad.mda-7 may be associated with a TH1 response. Furthermore, MDA-7/IL-24 has been shown to inhibit G2/M cell cycling [23,24,69] and has been shown to inhibit angiogenesis [66]. We also explored the effect of MDA-7/IL-24 on tumor suppressor genes such as h-catenin and iNOS. h-Catenin expression was reduced in six of nine patients and iNOS expression was reduced in four of nine patients tested. In conclusion, these initial clinical results reveal that Ad.mda-7 is well tolerated when administered via intratumoral injection. Repeat dosing with 2 x 1012 viral particles per injection could be utilized in subsequent clinical investigation via intratumor injection. It is likely that improved delivery vehicles, possibly conditional replicating viruses [86], will provide more efficient delivery of MDA-7/IL-24 and that this likely would be associated with greater clinical effect. Furthermore, combination therapy with radiation or chemotherapy may improve tumor response to MDA-7/IL-24 treatment.

Concluding Perspective on mda-7/IL-24: A Gene with a potentially Bright Future

As emphasized in this review, we have progressed very far in a relatively short time from the identification and cloning of mda-7/IL24 in 1993 as a novel gene induced in human melanoma cells as a consequence of induction of irreversible growth arrest and terminal differentiation [27,28] to an exciting new therapeutic for cancer [6].

Initial studies provided support for the concept of differentiation therapy of cancer, suggesting that genetic elements exist that can selectively regulate cancer cell growth and survival [33]. Progressively, studies have confirmed discriminating growth-suppressive activity in cancer cells, an ability to induce apoptosis selectively in multiple cancer cell types in vitro, anti-tumor activity in animal models, an ability to synergize with radiation to kill cancer cells, an ability to inhibit angiogenesis, and finally direct clinical applications that indicate safety, an ability to induce apoptosis, immune functions, and objective clinical responses in patients. Clearly, a lot remains to be learned about this interesting molecule and the mechanism by which it exerts its physiological function(s) and how supraphysiological expression (or treatment with GST-MDA-7 fusion protein) selectively elicits apoptosis in cancer cells. Interesting insights have now surfaced suggesting a potential novel means of selective anti-cancer action, i.e., induction of ER stress, for tumor cell killing that can occur independent of its proposed cytokine-like properties involving interactions with surface IL-20/IL-22 receptor complexes and activation of JAK/STAT signal pathways. At the current pace of discovery, we are optimistic that our future understanding of mda-7/IL-24 will increase rapidly and this information will pave the way for enhancing clinical applications of this significant molecule for treating patients with diverse cancers.

Acknowledgments

The present studies were supported in part by National Institutes of Health Grants CA35675, CA88906, CA97318, CA98172, NS31492, and DK52825; DOD Breast Cancer Grant DAMD17-03-1-0290; the Lustgarten Foundation for Pancreatic Cancer Research; the Samuel Waxman Cancer Research Foundation; and the Chernow Endowment. P.B.F. is the Michael and Stella Chernow Urological Cancer Research Scientist in the Departments of Pathology, Urology, and Neurosurgery, Columbia University Medical Center, College of Physicians and Surgeons, and a SWCRF Investigator. P.D. is the Universal, Inc., Professor in Signal Transduction Research at Virginia Commonwealth University. We are indebted to the numerous contributions of our colleagues who have contributed to our current understanding of the anti-tumor properties and mechanism of action of mda-7/IL-24.

RECEIVED FOR PUBLICATION JUNE 14, 2004; ACCEPTED AUGUST 12, 2004.

References

1. Sauane, M., et al. (2004). Melanoma differentiation associated gene-7/interleukin-24 promotes tumor cell-specific apoptosis through both secretory and nonsecretory pathways. Cancer Res. 64: 2988-2993.

2. Sieger, K. A., et al. (2004). The tumor suppressor activity of MDA-7/IL-24 is mediated by intracellular protein expression in NSCLC cells. Mol. Ther. 9: 355-367.

3. Sauane, M., et al. (2003). Mda-7/IL-24 induces apoptosis of diverse cancer cell lines through JAK/STAT-independent pathways. J. Cell. Physiol. 196: 334-345.

4. Madireddi, M. T., Su, Z. Z., Young, C. S., Goldstein, N. I., and Fisher, P. B. (2000). Mda-7, a novel melanoma differentiation associated gene with promise for cancer gene therapy. Adv. Exp. Med. Biol. 465: 239 - 261.

5. Sarkar, D., et al. (2002). mda-7 (IL-24): signaling and functional roles. Biotechniques Suppl.: 30-39.

6. Fisher, P. B., et al. (2003). mda-7/IL-24, a novel cancer selective apoptosis inducing cytokine gene: from the laboratory into the clinic. Cancer Biol. Ther. 2: S23-S37.

7. Lebedeva, I. V., Su, Z. Z., Sarkar, D., and Fisher, P. B. (2003). Restoring apoptosis as a strategy for cancer gene therapy: focus on p53 and mda-7. Sem/n. Cancer Bio/. 13: 169-178.

8. Sauane, M., ei a/. (2003). MDA-7/IL-24: novel cancer growth suppressing and apoptosis inducing cytokine. Cytokine Growth Factor Rev. 14: 35-51.

9. Chada, S., et a/. (2004). MDA-7/IL-24 is a unique cytokine-tumor suppressor in the IL-10 family. /nt. /mmunopharmaco/. 4: 649-667.

10. Gopalkrishnan, R. (2002). INGN-241. /ntrogen. Curr. Op/n. /nvest. Drugs 3:1773-1777.

11. Nemunaitis, J. (2003). MDA7 Phase I clinical trial results. Cancer Gene Ther. 10: S106.

12. Cunningham, C., et a/. (2004). A Phase I study of the clinical and local biological effects of an intratumoral injection of mda-7 (INGN 241) in patients with advanced carcinoma. Mo/. Ther. (in press).

13. Tong, A. W., et a/. (2004). Intratumoral injection of INGN-241, a non-replicating adenovector expressing the melanoma-differentiation associated antigen-7 (MDA-7/IL-24): biologic outcome in advanced cancer patients. Mo/. Ther. (in press).

14. Su, Z., et a/. (2001). A combinatorial approach for selectively inducing programmed cell death in human pancreatic cancer cells. Proc. Nat/. Acad. Sc/. USA 98: 10332-10337.

15. Jiang, H., et a/. (1996). The melanoma differentiation associated gene mda-7 suppresses cancer cell growth. Proc. Nat/. Acad. Sc/. USA 93: 9160-9165.

16. Su, Z. Z., et a/. (1998). The cancer growth suppressor gene mda-7 selectively induces apoptosis in human breast cancer cells and inhibits tumor growth in nude mice. Proc. Nat/. Acad. Sc/. USA 95: 14400-14405.

17. Lebedeva, I. V., et a/. (2003). Bcl-2 and Bcl-x(L) differentially protect human prostate cancer cells from induction of apoptosis by melanoma differentiation associated gene-7, mda-7/IL-24. Oncogene 22: 8758-8773.

18. Su, Z. Z., et a/. (2003). Melanoma differentiation associated gene-7, mda-7/IL-24, selectively induces growth suppression, apoptosis and radiosensitization in malignant gliomas in a p53-independent manner. Oncogene 22: 1164-1180.

19. Kawabe, S., et a/. (2002). Adenovirus-mediated mda-7 gene expression radiosensitizes non-small cell lung cancer cells via TP53-independent mechanisms. Mo/. Ther. 6: 637-644.

20. Yacoub, A., et a/. (2003). mda-7 (IL-24) inhibits growth and enhances radiosensitivity of glioma cells in vitro via JNK signaling. Cancer B/o/. Ther. 2: 347-353.

21. Yacoub, A., et a/. (2003). Melanoma differentiation-associated 7 (interleukin 24) inhibits growth and enhances radiosensitivity of glioma cells in vitro and in vivo. C//n. Cancer Res. 9: 3272-3281.

22. Yacoub, A., et a/. (2004). MDA-7 regulates cell growth and radiosensitivity in vitro of primary (non-established) human glioma cells. Cancer B/o/. Ther. (in press).

23. Lebedeva, I. V., et a/. (2002). The cancer growth suppressing gene mda-7 induces apoptosis selectively in human melanoma cells. Oncogene 21: 708-718.

24. Sarkar, D., et a/. (2002). Mda-7 (IL-24) mediates selective apoptosis in human melanoma cells by inducing the coordinated overexpression of the GADD family of genes by means of p38 MAPK. Proc. Nat/. Acad. Sc/. USA 99: 10054-10059.

25. Mhashilkar, A. M., et a/. (2003). MDA-7 negatively regulates the beta-catenin and PI3K signaling pathways in breast and lung tumor cells. Mo/. Ther. 8: 207-219.

26. Gopalkrishnan, R. V., Sauane, M., and Fisher, P. B. (2004). Cytokine and tumor cell apoptosis inducing activity of mda-7/IL-24. /nt. /mmunopharmaco/. 4: 635-647.

27. Jiang, H., and Fisher, P. B. (1993). Use of a sensitive and efficient subtraction hybridization protocol for the identification of genes differentially regulated during the induction of differentiation in human melanoma cells. Mo/. Ce//. D/ffer. 1: 285-299.

28. Jiang, H., Lin, J. J., Su, Z. Z., Goldstein, N. I., and Fisher, P. B. (1995). Subtraction hybridization identifies a novel melanoma differentiation associated gene, mda-7, modulated during human melanoma differentiation, growth and progression. Oncogene 11: 2477-2486.

29. Huang, F., Adelman, J., Jiang, H., Goldstein, N. I., and Fisher, P. B. (1999). Identification and temporal expression pattern of genes modulated during irreversible growth arrest and terminal differentiation in human melanoma cells. Oncogene 18: 3546-3552.

30. Fisher, P. B., Prignoli, D. R., Jr.Hermo, H., , Weinstein, I. B., and Pestka, S. (1985). Effects of combined treatment with interferon and mezerein on melanogenesis and growth in human melanoma cells. /. /nterferon Res. 5: 11 -22.

31. Fisher, P. B., et a/. (1986). Effect of recombinant human fibroblast interferon and mezerein on growth, differentiation, immune interferon binding and tumor associated antigen expression in human melanoma cells. Ant/cancer Res. 6: 765-774.

32. Jiang, H., Kang, D. C., Alexandre, D., and Fisher, P. B. (2000). RaSH, a rapid subtraction hybridization approach for identifying and cloning differentially expressed genes. Proc. Nat/. Acad. Sc/. USA 97: 12684-12689.

33. Jiang, H., Lin, J., and Fisher, P. B. (1994). A molecular definition of terminal cell differentiation in human melanoma cells. Mo/. Ce//. D/ffer. 2: 221 -239.

34. Jiang, H., et a/. (1995). The melanoma differentiation-associated gene mda-6, which encodes the cyclin-dependent kinase inhibitor p21, is differentially expressed during growth, differentiation and progression in human melanoma cells. Oncogene 10: 1855-1864.

35. Huang, F., Adelman, J., Jiang, H., Goldstein, N. I., and Fisher, P. B. (1999). Differentiation induction subtraction hybridization (DISH): a strategy for cloning genes

displaying differential expression during growth arrest and terminal differentiation. Gene 236: 125-131.

36. Lin, J. J., Jiang, H., and Fisher, P. B. (1998). Melanoma differentiation associated gene-9, mda-9, is a human gamma interferon responsive gene. Gene 207: 105-110.

37. Jiang, H., Su, Z. -z., Boyd, J., and Fisher, P. B. (1993). Gene expression changes induced in human melanoma cells undergoing reversible growth suppression and terminal cell differentiation. Mo/. Ce//. D/ffer. 1: 41-66.

38. Huang, E. Y., et a/. (2001). Genomic structure, chromosomal localization and expression profile of a novel melanoma differentiation associated (mda-7) gene with cancer specific growth suppressing and apoptosis inducing properties. Oncogene 20: 7051 -7063.

39. Caudell, E. G., et a/. (2002). The protein product of the tumor suppressor gene, melanoma differentiation-associated gene 7, exhibits immunostimulatory activity and is designated IL-24. J. /mmuno/. 168: 6041-6046.

40. Ekmekcioglu, S., et a/. (2001). Down-regulated melanoma differentiation associated gene (mda-7) expression in human melanomas. /nt. J. Cancer 94: 54-59.

41. Ellerhorst, J. A., et a/. (2002). Loss of MDA-7 expression with progression of melanoma. J. C//n. Onco/. 20: 1069-1074.

42. Garn, H., et a/. (2002). IL-24 is expressed by rat and human macrophages. /mmunob/o/ogy 205: 321 - 334.

43. Nakai, K. (2000). Protein sorting signals and prediction of subcellular localization. Adv. Prote/n Chem. 54: 277-344.

44. Nielsen, H., Engelbrecht, J., Brunak, S., and von Heijne, G. (1997). Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites. Protein Eng. 10: 1-6.

45. Dumoutier, L., Leemans, C., Lejeune, D., Kotenko, S. V., and Renauld, J. C. (2001). Cutting edge: STAT activation by IL-19, IL-20 and mda-7 through IL-20 receptor complexes of two types. J. /mmuno/. 167: 3545-3549.

46. Wang, M., Tan, Z., Zhang, R., Kotenko, S. V., and Liang, P. (2002). Interleukin 24 (MDA-7/MOB-5) signals through two heterodimeric receptors, IL-22R1/IL-20R2 and IL-20R1/IL-20R2. J. B/o/. Chem. 277: 7341-7347.

47. Blumberg, H., et a/. (2001). Interleukin 20: discovery, receptor identification, and role in epidermal function. Ce//104: 9-19.

48. Takekawa, M., and Saito, H. (1998). A family of stress-inducible GADD45-like proteins mediate activation of the stress-responsive MTK1/MEKK4 MAPKKK. Ce// 95: 521 -530.

49. Zhan, Q., et a/. (1994). The gadd and MyD genes define a novel set of mammalian genes encoding acidic proteins that synergistically suppress cell growth. Mo/. Ce//. Bio/. 14: 2361-2371.

50. McCullough, K. D., Martindale, J. L., Klotz, L. O., Aw, T. Y., and Holbrook, N.J. (2001). Gadd153 sensitizes cells to endoplasmic reticulum stress by down-regulating Bcl2 and perturbing the cellular redox state. Mo/. Ce//. B/o/. 21: 1249-1259.

51. Pataer, A., et a/. (2002). Adenoviral transfer of the melanoma differentiation-associated gene 7 (mda7) induces apoptosis of lung cancer cells via up-regulation of the double-stranded RNA-dependent protein kinase (PKR). Cancer Res. 62: 2239-2243.

52. Der, S. D., Yang, Y. L., Weissmann, C., and Williams, B. R. (1997). A double-stranded RNA-activated protein kinase-dependent pathway mediating stress-induced apoptosis. Proc. Nat/. Acad. Sc/. USA 94: 3279-3283.

53. Fawcett, T. W., Martindale, J. L., Guyton, K. Z., Hai, T., and Holbrook, N. J. (1999). Complexes containing activating transcription factor (ATF)/cAMP-responsive-element-binding protein (CREB) interact with the CCAAT/enhancer-binding protein (C/EBP)-ATF composite site to regulate Gadd153 expression during the stress response. B/ochem. J. 339(Pt 1): 135-141.

54. Madireddi, M. T., Dent, P., and Fisher, P. B. (2000). Regulation of mda-7 gene expression during human melanoma differentiation. Oncogene 19: 1362-1368.

55. Saeki, T., et a/. (2000). Tumor-suppressive effects by adenovirus-mediated mda-7 gene transfer in non-small cell lung cancer cell in vitro. Gene Ther. 7: 2051 -2057.

56. Leath, C. A., et a/. (2004). Infectivity enhanced adenoviral mediated mäa-7/IL-24 gene therapy for ovarian carcinoma. Gyneco/. Onco/. 94: 352-362.

57. Mhashilkar, A. M., et a/. (2001). Melanoma differentiation associated gene-7 (mda-7): a novel anti-tumor gene for cancer gene therapy. Mo/. Med. 7: 271 -282.

58. Lebedeva, I. V., et a/. (2003). Melanoma differentiation associated gene-7, mda-7/ interleukin-24, induces apoptosis in prostate cancer cells by promoting mitochondrial dysfunction and inducing reactive oxygen species. Cancer Res. 63: 8138-8144.

59. Cao, X. X., et a/. (2002). Adenoviral transfer of mda-7 leads to BAX up-regulation and apoptosis in mesothelioma cells, and is abrogated by over-expression of BCL-XL. Mo/. Med. 8: 869 -876.

60. Pataer, A., Chada, S., Hunt, K. K., Roth, J. A., and Swisher, S. G. (2003). Adenoviral melanoma differentiation-associated gene 7 induces apoptosis in lung cancer cells through mitochondrial permeability transition-independent cytochrome c release. J. Thorac/c Card/ovasc. Surg. 125: 1328-1335.

61. Yacoub, A., et a/. (2003). MDA-7 (interleukin-24) inhibits the proliferation of renal carcinoma cells and interacts with free radicals to promote cell death and loss of reproductive capacity. Mo/. Cancer Ther. 2: 623-632.

62. Bardeesy, N., and DePinho, R. A. (2002). Pancreatic cancer biology and genetics. Nat. Rev. Cancer2: 897-909.

63. Hruban, R. H., Iacobuzio-Donahue, C., Wilentz, R. E., Goggins, M., and Kern, S. E. (2001). Molecular pathology of pancreatic cancer. J. Cancer 7: 251 -258.

64. Gazdar, A. F., and Minna, J. D. (2001). Targeted therapies for killing tumor cells. Proc. Natl. Acad. Sci. USA 98: 10028-10030.

65. Lebedeva, I.V., et al. (2004). Induction of reactive oxygen species renders mutant and wild type K-ras pancreatic carcinoma cells susceptible to Ad. mda-7-induced apoptosis. Oncogene (in press).

66. Ramesh, R., et al. (2003). Melanoma differentiation-associated gene 7/interleukin (IL)-24 is a novel ligand that regulates angiogenesis via the IL-22 receptor. Cancer Res. 63: 5105-5113.

67. Parrish-Novak, J., et al. (2002). Interleukins 19, 20, and 24 signal through two distinct receptor complexes: differences in receptor-ligand interactions mediate unique biological functions. J. Biol. Chem. 277: 47517-47523.

68. Sauane, M., et al. (2004). Mechanistic aspects of mda-7/IL-24 selectivity analyzed via a bacterial fusion protein. Oncogene (in press).

69. Saeki, T., et al. (2002). Inhibition of human lung cancer growth following adenovirus-mediated mda-7 gene expression in vivo. Oncogene 21: 4558-4566.

70. Nishikawa, T., Ramesh, R., Munshi, A., Chada, S., and Meyn, R. E. (2004). Adenovirus-mediated mda-7 (IL-24) gene therapy suppresses angiogenesis and sensitizes NSCLC xenograft tumors to radiation. Mol. Ther. 9: 818-828.

71. Ramesh, R., et al. (2004). Ectopic production of MDA-7/IL-24 inhibits invasion and migration of human lung cancer cells. Mol. Ther. 9: 510-518.

72. Su, Z. -Z., et al. (2004). Radiation reverses resistance to mda-7/IL-24 in prostate cancer cells over-expressing the anti-apoptotic proteins Bcl-2 or Bcl-xL. 95th AACR Annual Meeting 45, Philadelphia, PA.

73. Yacoub, A., Park, J. S., Qiao, L., Dent, P., and Hagan, M. P. (2001). MAPK dependence of DNA damage repair: ionizing radiation and the induction of expression of the DNA repair genes XRCC1 and ERCC1 in DU145 human prostate carcinoma cells in a MEK1/2 dependent fashion. Int. J. Radiat. Biol. 77: 1067-1078.

74. Hagan, M., Wang, L., Hanley, J. R., Park, J. S., and Dent, P. (2000). Ionizing radiation-induced mitogen-activated protein (MAP) kinase activation in DU145 prostate

carcinoma cells: MAP kinase inhibition enhances radiation-induced cell killing and G2/M-phase arrest. Radiat. Res. 153: 371-383.

75. Dent, P., et al. (1999). Radiation-induced release of transforming growth factor alpha activates the epidermal growth factor receptor and mitogen-activated protein kinase pathway in carcinoma cells, leading to increased proliferation and protection from radiation-induced cell death. Mol. Biol. Cell. 10: 2493-2506.

76. Dent, P., Yacoub, A., Fisher, P. B., Hagan, M. P., and Grant, S. (2003). MAPK pathways in radiation responses. Oncogene 22: 5885-5896.

77. Kotenko, S. V. (2002). The family of IL-10-related cytokines and their receptors: related, but to what extent? Cytokine Growth Factor Rev. 13: 223-240.

78. Pestka, S., et al. (2004). Interleukin-10 and related cytokines and receptors. Annu. Rev. Immunol. 22: 929-979.

79. Zhang, K., and Kaufman, R. J. (2004). Signaling the unfolded protein response from the endoplasmic reticulum.j. Biol. Chem. 279: 25935-25938.

80. Berridge, M. J., Lipp, P., and Bootman, M. D. (2002). The versatility and universality of calcium signalling. Nat. Rev. Mol. Cell. Biol. 1: 11 -21.

81. Berridge, M. J. (2002). The endoplasmic reticulum: a multifunctional signaling organelle. Cell Calcium 32: 235 -249.

82. Tong, A. W., et al. (2002). Immune activation by Ad.mda-7 (INGN 241) gene transfer in advanced cancer patients. ASCO, Annual Meeting.

83. Chada, S., et al. (2003). INGN 241 (Ad.mda-7) induces widespread apoptosis and activates the immune system in patients with advanced cancer. Mol. Ther. 7: S446.

84. Swisher, S. G., et al. (1999). Adenovirus-mediated p53 gene transfer in advanced non-small-cell lung cancer. j. Natl. Cancer Inst. 91: 763-771.

85. Nemunaitis, J., et al. (2000). Adenovirus-mediated p53 gene transfer in sequence with cisplatin to tumors of patients with non-small-cell lung cancer. j. Clin. Oncol. 18: 609-622.

86. Lin, E., and Nemunaitis, J. (2004). Oncolytic viral therapies. Cancer Gene Ther. (in press).