WO2007143538A2

WO2007143538A2 - Pike-a enhances infection by myxoma virus

Info

Publication number: WO2007143538A2
Application number: PCT/US2007/070199
Authority: WO
Inventors: Grant Mcfadden; Steven Werden
Original assignee: Robarts Research Institute
Priority date: 2006-06-01
Filing date: 2007-06-01
Publication date: 2007-12-13
Also published as: WO2007143538A3

Abstract

Cellular Akt activation is increased by transducing an Akt-containing cell with a vector that expresses exogenous PIKE-A (PIl 3 -kinase enhancer activating Akt) in the cell. Expression of PIKE-A increases the susceptibility of cancer cells to myxoma virus infection. Cancer is treated by administering a vector that infects cancer cells and expresses exogenous PIKE-A; and a myxoma virus. Cancer is treated by taking cancer cells from a subject, transducing them ex vivo with a vector that expresses exogenous PIKE-A in the cells, treating the transduced cells with a myxoma virus, and then returning the transduced cells to the subject. Alternatively, myxoma virus can be administered to the patient after the transduced cells have been returned to the patient. The susceptibility of cells to infection with a myxoma virus can be determined by assaying for PIKE-A expression. Cellular expression of PIKE-A in the cell indicates susceptibility to myxoma virus infection.

Description

PIKE-A ENHANCES INFECTION BY MYXOMA VIRUS

BACKGROUND OF THE INVENTION

Myxoma virus (MV) is a rabbit-specific poxvirus pathogen that is being developed as an oncolytic therapeutic because it is non-pathogenic in man but nevertheless can infect and kill a wide spectrum of human cancer cells (1, 2). MV permissiveness in a subset of human cancer cells is critically dependent upon the expression of a viral host range protein called M-T5. M-T5 has no significant sequence homology to non-viral proteins in the sequence database but is categorized as a member of a larger poxviral superfamily designated as ankyrin- repeat (ANK) host range proteins (3). M-T5 was first identified as a virulence factor that is critical for productive myxomatosis in infected rabbits, based on its ability to inhibit apoptosis in rabbit T lymphocytes (4). More recently, M-T5 was shown to regulate cell cycle progression at the G0/G1 checkpoint through interactions with cellular cullin-1, which directly enhances ubiquitination of Kip p27 and subsequent degradation through the 26S proteasome pathway (5). MV exhibits strict species specificity for rabbits, causing a lethal disease called myxomatosis in European rabbits (Oryctolagus cuniculus) and localized fibromas that resolve in rabbits (Sylvilagus sp.) of the Americas (6). MV is non-pathogenic for all other tested vertebrates, however MV can productively infect a variety of human cancer cells that either possess activated Akt or can permit Akt activation following MV infection (7).

In virus-infected human tumor cells, M-T5 forms a complex with cellular Akt and upregulates its kinase activity (7). However some cancers are not susceptible, or have only low susceptibility, to infection by MV. Techniques to broaden the applicability of MV-treatment to additional cancer cell types would be desirable.

A recently identified cellular protein, PIKE-A (PB -kinase enhancer activating AKT), has been shown to bind directly to activated Akt in a guanine nucleo tide- dependent manner, stimulating the kinase activity of Akt and promoting the invasiveness of cancer cell lines (8) . PIKE-A exhibits broad tissue specificity and contains an N-terminal GTPase domain and a C-terminal ankyrin repeat motif which both associate with the regulatory and partial catalytic domains of Akt (9). Over-expression of PIKE-A in human cancer cells inhibits apoptosis by enhancing the kinase activity of Akt whereas rapid apoptosis and a loss of Akt activity is observed when PIKE-A is knocked-down by siRNA (8).

SUMMARY OF THE INVENTION

This invention provides a method of increasing Akt activation in a cell, comprising transducing an Akt-containing cell with a vector that expresses exogenous PIKE-A in the cell. When applied to cancer cells, this method increases the susceptibility of the cancer cell to infection by a myxoma virus and replication of myxoma virus in the cell.

This invention provides a method of treating a subject having cancer, comprising administering to the subject: (a) a vector that infects and expresses exogenous

PIKE-A in cancer cells in the subject; and (b) a myxoma virus, in a combined amount effective to treat the subject. This invention provides a method of treating a subject having cancer, comprising: (a) transducing cancer cells from the subject ex vivo with a vector that expresses exogenous PIKE-A in the cells; (b) administering a myxoma virus to the transduced cells; and (c) returning the transduced cells to the subject.

This invention provides a method of identifying cells susceptible to infection with a myxoma virus, comprising assaying a test cell for PIKE-A expression, wherein the expression of PIKE-A in the cell indicates susceptibility to myxoma virus infection.

DESCRIPTION OF THE FIGURES

Fig. 1. M-T5 exhibits sequence similarity to PIKE-A. (A) Alignment of the N- terminal sequences from MV M-T5 (1-105 aa) and PIKE-A (1-129 aa). Conserved residues are boxed. Dark shading indicates identical resides and light shading indicates similar residues. The bars above the M-T5 sequence define the predicted ankyrin repeats I and II. (B) M-T5 features including the predicted ankyrin repeats (I-VII) and F-box; compared to PIKE-A structure (9). The underlined sections indicate the regions of PIKE-A sufficient to bind Akt. The left line matches the aa sequence alignment in part A.

Fig. 2. Expression of cellular PIKE-A rescues MV infection in restrictive tumor cells. (A) The MV restrictive Type II human renal cancer cells (786-0) and the MV-nonpermissive Type III abortive breast cancer cells (MDA-MB435) were transfected with a Myc-tagged PIKE-A expressing plasmid (lanes 2, 4, 7 and 9) or mock transfected (lanes 1, 3, 5, 6, 8 and 10) for 12 hours and then mock infected (lane 1) or infected with either vMyxlac (lanes 2,3, 7 and 8) or vMyxT5KO (lanes 4, 5, 9 and 10) at an MOI of 5. Cell samples were collected at 48 hpi and cell lysates were examined by immunoblotting with anti-Serp-1 (late viral gene) and anti-Myc (PIKE-A). Single-step growth analysis of (B) HOS (C) 786-0 and (D) MDA-MB435 cells were transfected with PIKE-A (D) for 12 hours or mock transfected (■) and then infected with either vMyxlac (— ) or vMyxTSKO ( — ) at a MOI of 5. Cells were harvested at the indicated times post infection and infectious virus titers were determined on BGMK cells. Each viral growth analysis was performed in triplicate.

Fig. 3. PIKE-A upregulates Akt phosphorylation in Type II (786-0) cells infected with vMyxT5KO and stimulates Akt phosphorylation in Type III (MDA-MB435) cells infected with either vMyxlac or vMyxT5KO. Human 786-0 (Type II) cancer cells (A and B) and breast cancer MDA-MB435 (Type III) cells (C and D) were either transfected with PIKE-A plasmid (B and D) or mock transfected (A and C) and 12 hours later were infected with either vMyxlac (lanes 1-6) or vMyxT5KO (lanes 7-12) at an MOI of 5. Cells were harvested at the indicated times post infection and Akt phosphorylation, at both p-Akt Ser-473 and p-Akt Thr-308 sites, was detected in cell lysates (50 μg per lane). Total Akt protein levels are shown in the bottom lines.

Fig. 4. PIKE-A inhibits activation of apoptosis in Type II (786-0) cells infected with vMyxT5KO and Type III (MDA-MB435) cells infected with vMyxlac and vMyxT5KO. Human renal cancer 786-0 (Type II) cells (A and B) and breast cancer MDA-MB435 (Type III) cells (C and D) were transfected with PIKE-A plasmid (B and D) or mock transfected (A and C) and 12 hours later were infected with either vMyxlac (lanes 1-6) or vMyxT5K0 (lanes 7-12) at an MOI of 5. Cells were harvested at the indicated times post infection and cell lysates (50 μg per lane) were probed for pro-caspase-3 degradation by immunoblotting.

Fig. 5. Exogenous over-expression of PIKE-A induces MV replication in previously non-permissive human cancer cells. Human Type II (786-0) and Type III (MDA-MB435) cancer cells were either transfected with PIKE-A plasmid or mock transfected and 12 hours later were infected with either vMyxgfp or vMyxT5KOgfp at an MOI of 0.01. Foci were viewed under a Leica fluorescent microscope at 48 hpi to assess viral replication. BGMK cells infected with vMyxgfp or vMyxT5KOgfp at an MOI of 0.01 were used as positive controls. Expected (+) or not expected (-) MV replication is indicted below individual panels.

Fig. 6. Induction of endogenous Akt phosphorylation following transfection of PIKE-A in human cancer cells. The PIKE-A plasmid was transfected into (a) 786- 0 and (b) MDA-MB435 cells and Akt phosphorylation at both p-Akt Ser-473 and p-Akt Thr-308 sites were detected in cell lysates (50 μg per lane) by using immunoblotting at various time points (0-48 h). Total Akt protein levels are shown in the bottom lines.

DETAILED DESCRIPTION

In the method of this invention for increasing Akt activation in a cell or for increasing the susceptibility of a cancer cell to myxoma virus infection comprising transducing an Akt-containing cell with a vector that expresses exogenous PIKE- A in the cell, any conventional type of vector can be used. Examples of suitable vectors include a plasmid or a virus. For in vivo transduction a virus is preferred, for example a myxoma virus. When a myxoma virus is to be administered as well, it is preferred that the myxoma virus itself also serve as the vector to introduce exogenous PIKE-A in to the cells. The transduction can be performed either ex vivo or in vivo. In vivo transduction can be performed in a human or in a non-human mammal.

Although the method of this invention can increase Akt activation in all types of cells, it is most useful in cases where the cell has low levels of Akt activation prior to expression of the exogenous PIKE-A. In a more specific embodiment the cell has substantially no Akt activation prior to expression of the exogenous PIKE-A. As used herein "low levels of Akt activation" means cells that are permissive to myxoma virus that is positive for M-T5 but not to M-T5 knockkout myxoma virus. Such cells are also referred to herein as Type II cells. "Substantially no Akt activation" refers to cells that are non-permissive to both M-T5 positive myxoma virus and M-T5 knockkout myxoma virus. Such cells are also referred to herein as Type III cells. The cells used in this method can be normal cells, but preferably are cancer cells, still more preferably human cancer cells. Examples of cells that have high endogenous p-Akt and are permissive for both M-T5 positive and M-T5 knockout myxoma viruses (also referred to herein as Type I cells) include normal cell lines RX-13 (kidney, rabbit), BGMK (kidney, primate), HEK293 (kidney, human), and the cancer cell lines HOS (osteocaroma), Caki-1 (renal cancer), PC3 (prostate cancer) and T47D (breast cancer). Examples of cells that have low levels of Akt activation include HCTl 16 (colon cancer), 786-D (renal cancer), ACHN (renal cancer), SK-OV-3 (ovarian cancer) and U373 (glioma). Examples of cells that have substantially no Akt activation include MCF-7 (breast cancer), COLO205 (colon cancer), MDA-MB435 (breast cancer) and SK-MEL5 (melanoma).

The methods of this invention for treating a subject having cancer are useful for treating any subject. In a more specific embodiment of this invention the subject is a mammal, either a human or a non-human mammalian subject.

In the method of this invention for treating a subject having cancer, comprising: (a) transducing cancer cells from the subject ex vivo with a vector that expresses exogenous PIKE-A in the cells; (b) administering a myxoma virus to the transduced cells; and (c) returning the transduced cells to the subject, step (b) can be performed either before or after step (c). The method of this invention for identifying cells susceptible to infection with a myxoma virus, is applicable to normal cells as well as cancer cells. In a specific embodiment the cancer cell is a human cancer cell.

A number of cellular pathways, frequently mutated in cancer, were examined in MV permissive human cancer cells and the Akt pathway was identified to be a key restriction determinant for virus replication (7). The oncogene, Akt is a critical regulator of diverse cellular processes and has been demonstrated to contribute to cancer progression through stimulation of proliferation and inhibition of apoptosis (12, 13). The critical role of Akt in the regulation of multiple cellular functions makes it a central manipulator of cellular signaling and therefore it is not surprising a number of viruses have developed sophisticated strategies to manipulate the activation of Akt (14). For example, respiratory syncytial virus (RSV) induces activation of the PI-3K/Akt signaling pathway during early viral infection (15), thereby increasing cell survival and ensuring the virus has sufficient time to complete its replicative cycle (16). MV also manipulates the Akt pathway through the actions the protein M-T5 which has been shown to bind and upregulate the kinase activity of Akt during MV infection (7). Akt kinase activity is upregulated through direct binding to a recently identified cellular protein, PIKE-A. Over expression of PIKE-A stimulates Akt activity promoting cellular transformation leading to the development of cancer, while knock-down of PIKE-A diminishes Akt activity and increases apoptosis. Amplified PIKE-A has been identified in a number of human glioma cancer cells which express increased levels of Akt phosphorylation and reduced activation of apoptosis (10).

Analysis of permissiveness of human cancer cells to MV indicated that the cells fell into three categories (a) Type I cells possessed endogenous activated Akt and were permissive to MV and vMyxT5KO, (b) Type II cells had low levels of Akt activation and were permissive to MV, but not to vMyxT5KO, and (c) Type III cells had no activated Akt and were non-permissive to both MV and vMyxT5K0. Thus, if Akt was pre-activated or could be activated by MV infection via M-T5, the cancer cells were permissive but if Akt remained unactivated the cells were non-permissive for MV infection.

Restrictive Type II human cancer cells will switch from resistant to susceptible for vMyxT5KO infection following transient expression of PIKE-A. In the absence of M-T5, MV is unable to stimulate kinase activity of Akt, however over-expression of exogenous PIKE-A in Type II cells considerably increased levels of Akt phosphorylation at both Ser-473 and Thr-308 sites (Fig. 3). Type III human cancer cells are non-permissive to both vMyxlac and vMyxTSKO infection and Type III cells do not express basal levels of detectable, endogenous phosphorylated Akt (Sup. Fig. 2). Similar to the observation in Type II cells, expression of PIKE-A renders non-permissive Type III cells susceptible for both vMyxlac and vMyxT5K0 infection and upregulates Akt kinase activity (Fig. 2 and 3). The fact that exogenous PIKE-A rescues MV replication in previously non-premissive human cancer cells only strengthens the argument that the Akt pathway is a key restriction determinant for permissiveness of human cancer cells by MV. In addition to stimulating Akt kinase activity, transfection of PIKE-A was responsible for inhibiting activation of viral-induced apoptosis following MV infection in Type II and III human cancer cells. M-T5 also inhibits MV-induced apoptosis by protecting MV-infected cells from cell cycle arrest which otherwise would promote the activation of the apoptotic cascade (5). Sequence similarity between MV M-T5 and cellular PIKE-A is limited to the previously identified region of PIKE-A necessary to bind Akt. However, both M-T5 and PIKE-A contain ANK repeats, share the ability to upregulate the Akt pathway, block apoptosis and to interact with Akt. Functionally, M-T5 and PIKE-A represent a viral and cellular molecule evolved to control Akt activation.

A number of virus encoded proteins, several of which are host-related immunomodulatory genes, share sequence and functional similarity with cellular proteins and are categorized as viral homologs. Many of these viral homologs are gene products which have been hijacked from their host, increasing the replicative ability of the virus (17). Although M-T5 and PIKE-A share similar functions, based on this preliminary study we would predict that M-T5 represents a viral strategy evolved to mimic the cellular activity of PIKE-A. Exogenous PIKE-A is able to upregulate Akt kinase activity and rescue MV replication in Type III human cancer cells. Interaction of Type III cells with MV does not produce a productive infection even though M-T5 is expressed. It is likely that M-T5 expression and localization is altered during infection of Type III cells (7). Therefore, the mechanism by which PIKE-A activates Akt may exhibit some differences from the method employed by M-T5.

EXAMPLES

Materials and Methods

Cell culture. Established cell lines used in this study included baby green monkey kidney (BGMK) cells and the human tumor cell lines; HOS, 786-0 and MDA- MB435 which were obtained from the NCI-reference collection. All cells were propagated in 5% CO₂ at 37°C in Dulbecco's Modified Eagle Medium (DMEM) completed with 10% fetal bovine serum (FBS, Sigma), 100 units penicillin/ml and 100 μg streptomycin/ml (Invitrogen).

Viruses and Infection. Recombinant viruses used in this study have been described previous and include; vMyxlac, a control MV (strain Lausanne) that expresses β- gal and wild-type M-T5 (18), and vMyxT5KO, which also expresses β-gal but fails to express M-T5 due to targeted disruption of both copies of the M-T5 open reading frame (ORF) (M005R/L) (4). Other myxoma viruses used in this study include vMyxgfp and vMyxT5KOgfp which both contain the EGFP cassette (19). All viruses were propagated and titrated by focus formation on BGMK cells as described previously (18). For infection studies, cells were incubated with the indicated MOI of either virus for 1 h at 37°C, infected cells were then washed three times with phosphate-buffered saline (PBS) to remove excess virus and cultured in normal medium until used in subsequent experiments, β-galactosidase staining has been described previously (18).

Transfection of PIKE-A. Cells were seeded in six-well plates at a density of 5 x 10⁵ cells per well in complete growth medium with 10% FBS. Transfections were performed with LipofectAMINE 2000 (Invitrogen) in accordance with the manufacturer's instructions. HOS, 786-0 or MDA-MB435 cells were transfected with the plasmid myc-PIKE-A which has previously been described and was the kind gift of Dr. Ye (8, 10) or the control vector pcDNA3 (5 μg). The cells were collected at various time points. The lysate was used for detection with appropriate antibodies.

Viral growth curves. For single-step growth analysis HOS, 786-0 and MDA- MB435 cells (5 X 10⁵) were either mock transfected or transfected the myc-PIKE- A plasmid. The following day cells were infected with vMyxlac or vMyxT5KO at an MOI of 5 for I h.

Unabsorbed virus was removed by washing the cells with serum-free medium three times, and cells were grown in complete growth medium supplemented with 10% FBS. Cells were harvested following infection at the indicated time points: 0, 4, 8, 12, 24 and 48 hours. Virus titers were determined by serial dilution and infection of BGMK cells followed by X-GaI staining of fixed monolayers, as outlined previously (18). All growth analyses were performed in triplicate, and data were expressed as loglO FFU per 10⁵ cells.

Immunoblot analysis. Cultured cells were collected and cell lysis was prepared as previously described (5). Samples were separated by sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE) and immunoreactive proteins were detected by chemiluminescence (Perkin-Elmer, Boston, MA). Antibodies used included mouse monoclonal antibody of anti-Serpl, and rabbit polyclonal antibody of T-7 which have been described (5, 20, 21) Rabbit polyclonal phospho- Akt (Thr-308), mouse monoclonal phospho-Akt (Ser-473) (587Fl 1) antibody, and polyclonal Akt antibody that detects total levels of endogenous Aktl, Akt2, and Akt3 proteins (Cell Signaling Technology). Horseradish peroxidase-conjugated goat-anti-mouse and goat-anti-rabbit secondary antibodies were obtained from Jackson ImmunoResearch Laboratories. Results

M-T5 sequence exhibits similarity to cellular PIKE-A.

It has been demonstrated that M-T5 forms a complex with cellular Akt and as a result upregulates its kinase activity in MV infected human tumor cells (7). A database search identified the cellular protein PIKE-A, a physiological mediator of Akt which functions to stimulate Akt activation through direct binding (8). Sequence alignment between MV M-T5 and PIKE-A identified some sequence similarity between M-T5 and the region necessary for PIKE-A binding of Akt (Fig. IA). PIKE-A has two domains that contribute to Akt binding. The carboxyl terminus, which has two ANK repeats and the NH2 terminus 128 residues both interact with Akt independently (Fig. IB). The fact that PIKE-A contains ANK repeats, binds to Akt, blocks apoptosis in cancer cells were all features we have observed for MV M-T5 and this led us to investigate whether M-T5 represented a viral molecule that had adopted functions similar to cellular PIKE-A.

Transient expression of PIKE-A rescues MV replication in restrictive human tumor cells.

A wide spectrum of human tumor cell lines were screened for their ability to support MV replication and we observed that MV permissivity is dependent on the basal level of endogenous phosphorylated Akt (1, 7). Since PIKE-A has been demonstrated to bind and upregulate the kinase activity of Akt we wanted to determine if PIKE-A was able to influence the permissiveness of MV in various cancer cell types. For this study the following human cancer cells lines were used as representatives for the three cell types; HOS, human osteocarcoma (Type I), 786-0, renal cancer (Type II) and MDA-435, breast cancer (Type III). 786-0 and MDA-MB435 cells were mock transfected or transfected with a plasmid containing PIKE-A for 12 hours and then mock infected or infected with either vMyxlac or vMyxT5KO at a multiplicity of infection (MOI) of 5. Cell lysates were collected at 48 hours post infection (hpi) and expression levels of the late MV gene Serp-1 were assessed by Western blotting. Because poxvirus late genes require active virus replication to undergo expression our lab uses the MV late gene Serp-1 as a marker to represent successful virus replication. Based on the presence of Serp-1, 786-0 cells supported replication of vMyxlac (Fig 2A, lane 3) however 786-0 cells did not support vMyxT5KO replication (Fig 2A, lane 5) because the late gene product, Serp-1 was not detected. MDA-MB435 cells did not support replication of either virus (Fig 2A, lane 8 and 10). When both 786-0 (Fig 2A, lanes 2 and 4) and MDA-MB435 (Fig 2 A, lanes 7 and 9) cells were transfected with PIKE-A and infected with either vMyxlac or vMyxT5K0 expression of Serp-I was detected, suggesting that expression of PIKE-A preceding MV infection was able to rescue previously non-productive infection and allow virus replication. Expression of Serp-1 was not detected in the mock- infected cells (Fig 2A, lanes 1 and 6) for either cell line. Samples were probed with an antibody against Myc to demonstrate expression of the transfected Myc- tagged PIKE-A. Myc epitope was only detected in cells transfected with the PIKE-A plasmid (Fig 2A, lanes 2, 4, 7 and 9). Expression of MV Serp-1 protein was therefore observed in PIKE-A expressing cells which were previously non- permissive to MV-infection.

To quantitatively assess the ability of the virus to replicate, single-step growth curves on representative Type I, II and III cells were performed. Each cell type was mock transfected or transfected with PIKE-A and 12 hours later were infected with either vMyxlac or vMyxT5KO at an MOI of 5. Samples were harvested for infectious virus particles at 0, 4, 8, 12, 24 and 48 hpi, and all time point samples were titrated on BGMK cells by serial dilutions and stained with X-gal at 48 hpi to visualize foci. Infection of HOS cells (Type I) with vMyxlac and vMyxT5K0 produced growth curves characteristic of a classical poxvirus replication kinetics. A minimum virus titer was reached at approximately 4 hpi followed by a continuous increase up to 48 hpi, at which point the virus yield reached maximal levels. Identical replication curves were generated for vMyxlac infection of HOS cells regardless of the expression of PIKE-A. However, HOS cells infected with the knockout virus (vMyxT5K0) produced a slightly lower yield, nevertheless transfection of PIKE-A before infection restored virus titer to a level similar to that of cells infected with vMyxlac (Fig. 2B). Type II cells (786-0) completely supported vMyxlac infection but were non- permissive to vMyxT5KO. When 786-0 cells were transfected with PIKE-A and then infected with vMyxT5KO, the viral titers indicated that transfection of PIKE- A, before infection, was able to rescue vMyxT5K0 replication to levels similar to cells infected with vMyxlac (Fig. 2C).

Virus replication of either vMyxlac or vMyxT5KO was not supported in the Type III cells (MDA-MB435) and as a result little or no viral amplification was observed overtime. Viral titers were increased considerably when cells were transfected with PIKE-A prior to infection indicating rescue of virus replication (Fig 2D). The ability of PIKE-A to rescue viral replication and focus formation of vMyxT5KO in the restrictive Type II cells and restore permissiveness of both vMyxlac and vMyxT5K0 in the abortive Type III cells was also observed when cells were transfected with PIKE-A and infected with viruses which express vMyxgfp or vMyxT5KOgfp (Fig. 5.). Theses data indicate that over-expression of exogenous PIKE-A has the ability to render previously restricted human cancer cells permissive to MV infection.

Transient expression of PIKE-A upregulates the kinase activity of Akt in Type II and Type III human cancer cells.

Amplification of PIKE-A has been observed in a variety of human glioblastoma cells which coincidently results in the upregulation of Akt kinase activity (10). To determine if over-expression of PIKE-A would have the ability to increase kinase activity of Akt, Type II cells (786-0) and Type III cells (MDA-MB435) were transfected with PIKE-A. Cell lysates were collected at various time points following transfection of PIKE-A and Akt phosphorylation was assessed by Western blotting. Expression of exogenous PIKE-A induced the phosphorylation of Akt at both Ser-473 and Thr-308 sites in 786-0 and MDA-MB435 cell lines (Fig. 6.).

Susceptibility of human cancer cells to MV infection is dependent upon the basal level of endogenous phosphorylated Akt (7). In the 786-0 cells (Type II) the level of endogenous phosphorylated Akt was shown to be very low, however infection with vMyxlac dramatically induced Akt phosphorylation. The viral protein M-T5 is a critical determinant of MV tropism in human cancer cells and in its absence endogenous levels of phosphorylated Akt remained relatively unchanged in vMyxT5KO infected 786-0 cells. As over-expression of PIKE-A increases Akt kinase activity we wanted to examine levels of phosphorylated Akt following vMyxlac or vMyxT5KO infection in the presence or absence of exogenous PIKE- A. Cells were mock transfected or transfected with the PIKE-A plasmid for 12 hours and then infected with either vMyxlac or vMyxT5K0. Cell lysates were collected at various time points and phosphorylation of Akt was assessed by Western blotting. Increased levels of endogenous phosphorylated Akt at both Ser- 473 and Thr-308 sites were detected at 8 hpi in 786-0 cells infected with vMyxlac (Fig. 3 A and B, lanes 1-6). 786-0 cells infected with vMyxT5K0 exhibited very little Akt phosphorylation (Fig. 3A, lanes 7-12) yet transfection of PIKE-A prior to infection by vMyxT5KO considerably induced phosphorylation of Akt at both Ser-473 and Thr-308 sites compared to levels observed in the absence of exogenous PIKE-A (Fig. 3B, lanes 7-12). The levels of total Akt protein remained relatively constant following vMxylac and vMyxT5KO infection (Fig. 3A and B). Over-expression of PIKE-A, in Type II cells (786-0), was able to upregulate the kinase activity of Akt, which is critical for the replication of MV even in the absence of M-T5.

The basal level of endogenous phosphorylated Akt was undetectable in Type III cells (MDA-MB435), which do not support replication of either vMyxlac or vMyxT5KO. Levels of Akt phosphorylation remained undetectable following vMyxlac and vMyxT5K0 infection (Fig. 3 C, lanes 1-12) suggesting that M-T5 is unable to activate Akt kinase activity in Type III cells. Transfection of PIKE-A in MDA-MB435 cells prior to infection with either vMyxlac (Fig 3D, lanes 1-6) or vMyxT5KO (Fig 3D, lanes 7-12) dramatically induced phosphorylation of endogenous Akt at Ser-473 and Thr-308 sites at 8 hpi. Total Akt protein levels remained relatively unchanged (Fig. 3D) as observed in the 786-0 cells. Together these data indicate that in cell lines infected with MV, in which either M-T5 is not present (vMyxT5K0) or is present but unable to activate Akt (Type III cells), then ectopic expression of PIKE-A will activate Akt and rescue MV infection. These results explain the rescue of viral infection observed in Figures 2C and 2D.

PIKE-A inhibition of apoptosis in MV-infected human cancer cells

Following MV infection of human cancer cells a cascade of events, which induce apoptosis are initiated. When M-T5 is present and expressed it plays a critical role in preventing apoptosis through manipulation of the cell cycle (5). PIKE-A, a physiological regulator of Akt activation (11), is often amplified in human cancer cells and coincidently has been shown to promote cellular proliferation by inhibiting apoptosis through stimulation of Akt (10). Since transient expression of PIKE-A in Type II and Type III human cancer cells stimulates phosphorylation of Akt (Fig. 3) it was considered desirable to determine if over-expression of PIKE- A also functioned to inhibit apoptosis induction following MV infection. To investigate this possibility Type II (786-0) and Type III cells (MDA-MB435) were infected with either vMyxlac or vMyxT5KO and cleavage of procaspase-3 to its active form was assessed by Western blotting. As expected from our previous studies in which infection of 786-0 cells with MV did not induce apoptosis (5), when Type II cells (786-0) were infected with vMyxlac no cleavage of procaspsae-3 was observed (Fig. 4A, lanes 1 -6), in Type II cells (786-0) infected with vMyxT5KO induced activation of caspase-3 was observed as early as 8 hpi (Fig. 4A, lanes 7-12). However, when Type II cells were transfected with PIKE- A 12 hours prior to infection with vMyxT5KO, cleavage of caspase-3 was not detected, indicating that inhibition of caspase-mediated apoptosis even in the absence of M-T5 (Fig. 4B, lanes 7-12).

Induction of apoptosis as indicated by caspase-3 cleavage in Type III cells (MDA- MB435) was observed when cells were infected with either vMyxlac or vMyxT5K0 (Fig. 4C, lanes 1-12). Exogenous expression of PIKE-A was successful at inhibiting apoptosis following infection with either vMyxlac or vMyxT5KO by preventing the cleavage of procaspase-3 into its active form (Fig. 4D, lanes 1-12). Therefore, transfection of PIKE-A in Type II cells was able to inhibit vMyxT5KO-induced apoptosis in Type II cells. Viral-induced apoptosis was also inhibited in Type III cells infected with either vMyxlac or vMyxT5K0 when the cells had previously been transfected with PIKE-A. Stimulation of Akt kinase activity, in response to over-expression of PIKE-A, promotes the inhibition of the apoptotic signaling cascade, which would otherwise be activated in MV infected cells (Type III), especially in the absence of M-T5 (Type II and Type III cells).

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Claims

CLAIMSWhat is claimed is:

1. A method of increasing Akt activation in a cell, comprising transducing an Akt-containing cell with a vector that expresses exogenous PIKE-A in the cell.

2. The method of claim 1, wherein the vector is a plasmid.

3. The method of claim 1 , wherein the vector is a virus.

4. The method of claim 2, wherein the virus vector is a myxoma virus.

5. The method of claim 1 , wherein the transduction is performed ex vivo.

6. The method of claim 1 , wherein the transduction is performed in vivo.

7. The method of claim 6, wherein the transduction is performed in a human.

8. The method of claim 1, wherein the cell has low levels of Akt activation prior to expression of the exogenous PIKE-A.

9. The method of claim 1, wherein the cell has substantially no Akt activation prior to expression of the exogenous PIKE-A.

10. A method of increasing the susceptibility of a cancer cell to myxoma virus infection and replication, comprising the method of any one of claims 1 to 8.

11. The method of claim 10, wherein the cancer cell is a human cancer cell.

12. A method of treating a subject having cancer, comprising administering to the subject: (a) a vector that infects and expresses exogenous PIKE-A in cancer cells in the subject; and (b) a myxoma virus, in a combined amount effective to treat the subject.

13. The method of claim 12, wherein the subject is a human.

14. A method of treating a subject having cancer, comprising: (a) transducing cancer cells from the subject ex vivo with a vector that expresses exogenous PIKE-A in the cells; (b) administering a myxoma virus to the transduced cells; and (c) returning the transduced cells to the subject.

15. The method of claim 14, wherein step (b) is performed before step (c).

16. The method of claim 14, wherein step (c) is performed before step (b).

17. A method of identifying cells susceptible to infection with a myxoma virus, comprising assaying a test cell for PIKE-A expression, wherein the expression of PIKE-A in the cell indicates susceptibility to myxoma virus infection.

18. The method of claim 17, wherein the cell is a cancer cell.

19. The method of claim 18, wherein the cell is a human cancer cell.