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Phenotypic reversion of pancreatic carcinoma cells

Phenotypic reversion of pancreatic carcinoma cells description/claims

This application is continuation application of U.S. Ser. No. 11/142,051, filed May 31, 2005, which claims the benefit of U.S. Provisional Application Serial No. 60/575,131, filed May 28, 2004, and U.S. Provisional Application Serial No. 60/575,846, filed June 1, 2004, both of which are incorporated by reference herein.

The present invention was funded in part by NIH Grant RO1 CA 42500; the government may have certain rights in the invention.

Oncogenic ras-p21 protein, but not its wild-type counterpart protein, induces malignant transformation of mammalian cell lines such as NIH 3T3 cells (1) and has been implicated as a major causative factor in a high proportion of human solid tissue tumors (2). In Xenopus laevis oocytes, microinjection of oncogenic (containing Val in place of Gly 12), but not wild-type, p21 induces oocyte maturation (3). Insulin induces oocyte maturation and requires activation of normal cellular ras-p21 (4).

Several agents that strongly block Val 12-p21-induced oocyte maturation have virtually no effect on insulin-induced maturation (5). Among these agents are specific peptides, identified from molecular modeling studies, that correspond to effector domains from both ras-p21 itself, such as the 35-47, 96-110 and 115-126 sequences (5) and from some of its target proteins such as the ras-binding domain of raf(residues 97-110) (6-8) and the SOS guanine nucleotide exchange protein (residues 994-1004) (9,10). These peptide domains were identified as those that change conformation in response to the presence of single oncogenic amino acid substitutions at positions 12 or 61 or multiple substitutions at positions 10, 12 and 59 when the computed average structures for these proteins either alone or in complex with target proteins were superimposed on that for the wild-type protein.

The finding that these peptides (in addition to other agents) block oncogenic ras-p21 selectively indicates that the oncogenic protein induces mitogenesis by pathways that may overlap with, but are also distinct from, pathways utilized by the wild-type protein. In studies designed to identify pathway differences, it was found that, in oocytes, oncogenic but not insulin-activated wild-type ras-p21 interacts with the transcriptional activating protein, jun and its kinase, jun kinase (JNK) (11,12), and requires the presence of protein kinase C (PKC) (13). In these studies, it was determined that the peptide whose sequence corresponds to p21 residues 96-110, called PNC-2, blocks the interaction of Val 12-p21 with JNK (11,12) in a dose-response curve that superimposes on that for its inhibition of Val 12-p21-induced oocyte maturation (5).

Additionally, the peptide whose sequence corresponds to p21 residues 35-47, called PNC-7, encompasses a domain of the protein implicated in its interacting with multiple targets including raf p74 protein, GTPase activating protein (GAP) and the guanine nucleotide exchange protein, SOS (reviewed in ref. 5). This peptide strongly inhibits c-raf-induced oocyte maturation but has no effect on oocyte maturation induced by an oncogenic mutant raf lacking the ras binding domain (RBD) in its amino terminal regulatory domain (14). Both PNC-2 and 7 appear to act on different steps on the oncogenic ras-p21 signal transduction pathway. For example, PNC-2 but not PNC-7 interferes with Val 12-p21-JNK interaction (11,12) while PNC-7 but not PNC-2 blocks signal transduction through c-raf(15).

Since various cancers involve expression of Val 12-p21 protein, as well as other oncogenic proteins, it would be useful to be able to inhibit expression of such proteins. For example, pancreatic cancer is a nearly always fatal disease with a median survival time of only 80-90 days for a patient diagnosed with the disease. Pancreatic cancer is one of the more lethal forms of cancer in numbers of patients killed in the U.S. Less than 4% of patients are alive 5 years from the time of diagnosis. The present invention provides peptides and pharmaceutical compositions comprising such peptides which when administered to pancreatic cancer cells, not only inhibit oncogenic Val 12-p21 but actually cause cancerous cells to phenotypically revert to non-cancerous cells. The present invention is therefore useful in treating various types of cancers which express Val 12-p21 protein and/or other oncogenic proteins. Treatment of ras-induced tumors converts malignant masses into benign ones, allowing for the halting of metastatic disease.

The present invention provides peptides comprising at least about ten contiguous amino acids of the amino acid sequence: YREQIKRVKDSDDVP (SEQ ID NO: 1), or an analog or derivative thereof, wherein said peptide, analog, or derivative thereof comprises a membrane-penetrating leader sequence attached thereto. Preferably, a peptide has the sequence set forth in SEQ ID NO: 1.

The present invention also provides peptides comprising at least about ten contiguous amino acids of the amino acid sequence: TEEDSYRKQVVID (SEQ ID NO:2) or an analog or derivative thereof wherein said peptide, analog, or derivative thereof comprises a membrane-penetrating leader sequence attached thereto. Preferably, a peptide has the sequence as set forth in SEQ ID NO:2.

The peptides of the present invention, including analogs and derivatives thereof, are useful in treating cancer. Preferably, a peptide, analog or derivative thereof as provided herein has the membrane-penetrating leader sequence located at the carboxy terminal end. In another preferred embodiment, the leader sequence comprises predominantly positively charged amino acid residues. Examples of leader sequences for practicing the present invention include but are not limited to penetratin, Arg8, TAT of HIV1, D-TAT, R-TAT, SV40-NLS, nucleoplasmin-NLS, HIV REV, FHV coat, BMV GAG, HTLV-II (REX), CCMV GAG, P22N, Lambda N, Delta N, yeast PRP6, human U2AF, human C-FOS, human C-JUN, yeast GCN4, or p-vec.

Also provided by the present invention are pharmaceutical compositions comprising at least one of the subject peptides or analogs or derivatives thereof comprising a membrane-penetrating leader sequence admixed with a pharmaceutically acceptable carrier.

The present invention also provides methods of treating a patient suffering from cancer. The method comprises administering to said patient a therapeutically effective amount of at least one subject peptide, analog or derivative thereof comprising a membrane penetrating leader sequence. In another embodiment of the invention, there is provided a method of treating a patient suffering from cancer by administering to said patient a therapeutically effective amount of a subject pharmaceutical composition. Preferably, the cancer to be treated is a ras-induced cancer.

In still another embodiment of the invention, there is provided a replication incompetent Adenovirus (AdV) vector comprising a promoter sequence operably linked to a nucleotide sequence encoding a peptide, wherein the peptide comprises at least about ten contiguous amino acids of the amino acid sequence: YREQIKRVKDSDDVP (SEQ ID NO: 1), or an analog or derivative thereof. A replication incompetent Adenovirus (AdV) vector comprising a promoter sequence operably linked to a nucleotide sequence encoding a peptide, wherein the peptide comprises at least about ten contiguous amino acids of the amino acid sequence: TIEDSYRKQVVID (SEQ ID NO:2), or an analog or derivative thereof is also provided. Preferably, the nucleotide sequence further encodes a leader sequence attached to the sequence set forth in SEQ ID NO: 1, 2, or an analog or derivative thereof.

The present invention also provides a method of treating a patient suffering from cancer by administering to the patient, a therapeutically effective amount of a subject AdV vector. A method of inducing phenotypic reversion of cancerous cells to non-cancerous cells in a subject, by administering to the subject, a therapeutically effective amount of a subject AdV vector is also provided.

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