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Materials and methods for inhibiting wip1

Materials and methods for inhibiting wip1 description/claims

This invention pertains to isolated or purified oligonucleotides, isolated or purified morpholino oligomers, a method of detecting cancer or a predisposition to cancer, as well as a method of treating cancer. The present invention further pertains to a method of screening an oligonucleotide or morpholino oligomer for the ability to inhibit Wip1 expression and a method of screening a compound for inhibiting Wip1 activity, in addition to a method of determining the efficacy with which an oligonucleotide or morpholino oligomer inhibits Wip1 expression and a method of determining the efficacy with which a test compound inhibits Wip1 phosphatase activity.

Wild-type p53-induced phosphatase 1 (Wip1) is a Mg2+-dependent serine/threonine protein phosphatase that is expressed in response to ionizing or ultra-violet (UV) radiation in a manner that is dependent on the tumor suppressor gene product p53. Its role in cancer was first suggested by Fiscella et al., Proceedings of the National Academy of Sciences, U.S.A. 94: 6048-6053 (1997), which reported Wip1 as an important inhibitor of growth, since ectopic expression of WIP1 (also known as PPMD1) in a human glioblastoma cell line (T98G) resulted in fewer colonies of cells. In contrast to these results, Wip1 was shown by Takekawa et al., EMBO Journal 19(23): 6517-6526 (2000), to dephosphorylate the kinase p38, which functions to activate p53 for the induction of apoptosis and transcription in response to environmental stress, thereby rendering Wip1 anti-apoptotic as opposed to anti-proliferative.

Consistent with Takekawa et al., Wip1 has been shown by the present inventors to be a positive regulator of tumorigenesis. WIP1, located at chromosome 17q22/q23 by FISH analysis, was found amplified in human breast tumor cell lines as well as in approximately 11% of primary breast tumors as determined by Northern blot analysis, Southern blot analysis, and tissue microarray analysis. Furthermore, exogeneous expression of WIP1 in cells expressing H-Ras-V12 resulted in a decrease in p53-mediated apoptosis and a partial rescue of these cells from cell cycle arrest. Moreover, Wip1 was demonstrated as a negative regulator of p38, since UV-induced activation of p38 kinase was significantly attenuated in breast cell lines in which the Wip1 gene was amplified and overexpressed (BT-474 and MCF7) compared to a breast line (MA-MB436) without Wip1 amplification. Taken together, these results indicate that WIP1 is a candidate proto-oncogene involved in tumorigenesis and, thus, represents an attractive new target for cancer therapy.

In view of the foregoing, the present invention provides materials and methods for treating cancer in a mammal that expresses elevated levels of Wip1. These and other advantages of the invention, as well as additional inventive features, will be apparent from the description of the invention provided herein.

The present invention provides an isolated or purified oligonucleotide consisting essentially of the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 2, as well as an isolated or purified morpholino oligomer consisting essentially of the sequence of SEQ ID NO: 1 or SEQ ID NO: 2.

The present invention further provides a method of detecting cancer or a predisposition to cancer in a mammal. The method comprises comparing the level of expression of Wip1 in a test sample comprising Wip1 obtained from the mammal to the level of expression of Wip1 in a control sample. A higher level of expression of Wip1 in the test sample as compared to the control sample is indicative of cancer or a predisposition to cancer in the mammal.

Further provided by the present invention is a method of treating cancer in a mammal that expresses the same level or a higher level of Wip1 as compared to a mammal of the same species that does not have cancer. The method comprises administering to the mammal a cancer-treating effective amount of a Wip1 inhibitor.

The present invention also provides a method of screening an oligonucleotide or morpholino oligomer for the ability to inhibit the expression of Wip1. The method comprises comparing the level of expression of Wip1 in a test sample obtained from Wip1-expressing cells that have been contacted with the oligonucleotide or morpholino oligomer to the level of expression of Wip1 in a control sample obtained from Wip1-expressing cells that have not been contacted with the oligonucleotide or morpholino oligomer. A lower level of expression of Wip1 in the test sample as compared to the control sample is indicative of the ability of the oligonucleotide or morpholino oligomer to inhibit the expression of Wip1.

A method of determining the efficacy with which a test oligonucleotide or morpholino oligomer inhibits Wip1 expression is further provided by the present invention. The method comprises comparing the level of expression of Wip1 in a test sample obtained from Wip1-expressing cells that have been contacted with the test oligonucleotide or morpholino oligomer to the level of expression of Wip1 in a control sample obtained from Wip1-expressing cells that have been contacted with an oligonucleotide or morpholino oligomer that is known to inhibit the expression of Wip1. A lower level of expression of Wip1 in the test sample as compared to the control sample is indicative of the test oligonucleotide or morpholino oligomer having a greater efficacy for inhibiting the expression of Wip1 than the known oligonucleotide or morpholino oligomer, whereas an higher level of expression of Wip1 in the test sample as compared to the control sample is indicative of the test oligonucleotide or morpholino oligomer having a lower efficacy for inhibiting the expression of Wip1 than the known oligonucleotide or morpholino oligomer.

Further provided is a method of screening a compound for Wip1-inhibiting activity. The method comprises comparing the level of Wip1 phosphatase activity in a test sample obtained from Wip1-expressing cells that have been contacted with the compound to the level of Wip1 phosphatase activity in a control sample obtained from Wip1-expressing cells that have not been contacted with the compound. A lower level of Wip1 phosphatase activity in the test sample as compared to the control sample is indicative of the ability of the compound to inhibit Wip1.

The present invention also provides a method of determining the efficacy with which a test compound inhibits Wip1. The method comprises comparing the level of Wip1 phosphatase activity in a test sample obtained from Wip1-expressing cells that have been contacted with the test compound to the level of Wip1 phosphatase activity in a control sample obtained from Wip1-expressing cells that have been contacted with a compound that is known to inhibit Wip1. A lower level of Wip1 phosphatase activity in the test sample as compared to the control sample is indicative of the test compound having a greater efficacy for inhibiting Wip1 than the known compound, whereas a higher level of Wip1 phosphatase activity in the test sample as compared to the control sample is indicative of the test compound having a lower efficacy for inhibiting Wip1 than the known compound.

FIG. 1 represents a table of sequences (5′→3′ when read from left to right) of the oligonucleotides or morpholino oligomers of the present invention and the regions to which they hybridize.

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