MicroRNA-Based Methods and Compositions For the Diagnosis, Prognosis and Treatment of Breast Cancer

Abstract

The present invention provides novel methods and compositions for the diagnosis, prognosis and treatment of breast cancer. The invention also provides methods of identifying anti-breast cancer agents.

What is claimed is: 
     
         1 . A method of diagnosing a breast cancer associated with one or more prognostic markers in a subject, comprising:
 measuring the level of at least one miR gene product in a breast cancer sample from said subject,   wherein an alteration in the level of the at least one miR gene product in the test sample, relative to the level of a corresponding miR gene product in a control sample, is indicative of the subject having a breast cancer associated with the one or more prognostic markers,   wherein the breast cancer associated with one or more prognostic markers and the at least one miR gene product are selected from the group consisting of:   (i) the breast cancer is a breast cancer associated with estrogen receptor expression and the miR gene product is selected from the group consisting of miR-26a, miR-26b, miR-102 (miR-29b), miR-30a-5p, miR-30b, miR-30c, miR-30d, miR-185, miR-191, miR-206, miR-212, and combinations thereof;   (ii) the breast cancer is a breast cancer associated with progesterone receptor expression and the miR gene product is selected from the group consisting of let-7c, miR-26a, miR-29b, miR-30a-5p, miR-30b, miR-30c, miR-30d, and combinations thereof;   (iii) the breast cancer is a breast cancer associated with positive lymph node metastasis and the miR gene product is selected from the group consisting of let-7f-1, let-7a-3, let-7a-2, miR-9-3, and combinations thereof;   (iv) the breast cancer is a breast cancer associated with a high proliferative index and the miR gene product is selected from the group consisting of let-7c, let-7d, miR-26a, miR-26b, miR-30a-5p, miR-102, miR-145, and combinations thereof;   (v) the breast cancer is a breast cancer associated with detectable p53 expression and the miR gene product is selected from the group consisting of miR-16a, miR-128b and a combination thereof;   (vi) the breast cancer is a breast cancer associated with high vascular invasion and the miR gene product is selected from the group consisting of miR-9-3, miR-10b, miR-27a, miR-29a, miR-123, miR-205 and combinations thereof; and   (vii) the breast cancer is a breast cancer associated with an advanced tumor stage and the miR gene product is selected from the group consisting of miR-9-2, miR-15-a, miR-21, miR-30a-s, miR-133a-1, miR-137, miR-153-2, miR-154, miR-181a, miR-203, miR-213, and combinations thereof.   
     
     
         2 . The method of  claim 1 , further comprising: administering, to the subject, a pharmaceutical composition for treating breast cancer, wherein the pharmaceutical composition comprises: at least one miR expression inhibitor compound or at least one miR gene product and a pharmaceutically-acceptable carrier. 
     
     
         3 . The method of  claim 1 , comprising:
 reverse transcribing RNA from a test sample obtained from the subject to provide a set of target oligodeoxynucleotides;   hybridizing the target oligodeoxynucleotides to a microarray comprising miRNA-specific probe oligonucleotides to provide a hybridization profile for the test sample; and   comparing the test sample hybridization profile to a hybridization profile generated from a control sample,   wherein an alteration in the signal of at least one miRNA is indicative of the subject either having, or being at risk for developing, breast cancer.   
     
     
         4 . A method of treating breast cancer in a subject who has a breast cancer in which at least one miR gene product is down-regulated or up-regulated in the cancer cells of the subject relative to control cells, comprising:
 (1) when the at least one miR gene product is down-regulated in the cancer cells, administering to the subject an effective amount of at least one isolated miR gene product, provided that the miR gene product is not miR-15a or miR-16-1, such that proliferation of cancer cells in the subject is inhibited; or   (2) when the at least one miR gene product is up-regulated in the cancer cells, administering to the subject an effective amount of at least one compound for inhibiting expression of the at least one miR gene product, such that proliferation of cancer cells in the subject is inhibited.   
     
     
         5 . The method of  claim 4 , wherein the at least one isolated miR gene product in step (1) is selected from the group consisting of miR-145, miR-10b, miR-123 (miR-126), miR-140-as, miR-125a, miR-125b-1, miR-125b-2, miR-194, miR-204, let-7a-2, let-7a-3, let-7d (let-7d-v1), let-7f-2, miR-101-1, miR-143 and combinations thereof. 
     
     
         6 . The method of  claim 4  wherein the at least one miR gene product in step (2) is selected from the group consisting of: miR-21, miR-155, miR-009-1 (miR131-1), miR-34 (miR-170), miR-102 (miR-29b), miR-213, let-7i (let-7d-v2), miR-122a, miR-128b, miR-136, miR-149, miR-191, miR-196-1, miR-196-2, miR-202, miR-203, miR-206, miR-210, miR-213 and combinations thereof. 
     
     
         7 . The method of  claim 4 , further comprising:
 determining the amount of at least one miR gene product in breast cancer cells, relative to control cells; and   altering the amount of miR gene product expressed in the breast cancer cells by:   (i) administering to the subject an effective amount of at least one isolated miR gene product, provided that the miR gene product is not miR-15a or miR-16-1, if the amount of the miR gene product expressed in the cancer cells is less than the amount of the miR gene product expressed in control cells; or   (ii) administering to the subject an effective amount of at least one compound for inhibiting expression of the at least one miR gene product, if the amount of the miR gene product expressed in the cancer cells is greater than the amount of the miR gene product expressed in control cells,   such that proliferation of cancer cells in the subject is inhibited.   
     
     
         8 . The method of  claim 7 , wherein the at least one isolated miR gene product in step (i) is selected from the group consisting of: miR-145, miR-10b, miR-123 (miR-126), miR-140-as, miR-125a, miR-125b-1, miR-125b-2, miR-194, miR-204, let-7a-2, let-7a-3, let-7d (let-7d-v1), let-7f-2, miR-101-1, miR-143 and combinations thereof. 
     
     
         9 . The method of  claim 7 , wherein the at least one miR gene product in step (ii) is selected from the group consisting of miR-21, miR-155, miR-009-1 (miR-131-1), miR-34 (miR-170), miR-102 (miR-29b), miR-213, let-7i (let-7d-v2), miR-122a, miR-128b, miR-136, miR-149, miR-191, miR-196-1, miR-196-2, miR-202, miR-203, miR-206, miR-210, miR-213 and combinations thereof. 
     
     
         10 . A pharmaceutical composition for treating breast cancer, comprising at least one isolated miR gene product of  claim 8  and a pharmaceutically-acceptable carrier. 
     
     
         11 . A pharmaceutical composition for treating breast cancer, comprising at least one miR expression inhibitor compound of  claim 9 , and a pharmaceutically-acceptable carrier. 
     
     
         12 . A method of diagnosing whether a subject has, or is at risk for developing, breast cancer, comprising
 measuring the level of at least one miR-125b gene product in a test sample from said subject,   wherein a decrease in the level of the miR-125b gene product in the test sample, relative to the level of a corresponding miR-155 gene product in a control sample, is indicative of the subject either having, or being at risk for developing, breast cancer.   
     
     
         13 . The method of  claim 12 , which further comprises measuring at least one miR-125b-1 gene product. 
     
     
         14 . The method of  claim 12 , which further comprises measuring at least one miR-125b-2 gene product. 
     
     
         15 . The method of  claim 12 , which further comprises measuring at least one miR-10b gene product. 
     
     
         16 . The method of  claim 12 , which further comprises measuring at least one miR-145 gene product. 
     
     
         17 . The method of  claim 12 , which further comprises measuring at least one miR-21 gene product. 
     
     
         18 . The method of  claim 12 , wherein the level of the at least one miR-125b gene product is measured using Northern blot analysis. 
     
     
         19 . The method of  claim 12 , wherein the level of the at least one miR-125b gene product in the test sample is less than the level of the corresponding miR-125b gene product in the control sample. 
     
     
         20 . The method of  claim 12 , wherein the level of the at least one miR-125b gene product in the test sample is greater than the level of the corresponding miR-125b gene product in the control sample. 
     
     
         21 . A method of treating cancer in a subject in need thereof, comprising
 administering to the subject an effective amount of a compound for inhibiting the expression of a gene encoding one or more gene products in the subject, wherein the gene products are selected from one or more of miR-155, miR-10b, and miR-125b;   thereby inhibiting the proliferation of cancer cells in the subject.   
     
     
         22 . The method of  claim 21 , wherein the compound for inhibiting the expression of a gene encoding the gene product is an antisense nucleic acid. 
     
     
         23 . The method of  claim 21 , wherein the antisense nucleic acid is selected from the group consisting of a single-stranded RNA, a single-stranded DNA, a single-stranded RNA-DNA chimera and a single-stranded PNA. 
     
     
         24 . The method of  claim 21 , wherein the antisense nucleic acid contains one or more modifications to the nucleic acid backbone, a sugar moiety, a base moiety or a combination thereof. 
     
     
         25 . The method of  claim 21 , wherein the compound for inhibiting the expression of a gene encoding the gene product is a double-stranded RNA molecule having at least 90% sequence homology with the mature miRNAs of: miR-10b in SEQ ID NO:31, miR-125b-1 in SEQ ID NO: 118, miR-125b-2 in SEQ ID NO: 121, and miR-155 in SEQ ID NO:183. 
     
     
         26 . The method of  claim 25 , wherein the double-stranded RNA molecule is about 17 to about 29 nucleotides in length. 
     
     
         27 . The method of  claim 21 , wherein the compound for inhibiting the expression of the gene encoding the gene product is a ribozyme. 
     
     
         28 . The method of  claim 21 , wherein the compound for inhibiting the expression of a gene encoding the gene product is formulated as a pharmaceutical composition comprising the compound or a pharmaceutically-acceptable salt thereof, and a pharmaceutically-acceptable carrier. 
     
     
         29 . The method of  claim 21 , wherein the compound for inhibiting the expression of a gene encoding the gene product is administered to the subject orally, parenterally, by injection or infusion. 
     
     
         30 . The method of  claim 21 , wherein the compound for inhibiting the expression of a gene encoding the gene product is administered to the subject by direct injection into a tumor in the subject. 
     
     
         31 . The method of  claim 21 , wherein the compound for inhibiting the expression of a gene encoding the gene product is administered in combination with a delivery reagent. 
     
     
         32 . The method of  claim 31 , wherein the delivery reagent is a liposome. 
     
     
         33 . The method of  claim 21 , wherein the nucleic acid encoding the compound for inhibiting the expression of a gene encoding the gene product is a recombinant plasmid or viral vector. 
     
     
         34 . A method for inhibiting the proliferation of cancer cells, comprising administering a compound for inhibiting the expression of a gene encoding a miR-155 gene product to the cancer cells, thereby arresting or slowing the growth of the cancer cells. 
     
     
         35 . The method of  claim 34 , wherein the compound for inhibiting the expression of a gene encoding a miR-155 gene product is an antisense nucleic acid. 
     
     
         36 . A method for inhibiting the proliferation of cancer cells, comprising administering a compound for increasing the expression of a gene encoding one or more of a miR-10b, miR-125b-1 or miR-125b-2 gene product to the cancer cells, thereby arresting or slowing the growth of the cancer cells. 
     
     
         37 . The method of  claim 36 , wherein the compound for increasing the expression of a gene encoding one or more of a miR-10b, miR-125b-1 or miR-125b-2g gene product is a sense nucleic acid. 
     
     
         38 . A method of treating a subject with a breast cancer, comprising: i) selecting a subject with the breast cancer; and, ii) administering to the subject an isolated nucleic acid molecule encoding at least one transcript of: miR-155, miR-10b, miR-125b-1, and miR-125b-2, thereby treating the subject.