WO2016141269A1 - Keratin 17 as a diagnostic and therapeutic target for cancer - Google Patents

Abstract

The present disclosure relates to the discovery, production and isolation of novel agents that bind to keratin 17 (K17). The present disclosure is also directed to the use of these novel agents, to identify the presence of K17, and to treat a subject for a keratin 17 mediated disease, such as cancer. More specifically, the present disclosure relates to the therapeutic effects of treating a subject with novel modulators of K17 activity.

Description

KERATIN 17 AS A DIAGNOSTIC AND THERAPEUTIC

TARGET FOR CANCER

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims benefit of U.S. Provisional Application No.

62/128,610 filed on March 5, 2015, the entire contents of which are incorporated herein by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0002] The present disclosure was made with government support under grant numbers AI091175 and CA140084 awarded by the National Institutes of Health. The government has certain rights in the disclosure.

FIELD OF THE DISCLOSURE

[0003] The present disclosure relates to protein and peptide chemistry. In particular, the present disclosure relates to the discovery, production and isolation of novel molecules, antibodies, or peptides that bind to keratin 17 (K17), inhibit trafficking of K17, and modulate cell cycle progression. The present disclosure is also directed to the use of these molecules, antibodies and peptides to identify the presence of K17, and to treat cancer. More specifically, the present disclosure relates to the therapeutic effects of treating a subject with novel modulators of K17 binding activity.

BACKGROUND OF THE DISCLOSURE

[0004] Keratins are intermediate filaments that display a broad range of molecular diversity and undergo tight regulation in a tissue-specific, differentiation-related, and context-dependent manner. Keratin 17, although not present in normal mature epithelia, is expressed in stem cells of embryonic ectoderm (J.E. Martens, et al., Anticancer Res. 24(2B) 771-775 (2004)), skin appendages (K M. McGowan, et al., J Invest. Dermatol, 114(6), 1101-1107 (2000)), and the endocervical mucosa as well as re-expressed in carcinomas. K. M. McGowan, et al., J Cell Biol 143, 469-486 (1998); L. F. Escobar- Hoyos, et al., Mod. Path, 27(4) 621-630 (2014); M. Dal Molin, et al., Clin Cancer Res 21, 1944-1950 (2015); L. F. Escobar-Hoyos, et al., Cancer Res 75, 3650-3662 (2015).

[0005] In normal, non-cancerous epithelial cells, p27^{KIP 1} (p27, ) localization is tightly regulated during cell-cycle progression, which plays a pivotal role in governing its function as a cell-cycle inhibitor, i.e., a tumor suppressor. As a negative regulator of Gl- phase progression, when localized in the nucleus, p27^KIP1 inhibits the activity of cyclin- dependent kinases in complex with cyclins, preventing GO/Gl to S-phase transitions. After mitogen signaling during early-Gl, p27^KIP1 is actively exported from the nucleus in a CRM1 -dependent manner and is degraded by the KIPl ubiquitination-promoting complex, triggering Gl/S transition. See M.K. Connor, et al. Mol Biol Cell 14(1):201-13 (2003).

[0006] Notably, one of ordinary skill in the art would recognize that p27^KIP1 is not a classic tumor suppressor because it is rarely mutated or deleted in human cancers.

However, p27^{KIP 1} is progressively lost or sequestered in the cytoplasm during malignant transfonnation. J.M. Slingerland, et al., J. Cell Physiol 183(1) 10-17 (2000); LM. Chu, et al. Nat. Rev. Cancer 8(4) 253-267 (2008).

[0007] The present disclosure identifies a unique role for K17 in cancer development by identifying specific regions within the K17 peptide, such as a nuclear localization signal, p27 binding domains, and nuclear export signals that modulate K17 transport and keratin 17's ability to interact with p27^KIP1.

SUMMARY OF THE DISCLOSURE

[0008] The present disclosure shows that that nuclear-localized K17 interacts with tumor suppressor, p27, and mediates the nuclear export of p27. The discovery that K17 mediates ρ27^KIP1 -nuclear export, and thus p27 mediated Gl -phase cell cycle progression shows that targeted therapy against K17 can inhibit tumor growth and development.

[0009] Generally, the present disclosure relates to compositions and methods of modulating keratin 17 binding activity in a subject by administering an agent that modulates the interaction of K17 with other proteins or molecules, e.g., p27, or chaperone proteins.

[0010] In one aspect of the present disclosure a nuclear localization signal (NLS) has been identified within the K17 coding sequence. The NLS region of K17 is shown to bind cytosolic chaperone proteins, such as transportin, which shuttle K17 to the nucleus. The present disclosure also shows that prohibiting the interaction of a chaperone protein with the NLS region of K17 reduces translocation of K17 into the nucleus and inhibits cancer progression. In one embodiment the K17 NLS is present from amino acid residues 350 to 425 of the K17 protein. In another embodiment the K17 NLS is located between from amino acid residues 390 to 410 of the K17 protein. In a specific embodiment the K17 NLS is located from amino acid residues 386-400 of the human K17 protein. In a preferred embodiment the K17 NLS is located at amino acid residues 395-400 of the human K17 protein. In a specific embodiment the K17 NLS is located at amino acid residues 399 to 400 of human Kl 7. In one embodiment the Kl 7 nuclear localization signal includes the amino acid sequence KRX₁₀_₁₂KK [SEQ ID NO: 1], where X is any amino acid. In yet another embodiment of the present disclosure the K17 nuclear localization signal includes the amino acid sequence KRX_10-12KR [SEQ ID NO: 2], where X is any amino acid. In a specific embodiment of the present disclosure the K17 nuclear localization signal includes the amino acid sequence RRX₁₂KK [SEQ ID NO: 3] or RRLLX₈KK [SEQ ID NO: 4], where X is any amino acid. In certain embodiments the K17 nuclear localization signal includes the amino acid sequence is RRLLEGEDAHLTQYKK [SEQ ID NO: 5].

[0011] In another aspect of the present disclosure a nuclear export signal (NES) has been identified within the K17 amino acid sequence. The NES of K17 is shown to bind nuclear chaperone proteins, such as exportin, which shuttle K17 from the nucleus to the cytosol of a cell. The present disclosure also shows that prohibiting the interaction between a nuclear chaperone protein and the NES region of K17 inhibits translocation of Kl 7 into the cytoplasm and inhibits cancer development and cell cycle progression. In one embodiment the K17 NES is present from amino acid residues 150 to 225 of K17. In certain embodiments the K17 NES is located between amino acid residues 175 to 210 of the K17 protein. In one embodiment the K17 NES is found at amino acid residues 193 to 201 of the human K17 protein. In a specific embodiment of the present disclosure the K17 NES is located at amino acids 194 to 199 of the human K17 protein. In yet another embodiment the NES includes amino acid 194, 197 and 199 of the human K17 peptide. In one embodiment the K17 nuclear export signal includes the amino acid sequence LXXLXL [SEQ ID NO: 6], where X is any amino acid. In yet another embodiment of the present disclosure the K17 NES includes the amino acid sequence LDELTL [SEQ ID NO: 7]. In a specific embodiment of the present disclosure the K17 NES includes the amino acid sequence LEELEL[SEQ ID NO: 8] or LERLTL [SEQ ID NO: 9].

[0012] In yet another aspect of the present disclosure a p27 protein binding domain (MRAIL) has been identified within the K17 amino acid sequence. The present disclosure shows that K17 binds directly to p27 in the nucleus of a cell during Gl phase of the cell cycle at hydrophobic domains in the Kl 7 protein, MRAILs. In one embodiment the p27 binding domain of K17 is located between amino acid residues 155 and 220 of the K17 protein. In certain embodiments the p27 binding domain of K17 is located between amino acid residues 162 and 178 of the K17 protein (MRAIL 1), inclusive. In yet another embodiment the p27 binding domain of K17 is located between amino acid residues 200 and 216 of the Kl 7 protein (MRAIL 2), inclusive. In a specific embodiment the p27 binding domain of K17 is located at amino acid residues 163 to 176. In one preferred embodiment of the present disclosure the p27 binding domain of K17 includes the amino acid sequence RX₄DX₇E [SEQ ID NO: 10]. In a preferred embodiment the p27 binding domain of K17 is located at amino acid residues 201 to 214 of the human K17 protein. In a specific embodiment of the present disclosure the p27 binding domain of K17 includes the amino acid sequence RAXLX₈EE [SEQ ID NO: 11]. In yet another embodiment of the present disclosure the p27 binding domain of K17 is RLAADDFRTKFETE [SEQ ID NO: 12], or ARLAADDFRTKFETEQA [SEQ ID NO: 23], In yet another embodiment of the present disclosure the MRAIL 2 p27 binding domain of K17 is RADLEMOIENLKEE [SEQ ID NO: 13] or

ARADLEMQIENLKEELA [SEQ ID NO: 24].

[0013] Accordingly, the present disclosure provides a method for the treatment of a subject having cancer that includes administering, to a subject, an agent that modulates binding of K17 to a cytosolic chaperone protein, a nuclear chaperone protein, or p27 tumor suppressor protein. The present disclosure also identifies specific domains with K17 that are particularly useful targets in modulating the binding activity of K17 to such proteins. For example, in each of the foregoing instances, inhibition of the keratin 17 NLS, NES and/or a p27^KIP1 protein binding domain reduces nuclear export of tumor suppressor p27, and thus inhibits cell cycle arrest by preventing degradation of K17 in the cytosol of cancer cells.

[0014] Another aspect of the present disclosure includes the development of agents (e.g., antibodies, proteins or small molecules) that recognize and bind to the Kl 7 NES, K17 NLS and/or the p27 binding domains of K17. In certain specific embodiments the above agents specifically bind to any of the NES, NLS or p27 binding domain of K17 and are used to identify the presence of the K17 protein. In yet another embodiment the agents of the present disclosure that specifically bind to any of the NES, NLS or p27 binding domain of K17 are used to track the location of a K17 peptide in the cell, e.g., nucleus of the cell or cytoplasm, In certain embodiments, the antibodies that bind Kl 7 can be used to quantify the amount of K17 present in a cell. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] The file of this patent contains at least one drawing executed in color. Copies of this patent with color drawing(s) will be provided by the Patent and Trademark Office upon request and payment of the necessary fee.

[0016] FIG. 1: Model of K17 interaction with Ρ27^KIP1. K17 is present in the cytoplasm where it binds to a chaperone protein (importin) that binds K17 at the nuclear localization signal (NLS). A chaperone protein traffics K17 to the nucleus where it binds to P27^KIP1 at an MRAIL domain of K17. K17 then binds to an second chaperone protein (e.g., CRM1, exportin), which traffics the K17/P27^KIP1 protein complex out of the nucleus into the cytoplasm where p27 is subsequently degraded preventing cell cycle arrest.

[0017] FIG.2: Keratin 17 enhances tumor growth in vivo. Growth curve obtained from cervical cancer xenograft mouse models. Xenografts were developed from CaSki cervical cancer cells expressing endogenous levels of K17 (control shRNA) and CaSki cervical cancer cells devoid of K17 (K17 shRNA) * p<0.05; ** p<0.01.

[0018] FIGS.3A-F: Keratin 17 knockdown induces cell cycle arrest (A) Effects of K17 knockdown in cell-proliferation of (A) SiHa and (B) CaSki cells after transfection with control siRNA or siRNA against keratin 17 (siKRT17). (C-E) Gl/S ratio in SiHa and CaSki cervical cancer cells (C), as well as C-33A cervical cancer cells (D); L3.6 pancreatic cancer cells and MDAMB-231 and MDA-MB-468 breast cancer cell lines (E), assessed by flow cytometry using propidium iodine. (F) Post-mitotic GIA/GIB ratio in cells transfected with siKRT17, compared to control siRNA assessed by flow cytometry using acridine orange staining. Statistical analyses were carried out by T-test or Mann-Whitney U. * p < 0.05, ** p < 0.01.

[0019] FIGS.4A-D: Keratin 17 knockdown correlates with nuclear p27^KIP1

accumulation and stability. (A-B). Expression of p27^KIP1 in SiHa (A) and CaSki (B) cervical cancer cells transfected with control siRNA or siRNA against K17 (siKRT17) at different time points. (C) p27^KIP1 expression in C33-A cervical cells transfected with empty vector or human K17. (D) Expression of p27^KIP1 in pancreatic (L3.6) and breast cancer (MDA-MB-231, MDA-MB-468) cell lines transfected with control shRNA or shRNA against KRT17 (shKRT17, shl).

[0020] FIGS. 5A-B: Keratin 17 knockdown results in an increase in nuclear ρ27^KIP1'

(A) p27^KIP1 expression in nuclear and cytosolic fractions of control shRN A or shRNAs against keratin 17 (shKRT17) (SiHa-shI and CaSki-shll). (B) Nuclear p27^KM positive cells after immunofluorescent staining of cervical cancer cells transfected with control siRNA or siKRT17.

[0021] FIGS. 6A-B: Keratin 17 and p27^KIP1 interact in the nucleus of cells. (A) 2D-

Structured Illumination Microscopy images from immunofluorescent nuclear colocalization of K17 with p27^KIP1 in cells. Scale bar, 5um. N: Nucleus, C: Cytoplasm.

(B) Percentage of cells with nuclear colocalization of K17 and p27^KIP1. Nuclear-K17 speckles are shown after 1 hour of serum-starvation release in SiHa and CaSki cervical cancer cells. Scale bar, 5μm.

[0022] FIGS. 7A-C: Identification and characterization of the Keratin 17 nuclear localization signal and nuclear export signal. (A) A cartoon diagram of general locations of the NES and NLS on the K17 protein. (B) K17 Leucine-rich nuclear export signal (NES) alignment of in type I keratins in humans (h) and other species. hMAPKK and HIV1 Rev are prototypes of NES. (C) K17 unique bipartite nuclear localization signal (NLS) among type I keratins in humans (h) and conserved only in primates. SV40 and nucleoplasmin are prototypes of the bipartite NLS.

[0023] FIGS. 8A-C: Functional mutations in the NLS and NES regions of K17 result in aberrant K17 trafficking of K17 to and from the nucleus. (A) Wild-type NLS and NES sequences are shown. * Denotes mutated amino acids from either leucine (NES) or lysine (NLS) to alanine. (B) Expression of wild type and mutant K17 in cervical cancer, C33 Cells, reveals that mutations in the Kl 7 NES causes retention of Kl 7 in the nucleus, while mutation of the K17 NLS causes an increase in cytosolic K17. Cytosolic and nuclear lysates of C33-A cells stably transfected with wild-type K17 (Wt), mutated K17-NLS (mNLS) and mutated NES (mNES), blotted from K17 (His-Tag) expression. (C) Relative expression of K17 in cytosolic and nucleus C33 cellular fractions.

[0024] FIG.9: K17 promotes nuclear export and degradation of ρ27^KIP1 in cancer cells. (Left) Cytosolic and nuclear lysates of C33-A cells stably transfected with wild- type Kl 7 (Wt), mutated Kl 7-NLS (mNLS) and mutated NES (mNES), blotted from p27^{KIP 1} expression. (Right) Relative expression of p27^KIP1 in cytosolic and nuclear fractions. Mutation of the K17 NES reduces the amount of p27^KIP1 found in the cytosol of C33 cancer cells. In contrast, mutation of the NES and NLS leads to an increase in the present of P27 in the nucleus [0025] FIGS. lOA-C: Nuclear K17 and ρ27^KIP1 bind during Gl phase of the cell cycle. (A) K17 and p27^KM levels at different time points of Gl in cancer cells cultured in fetal bovine serum (FBS) and cycloheximide (CHX). (B) K17 binds to p27^Klpl as shown by immunoprecipitation over nuclear fractions of cervical cancer cells. (C)

Recombinant human K17 (rHK17) was provided to nuclear lysates from CaSki cervical cancer cells devoid of K17 expression (CaSki, shKRT17) Beads containing either K17 or P27^KIP1 antigen identify binding of recombinant Kl 7 and p27^KIP1.

[0026] FIG 11: Nuclear retention of ρ27^KIP1, K17 and JAB1 after leptomycin B (LMB) treatment (Left) Treatment with a nuclear chaperone protein (exportin) inhibitor leptomycin B results in an increase in accumulation of K17 and p27^KIP1 in the nucleus of cancer cells. (Right) Relative expression of K17 and p27^KIP1 in nuclear fractions. Statistical analyses were carried out by T-test, Mann-Whitney U or one-way ANOVA. * p < 0.05, ** p < 0.01 and *** p < 0.001.

[002η FIG. 12: p27 binding domains in K17 regulate p27KTPl translocation and interaction with cyclin A. (A) Structural basis for p27 interaction with cyclin A and CDK2. Lilac highlights the MRAILl binding domain (alpha helix) in cyclin A. Magenta K/RXL domain identified in p27 is required for p27-cyclin interactions. (B) Alignment of p27 binding domain sequences of K17 and cyclin A. Human (h) and murine (m) sequences are identical. * Single fully conserved residue; + Similar polarity; : Strongly similar properties; . Weak similar properties. Hydrophobic, acidic, basic and hydroxyl+sulfhydryl+ amine. Pairwise score based on ClustalW. # mutated residues. Mutated residues in a first p27 binding domain of K17 (i.e., MRAIL 1) are R163A, D168A, F169A, E174A and E176A. Mutated residues in a second p27 binding domain of K17 (i.e., MRAIL 2) include R201A, D203A, M206A, Q207A, E213A and E214A. (C) BxPC3 prostate cancer cells exhibiting knockdown of endogenous K17 (shK17) due to transfection hairpin RNA against K17, and wild-type (WT) K17, mutated MRAIL 1 (mMRAILl), mutated MRAIL2 (mMRAIL2) or Empty vector. Mutation ofMRAILl and MRAIL2 in K17 contribute to loss of p27 in the cytoplasm of cells. ** p< 0.05; *** p< 0.01.

DETAILED DESCRIPTION OF THE DISCLOSURE

[0028] The present disclosure relates to methods for modulating keratin 17 in a subject by admmistering an agent that inhibits the interaction of K17 with other proteins or molecules in a cell, e.g., p27, or chaperone proteins.

[0029] As depicted in FIG. 1, the present disclosure is based upon the discovery that keratin 17 interacts with and binds to tumor suppressor protein, p27^KJP1 in the nucleus. More specifically, the instant application identifies a nuclear localization signal (NLS) within the K17 protein, which facilitates K17 to trafficking from the cytosol of a cell to the cell nucleus. The present disclosure also identifies a p27^KIP1 protein binding domain within the K17 amino acid sequence that, when blocked, prohibits the nuclear p27^KIP1 protein from being transported to the cytoplasm, thereby preventing p27^{KIP 1} mediated cell cycle arrest in cancer cells or other instance of cellular stress. The present disclosure further identifies a nuclear export signal (NES) within the K17 protein coding sequence that mediates the transfer of nuclear K17, either bound or unbound to nuclear p27^Klpl, to the cytoplasm of a cell. Taken together, the present disclosure shows that inhibition of the Kl 7 nuclear localization signal (NLS), nuclear export signal (NES) or the p27^KIP1 protein binding domain mediates the export of p27^KIP1 from the cell nucleus where p27 acts as a tumor suppressor by halting Gl-S phase cell cycle progression. In contrast, when p27^KIP1 is transported to the cytoplasm, p27^KIP1 is degraded and cell cycle progression and tumor development continues uninhibited. Taken together, the following shows that the antibodies, molecules or proteins that bind to the identified domains of K17 can be used to identify the location of K17 within a cell and/or be used as a targeted therapy against K17 mediated cancer development.

Terminology

[0030] The term "agent" as used herein refers to any kind of compound or combination of compounds. In one embodiment of the invention the agent is a small molecule. In another embodiment of the disclosure, the agent is a biological molecule including, but not limited to, an antibody, protein or a peptide, or a nucleic acid.

[0031] The term "interfering RNA" is employed herein to refer to small interfering RNAs (siRNAs), short hairpin RNAs (shRNAs), microRNAs (miRNAs), antisense oligonucleotides, ribozymes, or any RNA-based molecule that interferes with the expression of a protein from its corresponding gene.

[0032] In the context of this disclosure, the term "small molecule" refers to small organic compounds, such as heterocycles, peptides, saccharides, steroids, and the like. The small molecule modulators preferably have a molecular weight of less than about 1500 Daltons, and more preferably less than 500 Daltons. The compounds can be modified to enhance efficacy, stability, pharmaceutical compatibility, and the like.

Candidate modulator compounds from libraries of synthetic or natural compounds can be screened. Synthetic compound libraries are commercially available from a number of companies including Maybridge Chemical Co. (Trevillet, Cornwall, UK), Comgenex (Princeton, N. J.), Brandon Associates (Merrimack, N.H.), and Microsource (New Milford, Conn.). Combinatorial libraries are available or can be prepared according to known synthetic techniques. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available from e.g., Pan Laboratories (Bothell, Wash.) or MycoSearch (NC), or are readily producible by methods well known in the art. Additionally, natural and synthetically produced libraries and compounds can be further modified through conventional chemical and biochemical techniques.

[0033] As used herein, the term "derivative" refers to a compound having a structure derived from the structure of a parent compound (e.g., a compound disclosed herein) and whose structure is sufficiently similar to those disclosed herein and based upon that similarity, would be expected by one skilled in the art to exhibit the same or similar activities and utilities as the claimed compounds, or to induce, as a precursor, the same or similar activities and utilities as the claimed compounds. Exemplary derivatives include salts, esters, amides, salts of esters or amides, and N-oxides of a parent compound.

[0034] The term "peptide" or "protein" as used in the current disclosure refers to a linear series of amino acid residues linked to one another by peptide bonds between the alpha- amino and carboxy groups of adjacent amino acid residues. In one example the protein is keratin 17 or a portion thereof. In yet another embodiment the protein can be tumor suppressor, p27. In certain non-limiting examples a protein of the present disclosure can be the NES, NLS or p27 binding domain of human K17. Homologs, analogs and fragments of these peptides are also contemplated by the present disclosure. By "homologs" it is meant that the corresponding proteins of other vertebrate species are substantially homologous at the overall protein (i.e., mature protein) level to the human amino acid sequence. In certain embodiments, homologs of a protein have an amino acid sequence substantially identical to the human wild-type protein, i.e., at least 80- 85%, at least 90-95% or 99% or more sequence identity. The term "analogs" shall mean peptides that differ by one or more amino acids alterations, which alterations, e.g., substitutions, additions or deletions of amino acid residues, do not abolish the ability to positively regulate protein activity. Thus, an analog can comprise a peptide having a substantially identical amino acid sequence to a peptide provided herein and in which one or more amino acid residues have been conservatively or non-conservatively substituted. Examples of a conservative substitution include the substitution of a hydrophobic residue such as isoleucine, valine, leucine or methionine for another.

Likewise, the present disclosure contemplates the substitution of one aromatic residues such as phenylalanine, tryptophan or tyrosine for another. The substitution of a polar residue such as lysine, arginine, glutamine or asparagine for another or the substitution of a polar residue such as aspartate, glutamate, glutamine or asparagine for another.

Additionally, the present disclosure contemplates the substitution of a non-polar aliphatic residue, such as between glycine and alanine, or a polar aliphatic residue such as between serine and threonine. Examples of non-conservative substitutions include the substitution of a non-polar residue e.g., isoleucine, valine, leucine, alanine or methionine for a polar residue e.g., glutamine, glutamate, lysine, and/or a polar residue for a non- polar residue. [0035] The term "nucleic acid" as used herein refers to one or more nucleotide bases of any kind, including single- or double-stranded forms. In one aspect of the current disclosure a nucleic acid is DNA and in another aspect the nucleic acid is RNA. In the current disclosure, nucleic acids can be the coding sequence for K17, p27 or binding partners thereof.

[0036] The term "fragment" as used herein shall mean any portion of a molecule (e.g., peptide or antibody) that is, by some measure smaller than the whole including, but not limited to, a peptide that contains fewer amino acids than the protein or domain of said protein as a whole.

[0037] The term "isolated" and "purified", when used in reference to a molecule (such as a peptide, protein or polypeptide), means that the molecule has been removed from its naturally occurring environment and is substantially free of other molecules (such as other proteins). By "substantially free" of other proteins, it is meant that a protein of interest accounts for at least 60%, 70%, 80%, 90%, or 95% (by dry weight) of total proteins in a composition. When an isolated protein is recombinantly produced, it can also be substantially free of culture medium, i.e., culture medium represents less than about 20% of the volume of the protein preparation, less than about 10% of the volume of the protein preparation or less than about 5% of the volume of the protein preparation. For example, the proteins of the present disclosure can be purified to homogeneity or other varying degrees of purity. The level of purification can be based on the intended use. In certain non-limiting examples, isolated peptides of the present disclosure can be purified from cells that express such protein, as further described below, or can be synthetically made using known protein synthesis methods. The term "synthetic peptide" is intended to refer to a chemically derived chain of amino acid residues linked together by peptide bonds that are isolated or substantially isolated from other materials or elements. Specifically, the term "synthetic peptide" is intended to refer to

recombinantly produced peptides in accordance with the present disclosure.

[0038] The term "keratin 17", "K17" or "KRT17" as used herein interchangeably and refers to the human keratin, keratin, type II cytoskeletal 4 gene located on chromosome 17, as set forth in accession number NG_008625 or a product thereof, which encodes the type I intermediate filament chain keratin 17. Included within the intended meaning of KRT17 are mRNA transcripts of the keratin 17 cDNA sequence as set forth in accession number NM 000422, and proteins translated therefrom including for example, the keratin, type 1 cytoskeletal protein, 17 as set forth in accession number NP 000413 or homologs thereof.

[0039] The terms "p27 protein", "ρ27™" and "p27" are used herein interchangeably and specify a protein as described herein, which is encoded by the "p27^KIP1gene" or "p27 gene", which is located on human chromosome 12, as set forth in accession number NG_016341.1. The p27 protein is characterized as a cyclin-dependent kinase inhibitor IB (CKDN1B). The human p27^KIP1 protein has the 198 amino acid sequence set forth in accession number B AG70105.

[0040] The term "chaperone protein", "chaperone" or "transport protein" as used herein refers to any gene product (i.e., protein), capable of selectively binding to another molecule and translocating that molecule to a different intracellular compartment. The term "cytosolic chaperone" as used herein means a protein or group of proteins that bind to a another protein in the cytoplasm of a cell and traffic the bound proteins to another cellular compartment or organelle, such as the nucleus. The term "nuclear chaperones" as used herein means a protein or group of proteins that bind to another protein in the nucleus of a cell and traffic the bound proteins to another cellular compartment or organelle, such as the cytoplasm or a ribosome.

[0041] The phrase "subject" or "patient" as used herein refers to any mammal. In one embodiment the subject is a candidate for cancer diagnosis (e.g., cervical cancer, pancreatic cancer, lung cancer, breast cancer) or an individual diagnosed with cancer or the presence of a pre-cancerous lesion, such as HSIL or LSIL. In certain non-limiting embodiments of the present disclosure, the subject has been diagnosed with cervical cancer, pancreatic cancer, lung cancer, or breast cancer and the subject is a candidate for treatment or undergoing treatment thereof. The methods and compositions of the current disclosure can be used on any mammalian subject that has a risk of developing cancer or has been diagnosed with cancer. Particularly, the methods or compositions described herein are most useful when practiced on humans.

[0042] A "control sample" "non-cancerous sample" or "normal sample" as used herein is a sample which does not exhibit cancer and exhibits endogenous levels of K17 and p27. In certain embodiments, a control sample does not contain cancerous cells. Non-limiting examples of control samples for use in the current disclosure include, non-cancerous tissue extracts, surgical margins extracted from a subject, or samples obtained from other healthy individuals or a database thereof. In one aspect, the control sample of the present disclosure is benign tissue obtained from the subject in question.

[0043] The term "effective amount" refers to an amount that is sufficient to achieve the desired result or to have an effect on an undesired condition. For example, a "therapeutically effective amount" refers to an amount that is sufficient to achieve the desired therapeutic result or to have an effect on undesired symptoms, but is generally insufficient to cause adverse side effects. The specific therapeutically effective dose level for any particular patient will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration; the route of administration; the rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed and like factors well known in the medical arts. For example, it is well within the skill of the art to start doses of a agent at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. If desired, the effective daily dose can be divided into multiple doses for purposes of administration. Consequently, single dose agents can contain such amounts or submultiples thereof to make up the daily dose. The dosage can be adjusted by the individual physician in the event of any contraindications. Dosage can vary, and can be administered in one or more dose administrations daily, for one or several days. Guidance can be found in the literature for appropriate dosages for given classes of pharmaceutical products. In specific embodiments the amount of an agent that is effective in modulating the activity of K17 in a subject. In certain context of the present disclosure an effective amount of an agent inhibits the interaction or binding of K17 to another molecule or protein. In a preferred embodiment, the agent is effective if it reduces p27 degradation. In another

embodiment, an effective amount of an agent reduces the amount of cytosolic p27 protein. In another embodiment, an effective amount of an agent raises the cytosolic K17 compared to the untreated level, or control sample. In another embodiment, the agent reduces amount of nuclear K17 compared to that of an untreated subject or control sample. In one embodiment, an effective amount of an agent increases the amount of nuclear p27compared to that of an untreated subject or control sample.

[0044] The term "increase", "increases" or "greater" or "elevated" means at least more than the relative amount of an entity identified (e.g., p27 or K17 protein), measured, located in a particular location or analyzed in a control sample. Non-limiting examples include, an effective amount of an agent increases p27 degradation by 5-10%, 10-20% over that of a control sample, or at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200% or greater increase over that of a control sample, or at least a 0.25 fold, 0.5 fold, 1 fold, 1.5 fold, 2 fold, 3 fold, 4 fold, 5 fold, 10 fold, 11 fold or greater, increase relative to the entity being analyzing in the control sample. In certain embodiments the measured amount of K17 located in the nucleus of a cell is increased by , 5-10%, 10- 20% compared to that of a control sample, or at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200% or greater increase when compared to that of a control sample, or at least a 0.25 fold, 0.5 fold, 1 fold, 1.5 fold, 2 fold, 3 fold, 4 fold, 5 fold, 10 fold, 11 fold or greater, increase relative to the entity being analyzing in the control sample.

[0045] The term "decrease", "reduces" or "reduction" means at least lesser than the relative amount of an entity identified (e.g., p27 or K17 protein), measured, located in a particular location, or analyzed in a control sample. Non-limiting examples include, an effective amount of an agent reduces p27 degradation in a cell or subject by, including but not limited to, a 5-10%, 10-20% decrease over that of a control sample, or at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200% or greater decrease over that of a control sample, or at least a 0.25 fold, 0.5 fold, 1 fold, 1.5 fold, 2 fold, 3 fold, 4 fold, 5 fold, 10 fold, 11 fold or greater, decrease relative to the entity being analyzing in the control sample. In certain embodiments the measured amount of K17 located in the nucleus of a cell is reduced by , 5-10%, 10-20% compared to that of a control sample, or at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200% or greater decrease when compared to that of a control sample, or at least a 0.25 fold, 0.5 fold, 1 fold, 1.5 fold, 2 fold, 3 fold, 4 fold, 5 fold, 10 fold, 11 fold or greater, decrease relative to the entity being analyzing in the control sample.

[0046] The phrase "inhibits the activity" is employed herein to refer to any disruption, partial or total, of the natural effect of K17 or the transport thereof. In certain embodiments the activity is the ability of K17 to bind to another protein. In specific embodiments the activity is the ability of K17 to translocate to another intracellular compartment or organelle. In a preferred embodiment the activity is the ability of Kl 7 to bind to and traffic p27 from the nucleus to the cytoplasm.

[0047] As used herein, the term "treatment" refers to the medical management of a patient with the intent to cure, ameliorate, stabilize, or prevent a disease, pathological condition, or disorder. This term includes active treatment, that is, treatment directed specifically toward the improvement of a disease, pathological condition, or disorder, and also includes causal treatment, that is, treatment directed toward removal of the cause of the associated disease, pathological condition, or disorder. In addition, this term includes palliative treatment, that is, treatment designed for the relief of symptoms rather than the curing of the disease, pathological condition, or disorder; preventative treatment, that is, treatment directed to minimizing or partially or completely inhibiting the development of the associated disease, pathological condition, or disorder; and supportive treatment, that is, treatment employed to supplement another specific therapy directed toward the improvement of the associated disease, pathological condition, or disorder. In various aspects, the term covers any treatment of a subject, including a mammal (e.g., a human), and includes: (i) preventing the disease from occurring in a subject that can be predisposed to the disease but has not yet been diagnosed as having it; (ii) inhibiting the disease, i.e., arresting its development; or (iii) relieving the disease, i.e., causing regression of the disease.

[0048] As used herein, the terms "administering'' and "administration'' refer to any method of providing a pharmaceutical preparation to a subject. Such methods are well known to those skilled in the art and include, but are not limited to, oral administration, transdermal administration, administration by inhalation, nasal administration, topical administration, intravaginal administration, ophthalmic administration, intraaural administration, intracerebral administration, rectal administration, sublingual administration, buccal administration, and parenteral administration, including injectable such as intravenous administration, intra-arterial administration, intramuscular administration, and subcutaneous administration. Administration can be continuous or intermittent. In various aspects, a preparation can be administered therapeutically; that is, administered to treat an existing disease or condition. In further various aspects, a preparation can be administered prophylactically; that is, administered for prevention of a disease or condition. [0049] The term "pharmaceutically acceptable" describes a material that is not biologically or otherwise undesirable, i.e., without causing an unacceptable level of undesirable biological effects or interacting in a deleterious manner.

[0050] As used herein, the term "pharmaceutically acceptable carrier" refers to sterile aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, as well as sterile powders for reconstitution into sterile injectable solutions or dispersions just prior to use. Examples of suitable aqueous and nonaqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol and the like), carboxymethylcellulose and suitable mixtures thereof, vegetable oils (such as olive oil) and injectable organic esters such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants. These compositions can also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms can be ensured by the inclusion of various antibacterial and antifungal agents such as paraben, chlorobutanol, phenol, sorbic acid and the like. It can also be desirable to include isotonic agents such as sugars, sodium chloride and the like.

Prolonged absorption of the injectable pharmaceutical form can be brought about by the inclusion of agents, such as aluminum monostearate and gelatin, which delay absorption. Injectable depot forms are made by forming microencapsule matrices of the drug in biodegradable polymers such as polylactide-polyglycolide, poly(orthoesters) and poly(anhydrides). Depending upon the ratio of drug to polymer and the nature of the particular polymer employed, the rate of drug release can be controlled. Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions which are compatible with body tissues. The injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable media just prior to use. Suitable inert carriers can include sugars such as lactose. Desirably, at least 95% by weight of the particles of the active ingredient have an effective particle size in the range of 0.01 to 10 micrometers.

Compositions

[0051] In one aspect of the present disclosure agents, such as small molecules, peptides and antibodies are provided, which modulate keratin 17 activity mcluding the ability of K17 to bind to other proteins. Keratin 17 binding partners, include but are not limited to, p27 importin-a (accession no. Q 14974), importin-β (accession no. P52292), exportin, as set forth in accession number 014980), as well as p21 (accession no. P38936), RBI (accession no. P06400), RBL1 (accession no. P28749), RBL2 (accession no. Q08999), RPS8 (accession no. P62241) and RPS11 (accession no. P62280). However, other keratin 17 binding partners are also contemplated by the present disclosure, which can be identified or confirmed through the use of, for example, immunoprecipitation (e.g., pulldown assay), yeast two-hybrid, or co-localization studies, all of which are not considered undue experimentation by the skilled artisan.

[0052] In certain embodiments of the present disclosure novel peptides have been synthesized, which are derived from or correspond the nuclear export signal region (NES), the nuclear localization signal region (NLS), or p27 binding domain region (MRAIL 1, MRAIL 2) portions of the K17 protein. [0053] The peptides of the present disclosure, homologs, analogs and fragments thereof can be synthesized by a number of known techniques. For example, the peptides can be prepared using the solid-phase synthetic technique initially described by Merrifield, in J. Am. Chem. Soc. 85, pp. 2149-2154 (1963). Other peptide synthesis techniques can be found in M. Bodanszky, et al. Peptide Synthesis, John Wiley & Sons, 2d Ed., (1976) and other references readily available to those skilled in the art. A summary of polypeptide synthesis techniques can be found in J. Stuart and J. D. Young, Solid Phase Peptide Synthesis, Pierce Chemical Company, Rockford, HI., (1984). Peptides can also be synthesized by solution methods as described in The Proteins, Vol. Π. 3d Ed., Neurath, H. et al., Eds., p. 105-237, Academic Press, New York, N.Y. (1976). Appropriate protective groups for use in different peptide syntheses are described in the above- mentioned texts as well as in J. F. W. McOmie, Protective Groups in Organic Chemistry, Plenum Press, New York, N.Y. (1973). The peptides of the present disclosure can also be prepared by chemical or enzymatic cleavage from larger portions of cytosolic chaperone proteins that bind to the NLS of K17, such as importin

[0054] Additionally, the peptides of the present disclosure can be prepared by recombinant DNA techniques known by one of ordinary skill in the art. See, e.g., Current Protocols in Molecular Cloning Ausubel et al., 1995, John Wiley & Sons, New York); Sambrook et al., (1989), Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, New York; Coligan et al. Current Protocols in Immunology, John Wiley & Sons Inc., New York, N.Y. (1994). The skilled artisan understands that any of a wide variety of expression systems can be used to provide the recombinant peptides of the present DISCLOSURE. The precise host cell used is not critical to the present methods. The peptides of the present disclosure can be produced in a prokaryotic host (e.g. E. coli)„ or in a eukaryotic host (e.g., S. cerevisiae or mammalian cells, e.g. COS1, CHO, NIH3T3, and JEG3 cells, or in the cells of an arthropod, e.g. S. frugiperda). Such cells are available from, for example, the American Type Culture Collection, Manassas, Va. It is appreciated by the skilled artisan that the method of transfection and the choice of expression vehicle will depend on the host system selected. Transformation and transfection methods are described, e.g. in Sambrook et al., (1989); expression vehicles can be chosen from those provided. See, e.g., P. H. Powels et al., Cloning Vectors: A Laboratory Manual. (1985).

[0055] For most of the amino acids used to build proteins, more than one coding nucleotide triplet (codon) can code for a particular amino acid residue. This property of the genetic code is known as redundancy. Therefore, a number of different nucleotide sequences can code for a particular peptide corresponding to the NLS, NES or p27 binding domains of Kl 7.

[0056] The present disclosure also includes nucleic acid molecules (DNA) that define a gene coding for, i.e., capable of expressing, a subject peptide or a subject chimeric peptide that binds to and inhibits Kl 7 binding to a Kl 7 binding protein, such as p27, a cytosolic chaperone or nuclear chaperone.

[0057] DNA molecules that encode peptides of the present disclosure can be synthesized by chemical techniques, for example, the phosphotriester method of Matteuccie, et al., J. Am. Chem. Soc. 103:3185 (1981), which is incorporated herein by reference. Using a chemical DNA synthesis technique, desired modifications in the peptide sequence can be made by making substitutions for bases which code for the native amino acid sequence. Ribonucleic acid equivalents of the above described DNA molecules can also be used. [0058] A nucleic acid molecule comprising a vector capable of replication and expression of a DNA molecule defining coding sequence for a subject polypeptide or subject chimeric polypeptide is also contemplated.

[0059] Another aspect of the present disclosure is directed to antibodies raised against the K17 NLS, NES or p27 binding sequences or homologs, analogs thereof. In a specific embodiment antibodies of the present disclosure are raised against the K17 NLS. In other embodiments antibodies of the present disclosure can be raised against peptides whose sequences coincide with the corresponding sequences of a K17 NLS.

[0060] In certain embodiments antibodies of the present disclosure are raised against the K17 NES. In other embodiments antibodies of the present disclosure can be raised against peptides whose sequences coincide with the corresponding sequences of a K17 NES.

[0061] In certain embodiments antibodies of the present disclosure are raised against a K17 p27 binding domain. In a specific embodiment antibodies of the present disclosure are raised against MRAIL 1 sequence, p27 binding domain of K17. In another embodiment the antibodies of the present disclosure are raised against the MRAIL 2, p27 binding domain of K17. In other embodiments antibodies of the present disclosure can be raised against peptides whose sequences coincide with the corresponding sequences of a p27 binding domain within the K17 amino acid sequence.

[0062] In certain embodiments synthetic peptides of the present disclosure are produced based on the MRAIL 1 and MRAIL2 amino acid sequences within the K17 coding sequence, with (or without) additional modifications. In a specific embodiment synthetic peptides of the present disclosure were modified from the original sequence to increase peptide solubility and stability. For MRAIL1 a modified peptide is NH₂- ARLAADDFRTKFETEQA-CONH2 [SEQ ED NO: 21] and for MRAIL2 a modified peptide is NH₂-ARADLEMQffiNLKEELA-CONH₂ [SEQ ID NO: 22]. In other embodiments synthetic peptides of the present disclosure can be produced bases on such leading peptides.

[0063] By way of example, peptides can be coupled to a carrier protein such as KLH as described in Ausubel et al. (1989) Current Protocols in Molecular Biology, John Wiley & Sons, New York. The KLH-antagonist peptide is mixed with Freund's adjuvant and injected into guinea pigs, rats, donkeys and the like or preferably into rabbits.

Antibodies can be purified by peptide antigen affinity chromatography. More specifically, antibodies of the present disclosure can be prepared using K17 binding peptides and standard hybridoma technology. See, e.g,. Kohler et al., Nature 256:495 (1975)); Hammerling et al., (1981); and Monoclonal Antibodies and T Cell Hybridomas, Elsevier, N.Y.. For example, monoclonal antibodies to K17 NES, Kl 7 NLS, and K17 p27 biding domains or fragments thereof can be raised in Balb/C or other similar strains of mice by immunization with purified or partially purified preparations of K17 binding peptides. The spleens of the mice can be removed, and their lymphocytes fused to a mouse myeloma cell line. After screening of hybrids by known techniques, a stable hybrid will be isolated that produces antibodies against the desired K17 sequence (e.g., NES, NLS, p27 binding sequence).

[0064] The effective binding of antibodies can be examined by measuring the level of Kl 7 in the cytosol or nucleus, and/or by measuring the relative amounts of p27 in the cytosol or nucleus, as stated herein. For example, once produced, monoclonal antibodies can be tested for K17 recognition by Western blot or immunoprecipitation analysis. The antibodies or fragments thereof, can be used in diagnostic assays or to further characterize K17 function of the function of fragments thereof. Both, polyclonal antibodies and monoclonal antibodies are contemplated by the present disclosure.

Further, any of these approaches my used in connection with an in vivo, ex vivo, or in vitro experimental setup.

[0065] In a specific embodiment, the present disclosure identifies a nuclear localization signal (NLS) within the K17 protein. The NLS region of K17 is shown to bind cytosolic chaperone proteins, such as importin, which shuttle K17 to the nucleus. The present disclosure also shows that inhibiting the interaction of a cytosolic chaperone protein with the NLS region of K17 reduces translocation of K17 into the nucleus and cell cycle progression. Hence, in a specific embodiment the present disclosure provides an agent that binds to the nuclear localization signal of Kl 7 within the Kl 7 protein. In certain embodiments, interaction between the agent and the K17 NLS prohibits binding of Kl 7 to a cytosolic chaperone protein, e.g., importin, and reduces the amount of K17 in the nucleus of a cell. More specifically, the agent inhibits the interaction between keratin 17 and a cytosolic chaperone protein, prohibits the translocation of K17 from the cytoplasm of a cell to the cell nucleus.

[0066] Therefore, agents of the present disclosure include peptides, antibodies, small molecules, or fragments of any of the foregoing, which bind to the K17 nuclear localization sequence (NLS). In one embodiment the K17 NLS for which an agent binds is present from amino acid residues 350 to 425 of the K17 protein. In another embodiment the K17 NLS is located between from amino acid residues 390 to 410 of the K17 protein. In a specific embodiment the K17 NLS is located from amino acid residues 386-400 of the human K17 protein. In a preferred embodiment the K17 NLS is located at amino acid residues 395-400 of the human K17 protein. In a specific embodiment the K17 NLS is located at amino acid residues 399 to 400 of human K17.

[0067] In preferred embodiments of the present disclosure the K17 nuclear localization signal includes the amino acid sequence KRX_10-12KK [SEQ ID NO: 1], where X is any amino acid. In yet another embodiment of the present disclosure the K17 nuclear localization signal includes the amino acid sequence KRX_10-12KR [SEQ ID NO: 2], where X is any amino acid. In a specific embodiment of the present disclosure the K17 nuclear localization signal includes the amino acid sequence RRX₁₂KK [SEQ ID NO: 3] or RRLLX₈KK [SEQ ID NO: 4], where X is any amino acid. A listing of single letter and three letter amino acid codes are provided herein, for convenience: A=Ala=Alanine; R=Arg=Arginine; N=Asn=Asparagine; D=Asp=Aspartate; B=Asx=Asparagine or Aspartate; C=Cys=Cysteine; Q=Gln==Glutamine; E==Glu=Glutamate; Z=Glx=Gl utamine or Glutamate; G=Gly=Glycine; H=His=Histidine; I=Ile=Isoleucme; L=Leu=Leucine; K=Lys=Lysine; F=Phe=Phenylalanine; P=Pro=Proline; S=Ser=Serine;

T=Thr=Threonine; W=Trp=Tryptophan; Y=Tyr=Tyrosine; and V=Val=Valine.

[0068] In specific embodiments the Kl 7 nuclear localization signal includes the amino acid sequence RRLLEGEDAHLTQYKK [SEQ ID NO: 5].

[0069] In another embodiment of present disclosure a nuclear export signal (NES) has been identified within the Kl 7 protein. The NES of K17 is shown to bind nuclear chaperone proteins, such as exportin, which shuttle K17 from the nucleus to the cytosol of a cell. The present disclosure also shows that prohibiting the interaction between a nuclear chaperone protein and the NES region of K17 inhibits translocation of K17 into the cytoplasm and inhibits cancer development and cell cycle progression. Hence, in certain embodiments an agent of the present disclosure inhibits the interaction between keratin 17 and a nuclear chaperone protein, and prohibits the translocation of K17 from the nucleus to the cytoplasm of a cell.

[0070] In certain embodiments the K17 NES for which an agent of the present disclosure binds is present from amino acid residues 150 to 225 of K17. In certain embodiments the K17 NES is located between amino acid residues 175 to 210 of the Kl 7 protein. In one embodiment the K17 NES is found at amino acid residues 193 to 201 of the human Kl 7 protein. In a specific embodiment of the present disclosure the Kl 7 NES is located at amino acids 194 to 199 of the human K17 protein. In yet another embodiment the NES includes amino acid 194, 197 and 199 of the human K17 peptide. In one embodiment the Kl 7 nuclear export signal includes the amino acid sequence LXXLXL [SEQ ID NO: 6], where X is any amino acid. In yet another embodiment of the present disclosure the K17 NES includes the amino acid sequence LDELTL [SEQ ID NO: 7]. In a specific embodiment of the present disclosure the K17 NES includes the amino acid sequence LEELEL [SEQ ID NO: 8] or LERLTL [SEQ ID NO: 9].

[0071] In yet another embodiment of the present disclosure a p27 protein binding domain (MRAIL) has been identified within the Kl 7 amino acid sequence. The present disclosure shows that K17 binds directly to p27 in the nucleus of a cell during Gl phase of the cell cycle at hydrophobic domains in the Kl 7 protein, MRAILs. Hence, in certain embodiments an agent of the present disclosure inhibits the interaction between keratin 17 and p27 in the nucleus of a cell, and thus prohibits the transport of p27 from the nucleus to the cytoplasm and inhibits tumor progression by inducing cell cycle arrest.

[0072] In certain embodiments the p27 binding domain of K17 for which an agent of the present disclosure binds is located between amino acid residues 155 and 220 of the K17 protein. In certain embodiments the p27 binding domain of K17 for which an agent of the present disclosure binds is located between amino acid residues 162 and 178 of the K17 protein (MRAIL 1), inclusive. In yet another embodiment the p27 binding domain of K17 for which an agent of the present disclosure binds is located between amino acid residues 200 and 216 of the Kl 7 protein (MRAIL 2), inclusive. In a specific embodiment the p27 binding domain of K17 for which an agent of the present disclosure binds is located at amino acid residues 163 to 176. In one preferred embodiment of the present disclosure the p27 binding domain of K17 includes the amino acid sequence RX₄DX7E [SEQ ID NO: 10]. In a preferred embodiment the p27 binding domain of Kl 7 is located at amino acid residues 201 to 214 of the human K17 protein, In a specific embodiment of the present disclosure the p27 binding domain of K17 includes the amino acid sequence RAXLX₈EE [SEQ ID NO: 11]. In yet another embodiment of the present disclosure the MRAIL 1 p27 binding domain of K17 is RLAADDFRTKFETE [SEQ ID NO: 12], or ARLAADDFRTKFETEQA [SEQ ID NO: 23]. In yet another embodiment of the present disclosure the MRAIL 2 p27 binding domain of K17 is

RADLEMOIENLKEE [SEQ ID NO: 13] or ARADLEMQffiNLKEELA [SEQ ID NO: 24]. Methods of treating

[0073] It has been discovered herein that keratin 17 (K17) functions as an intracellular carrier that shuttles p27 out of the nucleus to the cytosol be degraded. Further, inhibiting the translocation of K17 from the cytosol to the nucleus, and/or from the nucleus to the cytosol, has been shown to result in cell-cycle arrest, and inhibit tumor cell progression. Unexpectedly, aberrant K17 trafficking results in the accumulation of p27 in the nucleus, and thus enables p27 to maintain control over cell cycle progression, i.e., maintains its tumor suppressor function, without being degraded in the cytosol. Stated another way, the present disclosure shows that inhibiting the interaction of K17 and p27 in the nucleus reduces the amount of cytosolic p27, and thus inhibits tumor growth and development.

[0074] In one aspect of the present disclosure a method of modulating keratin 17 function in a subject is provided. In certain embodiments the method includes administering to the subject an effective amount of an agent that modulates the interaction between K17 and a keratin 17 binding protein.

[0075] A subject of the present disclosure includes a cancer patient whom has been diagnosed with cancer, or a precancerous lesion. In a preferred embodiment the subject has been diagnosed with cervical cancer, pancreatic cancer, lung cancer, breast cancer, liver cancer, kidney cancer, stomach cancer, head and neck cancers and glioblastomas (i.e., brain cancer), as expression of Kl 7 (K17-positive) in these cancer types is associated with more aggressive behavior and shorten patient survival, compared to cancers that are K17-negative. [0076] The binding of K17 to proteins such as, cytosolic chaperones, p27, or nuclear chaperones may be detected and quantified according to methods commonly known in the art. See, e.g., Glatz et al., J. Biol. Chem., 259:4295-4300, 1984; and Morrow & Martin, J. Lipid Res., 24:324-331, 1983). Accordingly, examples of such methods may include or involve incubation of K17 with radio-labeled p27, importin, or exportin in the presence or absence of agents of the present disclosure, that effect binding or interaction with K17, such as antibodies, small molecules, peptides or antisense RNA. Examples of such methods may further include the subsequent separation of Kl 7-bound and unbound p27 or chaperones and quantification of such K17 or chaperones bound to K17 in the presence or absence of an agent, as shown, for example, in FIGS. 9A-C, 10, 12 and 13C, below.

[0077] K17 activity, in a subject or elsewhere, can be detected and their amount and concentration measured by any method commonly known in the art. Including, for example, methods involving mass spectrometry, high pressure liquid chromatography (HPLC), combined gas chromatography-mass spectrometry, and liquid chromatography- atmospheric pressure chemical ionization-mass spectrometry. See, for example, De Marchi et al, Lipids Health Dis.2:5, (2003).

[0078] By way of example, whole cell protein samples can be collected with RIPA buffer (Sigma-Aldrich) and subsequently sonicated. Nuclear and cytoplasmic proteins can then be extracted and protein concentration can be determined by, for example, the BCA protein assay (Pierce). Protein samples can be loaded to sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membrane. The membranes can then be blocked and then probed with antibody specific to the protein of interest. Secondary antibodies are then provided and antibodies are detected using methods known by one of ordinary skill in the art, and quantified.

Exemplary antibodies for use in the present methods include, but are not limited to, mouse anti-keratin 17 antibody (Cat # sc-101461, Santa Cruz Biotechnology, Santa Cruz, CA), mouse anti-human p27^KIP1 antibody (Cat # 610242, BD transduction Labs), rabbit anti-phospho p27^KIP1 SerlO (Cat # sc-12939-R, Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-cyclin A (Cat # sc-751 Santa Cruz Biotechnology, Santa Cruz, CA.

[0079] As shown in FIG. 11A-C, keratin 17 binding partners contemplated by the present methods can also be identified or confirmed through the use of, for example, immunoprecipitation, yeast two-hybrid, or co-localization studies, which are not considered undue experimentation by the skilled artisan. In a specific embodiment an agent that binds to the K17 NLS, p27 binding domain or K17 NES is used in the present methods.

[0080] As stated above, the present disclosure identifies a nuclear localization signal (NLS) within the K17 protein. The NLS region of K17 is shown to bind cytosolic chaperone proteins, such as importm, which shuttle Kl 7 to the nucleus. The present disclosure also shows that prohibiting the interaction of a cytosolic chaperone protein with the NLS region of K17 inhibits translocation of K17 into the nucleus and inhibits cancer progression. Hence, in a specific embodiment the instant method includes administering to a subject an effective amount of an agent that binds to the nuclear localization signal of Kl 7 within the Kl 7 protein. In certain embodiments, interaction between the agent and the K17 NLS prohibit binding of K17 to a cytosolic chaperone protein, e.g., importin, and reduces the amount of K17 in the nucleus of a cell. More specifically, the present methods inhibit the interaction between keratin 17 and a cytosolic chaperone protein, and thus prohibits the translocation of K17 from the cytoplasm of a cell to the cell nucleus. In certain embodiments the therapeutic efficacy of an agent can be realized by detecting the relative amount of K17 present in the cytoplasm of a cell, before and after administration of an agent. In other embodiments the therapeutic efficacy of an agent can be determined by detecting the relative amount of K17 present in the nucleus before and after administration of the agent. Here, an increase in cytosolic K17 and/or a reduction in nuclear Kl 7 will be therapeutically effective in treating a subject.

[0081] Agents of for use in the present methods include peptides, antibodies, small molecules or fragments of any of the foregoing, which bind to the K17 nuclear localization sequence (NLS). In one embodiment the K17 NLS for which an agent binds is present from amino acid residues 350 to 425 of the K17 protein. In another embodiment the K17 NLS is located between from amino acid residues 390 to 410 of the K17 protein. In a specific embodiment the K17 NLS is located from amino acid residues 386-400 of the human K17 protein. In a preferred embodiment the K17 NLS is located at amino acid residues 395-400 of the human K17 protein. In a specific embodiment the K17 NLS is located at amino acid residues 399 to 400 of human K17.

[0082] In preferred embodiments of the present disclosure the Kl 7 nuclear localization signal includes the amino acid sequence KRX_10-12KK [SEQ ID NO: 1], where X is any amino acid. In yet another embodiment of the present disclosure the K17 nuclear localization signal includes the amino acid sequence KRXjo-nKR [SEQ ID NO: 2], where X is any amino acid. In a specific embodiment of the present disclosure the K17 nuclear localization signal includes the amino acid sequence RRX^KK [SEQ ID NO: 3] or RRLLX₈KK [SEQ ID NO: 4], where X is any amino acid. In specific embodiments the K17 nuclear localization signal includes the amino acid sequence

RRLLEGEDAHLTQYKK [SEQ ID NO: 5].

[0083] In another embodiment present disclosure a nuclear export signal (NES) has been identified within the K17 protein. The NES of K17 is shown to bind nuclear chaperone proteins, such as exportin, which shuttle K17 from the nucleus to the cytosol of a cell. The present disclosure also shows that prohibiting the interaction between a nuclear chaperone protein and the NES region of K17 inhibits translocation of K17 into the cytoplasm and inhibits cancer development and cell cycle progression. Hence, in a specific embodiment the instant method includes administering to a subject an effective amount of an agent that binds to the nuclear export signal of K17 (NES) within the K17 protein. In certain embodiments, interaction between the NES binding agent and the Kl 7 NES prohibit binding of Kl 7 to a nuclear chaperone protein, e.g., exportion, and reduces the amount of Kl 7 in the cytoplasm of a cell. More specifically, the present methods inhibit the interaction between keratin 17 and a nuclear chaperone protein, thereby prohibiting the translocation of K17 from the nucleus of a cell to the cytosol.

[0084] In certain embodiments the therapeutic efficacy of an agent can be realized by detecting the relative amount of K17 present in the cytoplasm of a cell, before and after administration of an agent. In other embodiments the therapeutic efficacy of an agent can be determined by detecting the relative amount of K17 present in the nucleus before and after administration of the agent. Here, a reduction in cytosolic K17 and/or an increase in nuclear K17 accumulation will be therapeutically effective in treating a subject. In a specific embodiment, therapeutic efficacy of an agent can be determined by the presence of a K17 binding partner in the cytoplasm of a cell after administration of an NES binding agent. For example, a reduction in cytosolic p27 and/or an increase in nuclear p27 accumulation after administration with an NES binding agent will be therapeutically effective in treating a subject.

[0085] In certain embodiments the K17 NES for which an agent binds is present from amino acid residues 150 to 225 of K17. In certain embodiments the K17 NES is located between amino acid residues 175 to 210 of the Kl 7 protein. In one embodiment the K17 NES is found at amino acid residues 193 to 201 of the human K17 protein. In a specific embodiment of the present disclosure the K17 NES is located at amino acids 194 to 199 of the human K17 protein. In yet another embodiment the NES includes amino acid 194, 197 and 199 of the human K17 peptide. In one embodiment the K17 nuclear export signal includes the amino acid sequence LXXLXL [SEQ ID NO: 6], where X is any amino acid. In yet another embodiment of the present disclosure the K17 NES includes the amino acid sequence LDELTL [SEQ ED NO: 7]. In a specific embodiment of the present disclosure the K17 NES includes the amino acid sequence LEELEL [SEQ ID NO: 8] or LERLTL [SEQ ID NO: 9].

[0086] In yet another embodiment of the present disclosure a p27 protein binding domain (MRAIL) has been identified within the Kl 7 amino acid sequence. The present disclosure shows that K17 binds directly to p27 in the nucleus of a cell during Gl phase of the cell cycle at hydrophobic domains in the K17 protein, MRAILs. Hence, in a specific embodiment the instant method includes administering to a subject an effective amount of an agent that binds to and inhibits the interaction between keratin 17 and p27 in the nucleus of a cell, and thus prohibits the transport of p27 from the nucleus to the cytoplasm and inhibits tumor progression by inducing cell cycle arrest. More specifically, in certain embodiments, interaction between the agent and the K17 p27 binding domain prohibits binding of K17 to a p27 or a protein complex containing p27 and increases the amount of p27 in the nucleus of a cell. Stated another way, the present methods inhibit the interaction between keratin 17 and p27, and thus prohibits the translocation of p27 from the nucleus of a cell to the cytoplasm, where p27 is degraded.

[0087] In certain embodiments the therapeutic efficacy of an agent can be realized by detecting the relative amount of p27 present in the cytoplasm of a cell, before and after administration of a therapeutic agent. In other embodiments the therapeutic efficacy of an agent can be determined by detecting the relative amount of p27 present in the nucleus before and after administration of the agent. Here, an increase in nuclear p27 and/or a reduction in cytosolic p27 will be therapeutically effective in treating a subject.

[0088] In certain embodiments the p27 binding domain of Kl 7 is located between amino acid residues 155 and 220 of the K17 protein. In certain embodiments the p27 binding domain of K17 is located between amino acid residues 162 and 178 of the Kl 7 protein (MRAIL 1), inclusive. In yet another embodiment the p27 binding domain of Kl 7 is located between amino acid residues 200 and 216 of the Kl 7 protein (MRAIL 2), inclusive. In a specific embodiment the p27 binding domain of Kl 7 is located at amino acid residues 163 to 176. In one preferred embodiment of the present disclosure the p27 binding domain of K17 includes the amino acid sequence RX₄DX7E [SEQ ID NO: 10]. In a preferred embodiment the p27 binding domain of K17 is located at amino acid residues 201 to 214 of the human K17 protein. In a specific embodiment of the present disclosure the p27 binding domain of K17 includes the amino acid sequence RAXLX₈EE [SEQ ID NO: 11]. In yet another embodiment of the present disclosure the MRA1L 1 p27 binding domain of K17 is RLAADDFRTKFETE [SEQ ID NO: 12], or

ARLAADDFRTKFETEQA [SEQ ID NO: 23]. In yet another embodiment of the present disclosure the MRAIL 2 p27 binding domain of K17 is RADLEMOIENLKEE [SEQ ID NO: 13] or ARADLEMQIENLKEELA [SEQ ID NO: 24].

[0089] The dosage of an agent that is administered to a subject during therapy may vary, depending on the reason for use and the individual subject. The dosage may be adjusted based on the subject's weight, the age and health of the subject, and tolerance for the compound or composition. Non-limiting examples of effective dosages include about 2 mg/kg of bodyweight/day, about 5 mg/kg of bodyweight/day, about 10 mg/kg of bodyweight/day, about 15 mg/kg of bodyweight/day, about 20 mg/kg of bodyweight/day, about 25 mg/kg of bodyweight/day, about 30 mg/kg of bodyweight/day, about 40 mg/kg of bodyweight/day, about 50 mg/kg of bodyweight/day, about 60 mg/kg of

bodyweight/day, about 70 mg/kg of bodyweight/day, about 80 mg/kg of bodyweight/day, about 90 mg/kg of bodyweight/day, about 100 mg/kg of bodyweight/day, about 125 mg/kg of bodyweight/day, about 150 mg/kg of bodyweight/day, about 175 mg/kg of bodyweight/day, about 200 mg/kg of bodyweight/day, about 250 mg/kg of

bodyweight/day, about 300 mg/kg of bodyweight/day, about 350 mg/kg of

bodyweight/day, about 400 mg/kg of bodyweight/day, about 500 mg/kg of

bodyweight/day, about 600 mg/kg of bodyweiglnVday, about 700 mg/kg of

bodyweight/day, about 800 mg/kg of bodyweight/day, and about 900 mg/kg of bodyweight/day. Routine experimentation will determine the appropriate value for each patient by monitoring the agent's effect on K17 or p27 in a cell, which can be frequently and easily monitored. The agent can be administered once or multiple times per day. The frequency of administration may vary from a single dose per day to multiple doses per day. Preferred routes of administration include oral, intravenous and intraperitoneal, but other forms of administration may be chosen as well.

[0090] The effective amount of an agent according to the present methods may be administered along any of the routes commonly known in the art. This includes, for example, (1) oral administration; (2) parenteral administration, for example, by subcutaneous, intramuscular or intravenous injection; (3) topical administration; or (4) intravaginal or intrarectal administration; (5) sublingual or buccal administration; (6) ocular administration; (7) transdermal administration; (8) nasal administration; and (9) administration directly to the central nervous system (CNS).

[0091] In the context of the present disclosure, the effective amount of the agent modulating the K17 binding activity can be administered alone or in combination with one or more of other therapeutic agents. In a combination therapy, the effective amount of the agent modulating the binding activity of K17 can be administered before, during, or after commencing therapy with another agent, such as cisplatin or other

chemotherapeutic agent, as well as any combination thereof, i.e., before and during, before and after, during and after, or before, during and after commencing the additional therapy.

[0092] Consistent with the observed function of keratin 17, and more specifically the K17 NES, NLS and p27 binding domain(s) of the disclosure, the present methods can also be used other K17 mediated diseases. As such, the methods of the present disclosure are not intended to be limited by the foregoing description or examples that follow.

EXAMPLES

[0093] The following examples further illustrate the disclosure, but should not be construed to limit the scope of the disclosure in any way.

Example 1. Materials and Methods.

[0094] Xenograft models. In all, 10- to 12-week-old NOD/SCID female mice (Harlan laboratories, Dublin, VA), were used for tumor implantation. Cancer cells were subcutaneously injected into the lower back areas of the mice using 2 x 106 cells in 100 μL DMEM with 50% matrigel (BD Biosciences). As shown in FIG.2, the tumor size was measured over a month, using a caliper and tumor volume was calculated using the formula V=length x width2/2 (17). The Stony Brook University Institutional Animal Care and Use Committee approved all animal procedures. The mice were anesthetized by isoflurane inhalation.

[0095] Cell culture. The human cervical cancer cell lines SiHa, CaSki, C-33A, HT-3, ME- 180 and HeLa were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA), and pancreatic cancer cell line, L3.6 and breast cancer cell lines MBA-MD-231, MBA-MD-468 were obtained and cultured as recommended with RPMI1640, DMEM or McCoy's 5A medium (Gibco-Life Technologies) with 10% fetal bovine serum (Sigma-Aldrich, St Louis, MO, USA). Cells were grown at 37°C in a humidified atmosphere containing 5% C0₂. The medium was replaced every 48 hours. [0096] Small-interference RNA and short-hairpin RN A. For transient transfection, ON-TARGETplus Human KRT17 (3872) small-interference RNAs (siRNA)-SMART pool (Thermo Scientific, Waltham, MA, USA) of 4 siRNAs were used to knockdown KRT17 expression (siKRT17). The following KRT17 siRNA sequences were used to knockdown KRT17 expression: (5'-3') AGAAAGAACCGGUGACCAC [SEQ ID NO: 14], CGUCAGGUGCGUACCAUUG [SEQ ID NO: 15],

GGUCCAGGAUGGCAAGGUC [SEQ ID NO: 16], GGAGAGGAUGCCCACCUGA [SEQ ID NO: 17]. ON-TARGETplus Non-targeting Control siRNAs (Thermo

Scientific, Waltham, MA, USA) were used as RNA interference control (Negative siRNA). siRNAs were transfected into cancer cells using Oligofectamine™ 2000 (Life Technologies, Grand Island, NY, USA) according to the standard protocol. For stable knockdown of KRT17, three GEPZ Lentiviral shRNA (GE Dharmacon Lafayette, CO, USA) were used to screen for best knockdown efficiency. The following KRT shRNA sequences were used to knockdown KRT17 expression: (5 '-3') shl- TCTTGTACTGAGTCAGGTG [SEQ ID NO: 18], sh2-TCTTTCTTGTACTGAGTCA [SEQ ID NO: 19], and sh3-CTGTCTCAAACTTGGTGCG [SEQ ID NO: 20]. Negative GIPZ lentiviral shRNA controls were used as negative shRNA. Lentivirus production was carried out following manufactures' protocol. After cancer cell transduction, cells were selected with 10 μg/ml, and stable clones were produced for each cell line. For stable, expression of human K17, the human ORF of K17 was cloned into pcDNA4/TO mammalian expression vector. Empty vector was used as a control. Plasmids were transfected into cancer cells using Lipofectamine™ 2000 (Life Technologies, Grand Island, NY) according to standard protocol from the manufacturer. Cells were selected for using lOOug/ml of Zeocin. [0097] Site directed mutagenesis. Point mutations were introduced to the K17 amino acid sequence using functional site directed mutagenesis. Specifically, two point mutations within a second cluster of basic amino acids (i.e., K399A and K400A) in the NLS of K17 and three point mutations within the NES of K17 (i.e., L194A, L197A and LI 99 A) were, respectively, sufficient to abolish nuclear localization and nuclear accumulation of K17, respectively. In addition, cancer cells expressing K17 protein having such mutations exhibited nuclear accumulation of p27 and underwent cell cycle arrest. Additional point mutations in the first cluster of basic amino acids (e.g., R386C, R386H) in the Kl 7 NLS can also inhibit K17 trafficking to the nucleus. These data can be extrapolated to a potential inhibitory mutation that abolishes nuclear entry of K17 and degradation of p27 without undue experimentation.

[0098] Cell proliferation, cell cycle analysis and senescence assay. Twenty-four hours after transient transfection, SiHa and CaSki cells were seeded in 96-well plates at 4000 cells/well. The cell proliferation assay was performed on days 1, 3 and 5 by incubating 10 μl WST-1 (Roche Applied Science, Mannheim, Germany) in the culture medium for 2 h and reading the absorbance at 450 and 630 nm. The cell proliferation rate was calculated by subtracting the absorbance at 450 nm from the absorbance at 630 nm. A cell number absorbance curve was performed to calculate cell per well. Cell cycle analysis was performed by flow cytometry using propidium iodine and acridine orange stains. Three days or two weeks after transient and stable transfections, respectively, cells were harvested and resuspended at 0.5-1 x 10⁶ cells/ml in modified Krishan buffer with 0.02 mg/ml RNase H (Invitrogen) and 0.05 mg/ml propidium iodide (Sigma-Aldrich). Results were calculated with Modfit LT software version 3 (Verity Software House, Topsham, ME, USA). For acridine orange cell cycle stain and analyses were performed as previously described (DarzynMewicz et al., 1980; El-Naggar, 2004). All samples were analyzed in FACSCalibur™ (Becton Dickinson) at the Research Flow Cytometry core at Stony Brook University. The Senescence β-galactosidase staining kit (Cell Signaling, Danvers, MA, USA #9860) was used to determine percentage of senescent cells following the manufactures' instructions.

[0099] Serum Starvation Release, Cycloheximide Chase and leptomycin B treatment Serum starvation release was used to arrest cancer cells in Gl phase and stabilize p27. For protein stability analysis, cells were plated into 60-mm dishes at 50% confluence and serum starved for 48 h. After serum starvation, cell were restimulated with DMEM containing 20% FBS and cycloheximide at 40 μg/ml (CHX, catalog no. 239764; Calbiochem). At the indicated time points, whole cell extracts were prepared and western blotted. For nuclear-export inhibition assays, leptomycin B was added at 20nM (Cell Signaling, Beverly, MA) in a mixture containing 20% FBS DMEM for serum-starve release.

[00100] Western Blotting and Extraction of Nuclear Proteins. Whole cell protein samples were collected with RIP A buffer (Sigma- Aldrich) and subsequently sonicated. Nuclear and cytoplasmic proteins were extracted by NE-PER™ Protein Extraction Reagent (Pierce) according to the manufacturer's instructions. Protein concentration was determined by the BCA protein assay (Pierce). Equal amounts of samples were loaded to sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membrane. The membranes were blocked with 5% non-fat milk in TBS/0.5% Tween-20 (TBS-T) at room temperature for 30 min, then probed with: mouse anti-keratin 17 antibody (Cat # sc-101461, Santa Cruz Biotechnology, Santa Cruz, CA), mouse anti-human p27 antibody (Cat # 610242, BD transduction Labs), rabbit anti-human pRB antibody (Cat # 9313S, Cell Signaling, Danvers, MA, USA), rabbit anti-cyclin Dl (Cat # 2978S, Cell Signaling, Danvers, MA, USA), rabbit anti- SKP2 (Cat # 2652P, Cell Signaling, Danvers, MA, USA), rabbit anti-phospho p27^KIP1 SerlO (Cat # sc-12939-R, Santa Cruz Biotechnology, Santa Cruz, CA), mouse anti-JABl (Cat # sc-13157, Santa Cruz Biotechnology, Santa Cruz, CA), mouse anti-HPV16 E6/18E6 (Cat # sc-460, Santa Cruz Biotechnology, Santa Cruz, CA), mouse anti-HPV16 E7 (Cat # sc-6981, Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-cyclin A (Cat

# sc-751 Santa Cruz Biotechnology, Santa Cruz, CA), mouse anti-RNF123 (KPCl) (Cat

# sc-101122 Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-UBE3A (Cat # AP2154B ABGENT, San Diego, CA, USA), rabbit anti-pl30 (Cat # sc-317, Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-phospho keratin 17 Ser44 (Cat # 3519S, Cell Signaling, Danvers, MA, USA), rabbit anti-cytokeratin 17 (Cat # ab 109725 Abeam, Cambridge, MA, USA), mouse anti- p53 antibody (Cat # sc-126, Santa Cruz

Biotechnology, Santa Cruz, CA, USA), mouse anti-human p21 antibody (Cat #2946, Cell Signaling, Danvers, MA, USA), mouse anti- GAPDH antibody (Cat # sc-365062, Santa Cruz Biotechnology, Santa Cruz, CA, USA), mouse anti-human a-tubulin antibody (Cat # 05-829, Millipore, Temecula, CA, USA), mouse anti-Lamin Bl (Cat # ab90576 Abeam, Cambridge, MA, USA) overnight at 4 °C. Goat anti-rabbit and anti-mouse and rabbit anti-goat horseradish peroxidase-conjugated secondary antibodies (Jackson Immunoresearch, West Grove, PA, USA) were used at 1 :5000. Horseradish peroxidase activity was detected with SuperSignal West Pico Qiemiluminescent Substrate (Thermo Scientific, Waltham, MA, USA) and visualized in an UVP Bioimaging system (Upland, CA, USA). Expression levels were quantified using ImageJ software (National Institute of Health, Bethesda, MA, USA), and normalized to loading controls

[00101] Statistical Analysis. Each experiment was independently repeated three to four times with one to three replicates per experiment Categorical data are described using frequencies and percentages. Continuous data are described using means ± standard deviation or standard error of the mean. Statistical significance between the means of two groups was determined using Student's t tests or Mann-Whitney U tests. Statistical comparisons of the means of multiple groups were determined using one-way ANOVA or Kruskal-Wallis ANOVA by ranks. All analyses were performed using SAS 9.3 (SAS Institute, Inc., Cary, NC) and SigmaPlot 11 (Systat Software, San Jose, CA). Statistical significance was set at p < 0.05 (a).

[00102] Example 2: K17 regulates cell-cycle progression through p27 interaction. In order to determine whether K17 is directly involved in tumor progression the effect of K17 on tumor growth was evaluated in a cervical cancer subcutaneous xenograft model. The results exhibited in FIG. 2 how that K17-expressing tumors (control shRNA) doubled in size over a 30 day period compared to tumors not expressing K17 (Kl 7 shRNA). Hence, K17 confers a growth advantage to tumors. This finding in cervical cancer was also recognized in pancreatic cancer, where elevated levels of K17 lead to increased tumor growth and development.

[00103] To determine if K17 contributes to sustained-proliferation signaling, RNAi was employed to target the K17-expressing cervical cancer cell lines SiHa and CaSki, resulting in a 50% decrease in proliferation (FIGS. 3A-B). Cell-cycle analyses indicated that K17 silencing induces Gl -arrest (FIG. 3C). In contrast, forced expression of K17 in the non K17-expressing cervical cancer cell line (C-33A) significantly decreased the Gl/S-phase ratio (Fig. 3D). Furthermore, K17 knockdown in pancreatic- and breast- cancer cell lines increased Gl -phase accumulation (FIG. 3E). The effect of K17 appears to be early in Gl in cervical cancer cells as reflected by an increase in the G1A/G1B ratio shown in FIG. 3F, a decrease in total RNA and a decrease in cell size. No differences were found in sub-Gl/GO (apoptotic) percentage, mitotic entry or percentage of senescent cells. C33-A cells challenged with K17 RNAi did not arrest in Gl, providing evidence of on-target action for the effects observed for Kl 7 RNAi-mediated knockdown in the SiHa and CaSki cell lines.

[00104] Example 3: Keratin 17 knockdown promotes p27iapi-nuclear accumulation and stabilization. RNAi-targeting of K17 was accompanied by 3-5-fold increased p27 protein but no increase in its mRNA level (FIGS.4A-B). Elevation of p27 is known to inhibit Gl cyclin-dependent kinases. Consistent with this, K17-knockdown decreased Rb phosphorylation by 50% and decreased expression of cyclin A (an S/G2-associated cyclin). Changes in the GO marker pi 30 were not detected, demonstrating that K17 knockdown causes cell-cycle arrest rather than cell-cycle exit. In contrast, FIG.4C shows that expression of K17 in C33-A cells eliminated endogenous p27 protein expression. Similarly, p27 expression increased in pancreatic- and breast-cancer cells following K17 knockdown (FIG.4D). Expression of K17 in Hek293 cells, derived from benign tissue, also resulted in significantly decreased p27. Taken together, these data show that to act as a cell-cycle inhibitor, p27 must be located in the nucleus, whereas its cytoplasmic sequestration enables cell-cycle progression because in the absence of K17, nuclear but not cytoplasmic levels of p27 increased significantly (2- to 3-fold), as shown in FIGS. 5A-B. [00105] The present disclosure also reveals that in the absence of K17, export-tagged ρ27^KIP1 accumulated in the nucleus, correlating to early-Gl arrest. Although p27^KM is one of the key cyclin dependent kinase inhibitors (CKI) that ensure correct Gl phase timing, gene-expression of other CKIs in K17-knockdown cells were screened for.

Unexpectedly, shKRT17- treated cell levels of p21 were significantly decreased only in CaSki, indicating that Gl arrest was solely attributed to p27^KIP1.

[00106] It is well known that p27^KIP1 translation and protein stability are maximal during Gl and increased p27^KIP1 -nuclear export and degradation enables Gl/S transition.

Serum-starved control and K17-knockdown cells in Gl were stimulated with FBS to trigger the Gl/S transition in the presence of cycloheximide to block protein translation. p27^KIP1 levels decreased 40% in the K17-expressing control cells but were virtually unchanged in K17-knockdown cells. These findings shown that p27^KIP1 degradation at Gl is reduced in the absence of K17.

[00107] It is also known that p27^KIP1 is targeted by KPC1 and SKP2 ubiquitin-E3 ligases, which function at Gl- and S phases, respectively. Hence, the expression and

ubiquitination activity of

KPC1 and SKP2 was examined herein. Surprisingly, only in SiHa cells with K17 knockdown, there was an increase in SKP2 expression, due to Gl phase arrest, but notably, KPC1

not SKP2, co-immunoprecipitated with K17 and p27^KIP1 and colocalized with K17. Overall, these results reveal that p27^KIP1 stabilization is mediated by retention or delayed nuclear export, preventing degradation in the cytosol, rather than as a result of deficiency in the ubiquitin-mediated degradation by KPC1 or SKP2. [00108] Example 4: Keratin 17 colocalizes with p27 in the nucleus. In view of the foregoing, it was determined whether K17 binds to p27 in the nucleus or cytoplasm of a cell. FIGS. 6A-B demonstrate that nuclear-K.17 binds to nuclear p27^KM during early-Gl phase. The results herein show by immunoprecipitation and co-localization staining using super-resolution structured illumination analysis that more than half the cells had at least one co-localized signal for K17 and p27^KIP1 (FIG. 6B).

[00109] Example 5: Keratin 17 contains a nuclear localization signal (MLS). In silico analysis of the K17 coding sequence revealed a classical bipartite nuclear localization signal (NLS) at amino acid residues 385-401 of human K17 (FIGS. 7C and 8 A), specific among type I keratins, which was present only in primates and humans, but not in other species (FIG. 7C). The cNLS-Mapper cut-off score of 3.3 predicts that K17 should be found in both the nuclear and cytoplasmic compartments.

[00110] Example 6: Keratin 17 contains a nuclear export signal (NES). Nuclear export of ρ27ΚΠ^»1 involves an adaptor for CRMl-exportin binding. By in silico analysis, using ValidNESs, a putative leucine-rich, nuclear-export signal (NES) was identified. It was noted that K17 nucelar export was CRM 1 -binding dependant and carried out by an interaction at residues 194 and 199 of the K17 protein, as shown in FIGS. 7A and 8A.

[00111] FIG. 11 shows that treatment of SiHa and CaSki cells with leptomycin B (LMB), a chemical inhibitor of the CRM1 -dependent nuclear export, caused a > 2-fold retention of nuclear K17 and p27^KIP1. Additionally, JABl, a known adaptor between p27^KIP1 and CRM1 -exportin, accumulated in the nucleus after LMB treatment (FIG. 11). Additionally, immunoprecipitation analyses revealed that K17 and export-tagged p27^KIP1 binds to CRMl-exportin. These findings suggest that K17 and p27 are exported from the nucleus through CRMl-exportin interaction, as indicated in FIG. 1.

[00112] Example 7: Site directed mutagenesis of the K17 NES and NLS domains.

The three leucines within the K17 NES (L194A, L197A and L199A) and the two lysines within K17 NLS (K399A and K400A) were mutated to determine if manipulations of the NLS and/or NES affected p27^KM activity. C33-A cells were transfected with vectors encoding human wild-type K17 with the putative NES and NLS (Wt), or mutated NES (mNES), or mutated NLS (mNLS), and the p27^{KIP 1} levels in nuclear and cytoplasmic fractions were quantified. See FIGS. 8A-C and 9. Nuclear p27^KIP1 was lost in cells expressing Wt K17, as p27^KIP1 was trafficked to the cytosol and degraded. In contrast, nuclear p27^KIP1 levels were 3-fold higher in cells expressing either mNLS- or mNES-K17 proteins (FIG. 9). Furthermore, nuclear localization of K17 was abolished in mNLS cells (FIG. 9). Taken together, functional mutagenesis showed that that two point mutations on a cluster of basic amino acids in the NLS (mNLS) and three point mutations on the NES (all leucines, mNES) were, respectively, sufficient to abolish nuclear localization of K17 and nuclear accumulation of K17 (FIG. 8C)

[00113] Additionally, cells that expression of Kl 7 with a mutated NLS or NES signals increased nuclear p27, whereas wild-type K17 depleted total cellular p27 levels in numerous cancer types, such as cervical, pancreatic and breast cancer (FIG. 9). These studies revealed a novel and unexpected function for Kl 7 as an adaptor protein for p27- CRM1 nuclear export and degradation across cancer cell types.

[00114] Example 8: K17 promotes degradation of p27 through interaction with MRAIL binding domains. As discussed above, the present disclosure shows that that K17 harbors key functional domains that allow it to function as a nuclear chaperone for p27. FIGS. lOA-C 12A-C further shown that K17 directly binds to p27 during Gl. Here, amino acid sequences were compared between K17 and known binding partners of p27, including CDKs and cyclins. Surprisingly, the results shown in FIG. 12B reveal that K17 has two MRAIL sequences from amino acid residues 162-178 (MRAIL 1) and from amino acid residues 200-216 (MRAIL 2), that were identified using the crystal structure showing where p27 docks to cyclins, as set forth in FIG. 12A. Point mutations in key residues of either MRAIL 1 or MRAIL2 decreased Kl 7-mediated degradation of p27 in cancer cells (FIG. 12C) which shows that both MRAIL domains in Kl 7 contribute independently to the loss of p27 activity.

[00115] Example 9: Discussion. The results provided herein show, for the first time, that K17 deregulates key tumor suppressor programs in Gl, promoting p27^KIP1 -nuclear export, sustained proliferation, and tumor growth. p27^{KIP 1} lacks a classical leucine-rich NES and mutation of the suggested NES in K17 impair nuclear export (FIG. 8B).. Here, Kl 7 serves as a bridge between p27^KIP1 and CRM1, as point mutations in K17-NES lead to nuclear accumulation of both K17 and p27^KIP1, as does leptomycin B (LMB) treatment (FIG. 11). Although p27^KIP1 -nuclear export seems to be mediated by several mechanisms, the results herein teach that K17 promotes p27^Klp1 -nuclear export in cervical, pancreatic and breast cancer cells.

[00116] Additionally, this is the first report of the existence and mechanistic role of the Kl 7 NLS, a sequence that is found only among primates and humans (FIG. 8C).

Mutations of the NLS prevent K17-nuclear localization and result in p27KIPl -nuclear accumulation (FIGS. 8A-C and 9). Hence, it can be determined that mitogen stimulation of K17-expressing tumor cells results in post-translational modifications of K17 that increase its nuclear targeting and binding of p27°^?1 and CRM1, leading to p27^KIP1 export from the nucleus.

Claims

WHAT IS CLAIMED IS:

1. An agent that inhibits keratin 17 (K17) binding, wherein said agent binds to a portion of a keratin 17 protein selected from the group consisting of the keratin 17 nuclear localization signal (NLS), the keratin 17 nuclear export signal (NES) and a p27 binding sequence.

2. The agent of Claim 1, wherein said agent is selected from a group consisting of an antibody, a peptide, a small molecule and fragments thereof.

3. The agent of Claim 1, wherein said agent binds to the NLS of K17.

4. The agent of Claim 3, wherein said NLS comprises the amino acid sequence KRX₁₀- 12KK, as set forth in SEQ ID NO: 1.

5. The agent of Claim 4, wherein said amino acid sequence is RRX₁₂KK, as set forth in SEQ ID NO: 3.

6. The agent of Claim 5, wherein said amino acid sequence is or RRLLX₈KK, as set forth in SEQ ID NO: 4]

7. The agent of Claim 6, wherein said amino acid sequence is RRLLEGEDAHLTQYKK as set forth in SEQ ID NO: 5.

8. The agent of Claim 3, wherein said NLS comprises the amino acid sequence KRXio- 12KR as set forth in SEQ ID NO: 2.

9. The agent of Claim 1 , wherein said agent binds to the NES of K17.

10. The agent of Claim 9, wherein said NES comprises the amino acid sequence LXXLXL, as set forth in SEQ ED NO: 6.

11. The agent of Claim 10, wherein said NES comprises the amino acid sequence selected from the group consisting of LDELTL [SEQ ID NO: 7], LEELEL [SEQ ID NO: 8] and LERLTL [SEQ ID NO: 9].

12. The agent of Claim 1, wherein said agent binds to a p27 binding domain of K17.

13. The agent of Claim 12, wherein said p27 binding domain of K17 comprises the amino acid sequence RX₄DX7E, as set forth in SEQ ID NO: 10.

14. The agent of Claim 12, wherein said p27 binding domain of K17 comprises the amino acid sequence RAXLX₈EE, as set forth in SEQ ID NO: 11.

15. The agent of Claim 12, wherein said p27 binding domain of K17 comprises the amino acid sequence RLAADDFRTKFETE, as set forth in SEQ ID NO: 12 or

ARLAADDFRTKFETEQA [SEQ ID NO: 23]

16. The agent of Claim 12, wherein said p27 binding domain of K17 comprises the amino acid sequence is RADLEMOIENLKEE, as set forth in SEQ Π) NO: 13 or ARADLEMQIENLKEELA [SEQ ID NO: 24].

17. A method for treating a subject comprising administering an effective amount of an agent to a subject, wherein said agent inhibits the interaction of K17 with another molecule.

18. The method of Claim 17, wherein said agent reduces the amount of K17 protein in the nucleus of a cell of said subject.

19. The method of Claim 17, wherein said agent reduces the amount of p27 in the cytosol of a cell of said subject.

20. The method of Claim 19, wherein said agent increases the amount of p27in the nucleus of a cell of said patient.

21. The method of Claim 17, wherein said another molecule is a cytosolic chaperone, a nuclear chaperone or a p27 protein.

22. The method of Claim 21, wherein said another molecule is the cytosolic protein, importin.

23. The method of Claim 21 , wherein said another molecule is the nuclear chaperone protein, exportin.

24. The method of Claim 21, wherein said another molecule is p27.