Patent Application
20240368566
The present invention is generally directed to treatment of cancer. More specifically, the invention relates to treatment of cancer by inhibiting cancer metastasis.
Metastatic dissemination of cancer cells from the primary tumor and their spread to distant tissues and organs is the leading cause of mortality in breast cancer patients. Although cytotoxic drugs and hormone-blocking therapeutics are often used to shrink the primary tumor or prevent disease recurrence, no treatment to permanently eradicate metastatic breast cancer exists at present.
Metastatic cancer cells leaving the primary tumor use invasive feet-like structures called invadopodia, actin-rich protrusions with extracellular matrix (ECM) degrading activity, to invade through the basement membrane and to intravasate into blood vessels and spread to distant tissues and organs throughout the body. Invadopodia were identified in several invasive cancer cell lines, such as breast, head and neck, prostate, fibrosarcoma, and melanoma as well as in primary tumor cells. Moreover, direct molecular links between invadopodia assembly and cancer metastasis have previously been demonstrated in both mice models and human patients. Along these lines, the inventors have recently shown that therapeutic inhibition of invadopodia formation and function can block breast cancer metastasis in a xenograft mouse model.
The actin-nucleation promoting factor cortactin is a crucial regulator of actin cytoskeleton remodeling and an essential component of invadopodia. Cortactin plays an imperative role in actin polymerization-mediated membrane protrusions, cell migration, cell-cell adhesion, endocytosis, and neuronal synapse stabilization. Its five functional regions include an N-terminal acidic domain (NTA) that binds the Arp3 component of the Arp2/3 complex and synergizes with the N-WASP VCA domain to promote actin polymerization, six and a half F-actin-binding repeats of 37 amino acids each, an α-helical domain with unknown function, a proline-serine-threonine (PST) rich phosphorylation region enabling cortactin to integrate signals from diverse upstream signaling cascades, and a Src homology 3 (SH3) domain (residues 495-550). The SH3 domain mediates the association of cortactin with various signaling proteins such as dynamin, Wiskott-Aldrich Syndrome (WASp)-interacting protein (WIP), CortBP-1/Shank2, Shank-3, ZO-1, and faciogenital dysplasia protein (Fgd1). The association of cortactin with proline-rich regions of diverse proteins enables regulating diverse actin remodeling-mediated cellular processes, such as endocytosis, lamellipodial protrusions, and cell migration. Amplification of the CTTN gene (encoding cortactin) followed by overexpression of cortactin have been observed in various human cancers and are associated with poor patient prognosis. Importantly, cortactin is an essential component of invadopodia, regulating both their assembly and the trafficking and secretion of matrix metalloproteinases (MMPs) from invadopodia tips. Cortactin phosphorylation on tyrosine residues regulates an on-off switch which initiates actin polymerization in invadopodia, leading to their maturation and activation and consequent tumor cell invasion.
Complexes between SH3 domains and proline-rich polypeptide segments are well-known regulators of major intracellular signaling events. A key feature is the steric compatibility between a flat SH3 hydrophobic surface with three grooves delineated by conserved aromatic residues and a PxxP core adopting a left-handed helical conformation known as the polyproline II helix, recently also termed x-helix. Typically, two qP dipeptides (q being a hydrophobic residue) fill binding pockets between the conserved aromatic residues, and the inherent symmetry of the PxxP helix allows for binding in both possible orientations. The location of flanking basic residues and their interaction with negatively charged residues in the SH3 specificity pocket determines the preferred binding pose, roughly dividing SH3 ligands into class I and class II motifs, exhibiting N-terminal or C-terminal basic residues, respectively. Screening of a phage-displayed peptide library defined the cortactin-binding consensus motif as +PPΨPPxKP (‘Ψ’ is an aliphatic residue, >90% conserved positions in bold), a class II motif with an additional PxxP motif, sometimes termed a class VIII motif. However, structure determination of SH3/PxxP complexes is highly challenging due to their moderate affinity levels, which are in the micromolar range. Limited information at atomic resolution restricts a deeper structural understanding of cortactin SH3-PRR regulatory complexes.
Proline-rich tyrosine kinase 2 (Pyk2), along with focal adhesion kinase (FAK), include a subfamily of non-receptor protein tyrosine kinases that integrate signals from various receptors, activating signaling pathways that regulate proliferation, migration, and invasion in numerous cell types. Both share a conserved domain structure, consisting of an N-terminal FERM domain, a central catalytic kinase domain, three proline-rich sequences, and a C-terminal FAT domain. Two of the proline-rich sequences, proline-rich region 2 (713Pro-Pro-Pro-Lys-Pro-Ser-Arg-Pro721 of UniProt Acc. No. Q14289) (PRR2) and proline-rich region 3 (855Pro-Pro-Gln-Lys-Pro-Pro-Arg-Leu862) (PRR3) are major sites for SH3-mediated protein-protein interactions. The former exhibits high similarity between the two kinases, while the latter shows very little similarity, possibly accounting for their unique binding partners. Interestingly, a previous study showed that cortactin SH3 binds directly to both FAK-PRR2 and FAK-PRR3, an interaction followed by a sequence of events that promote focal adhesion turnover and cell migration.
Accordingly, the progress in understanding the interactions between cortactin and tyrosine kinases may be harnessed for the development of the much needed therapeutics for inhibiting cancer metastases.
The following embodiments and aspects thereof are described and illustrated in conjunction with compositions and methods which are meant to be exemplary and illustrative, not limiting in scope. In various embodiments, one or more of the above-described problems have been reduced or eliminated, while other embodiments are directed to other advantages or improvements.
The inventors designed a cell-permeable peptide inhibitor that contains the second proline-rich region (PRR2) sequence of Pyk2, which binds to the SH3 domain of cortactin and blocks spontaneous lung metastasis in immune-competent mice by inhibiting invadopodia maturation and function In some embodiments, the present invention provides an isolated Pyk2-derived peptide including a consensus proline-rich region 2 (PRR2) sequence, wherein the consensus PRR2 sequence is PxxPx(R/K)P(K/R)(Y/W/F) in which “x” stands for any amino acid; and the peptide is capable of inhibiting metastasis by binding to a Src homology 3 (SH3) domain of cortactin.
In some embodiments, the peptide includes a sequence at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical to a sequence including the second proline-rich region of Pyk2.
In some embodiments, the peptide has a length of about 9-100, 9-50, 10-30, or 15-25 amino acids.
In some embodiments, the consensus PRR2 sequence is PxxPxRPKY (SEQ ID NO: 12). In some embodiments, the consensus PRR2 sequence is PPKPSRPKY (SEQ ID NO: 24).
In some embodiments, the peptide includes a sequence at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical to the sequence set forth in SEQ ID NO: 1.
In some embodiments, the peptide does not include a complete Pyk2 sequence. In some embodiments, the peptide does not include a proline-rich region 3 (PRR3) sequence of a Pyk2 protein.
In some embodiments, the peptide includes at least one proline outside the consensus PRR2 sequence. In some embodiments, the peptide further includes a least one tag sequence selected from the group consisting of HIV-TAT, HAIYPRH, and combinations thereof. In some embodiments, the peptide includes at least one non-conventional amino acid and/or at least one modified amino acid. In some embodiments, the at least one non-conventional amino acid and/or modified amino acid is outside the consensus PRR2 sequence. In some embodiments, the peptide is a cyclic peptide. In some embodiments, the peptide is capped at the N-terminus, at the C-terminus, or at both the N- and the C-terminus.
In some embodiments, the peptide is not capable of inhibiting primary tumor growth. In some embodiments, the peptide does not inhibit primary tumor growth.
In some embodiments, the present invention provides a method of treating a cancer in a subject, the method including administering to the subject a therapeutically-effective dose of the isolated Pyk2-derived peptide disclosed herein.
In some embodiments, the treating includes inhibiting or preventing cancer invasion and/or cancer metastasis in the subject.
In some embodiments, the present invention provides a method of inhibiting or preventing cancer invasion and/or cancer metastasis in a subject, the method including administering to the subject a therapeutically-effective dose of the isolated Pyk2-derived peptide disclosed herein.
In some embodiments, the method further includes administering to the subject an anti-cancer treatment. In some embodiments, the anti-cancer treatment includes a chemotherapeutic agent and/or an immunotherapeutic agent.
In some embodiments, the cancer includes invadopodia.
In some embodiments, the cancer is invasive or metastatic.
In some embodiments, the cancer is not invasive or is not metastatic.
In some embodiments, the cancer is selected from the group consisting of breast cancer, pancreatic cancer, head and neck cancer, prostate cancer, fibrosarcoma, and melanoma.
In some embodiments, the administration is intravenous, intramuscular, subcutaneous, intratumoral, or intraperitoneal.
In addition to the exemplary aspects and embodiments described above, further aspects and embodiments will become apparent by reference to the figures and by study of the following detailed descriptions.
The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
The invention will now be described in relation to certain examples and embodiments with reference to the following illustrative figures.
In the following description, various aspects of the disclosure will be described. For the purpose of explanation, specific configurations and details are set forth in order to provide a thorough understanding of the different aspects of the disclosure. However, it will also be apparent to one skilled in the art that the disclosure may be practiced without specific details being presented herein. Furthermore, well-known features may be omitted or simplified in order not to obscure the disclosure.
Despite the high significance of cortactin for invadopodia-mediated tumor cell invasiveness and subsequent cancer metastasis, the design of a specific cortactin inhibitor has been challenging. Research to date has focused on disruption of the upstream signaling pathway EGFR-Src-Arg-cortactin or inhibition of cortactin protein-protein interactions. Indeed, inhibition of EGFR or Src suppresses invadopodia-mediated invasiveness of head-and-neck squamous cell carcinoma cell lines (Hayes et al. Ableson kinases negatively regulate invadopodia function and invasion in head and neck squamous cell carcinoma by inhibiting an HB-EGF autocrine loop. Oncogene 2013; 32: 4766-4777), while knockdown or selective inhibition of Arg kinase block in vivo breast cancer metastasis (Gil-Henn et al. Arg/Abl2 promotes invasion and attenuates proliferation of breast cancer in vivo. Oncogene 2013; 32: 2622-2630; Meirson et al. Targeting invadopodia-mediated breast cancer metastasis by using ABL kinase inhibitors. Oncotarget 2018; 9: 22158-22183). Also, inhibition of the interaction between the ARF-GAP AMAP1 and cortactin by an AMAP1-derived peptide blocked tumor metastasis and angiogenesis (Hashimoto et al. Targeting AMAP1 and cortactin binding bearing an atypical Src homology 3/proline interface for prevention of breast cancer invasion and metastasis. Proc Natl Acad Sci USA 2006; 103: 7036-7041; Hashimoto et al. GEP100-Arf6-AMAP1-cortactin pathway frequently used in cancer invasion is activated by VEGFR2 to promote angiogenesis. PLoS One 2011; 6: e23359).
The inventors have previously identified cortactin as a potential substrate of Pyk2 in invadopodia, by using high-throughput protein arrays combined with bioinformatics analysis. Pyk2 was found to bind cortactin via its second proline-rich region (PRR2) at invadopodia of invasive breast cancer cells, where it mediates EGF-induced phosphorylation both directly and indirectly via Src-mediated Arg activation, leading to free actin barbed end generation and subsequent actin polymerization in invadopodia, ECM degradation, and consequent tumor cell invasion (Genna et al., Pyk2 and FAK differentially regulate invadopodia formation and function in breast cancer cells. J Cell Biol 2018; 217: 375-395).
In the present invention, the inventors have designed a cell-permeable peptide including the second proline-rich region of Pyk2 (Pyk2-PRR2), with the idea that this peptide may be used to inhibit metastasis by competing with the native Pyk2 on binding to cortactin. This idea was based, inter alia, on an in silico analysis showing that the combined effect of PTK2B (the Pyk2 encoding gene) and CTTN (the cortactin encoding gene) overexpression considerably reduced distant metastasis free survival (DMFS) estimates, suggesting that Pyk2 and cortactin act synergistically to promote breast cancer metastasis (Example 1).
The inventors further demonstrated that the Pyk2-PRR2 peptide inhibits spontaneous lung metastasis in a xenograft mouse model by suppressing invadopodia-mediated invasiveness of breast tumor cells (Example 2). Additionally, the Pyk2-PRR2 peptide was shown to suppress motility of breast cancer cells in extracellular matrix, and specifically matrix metalloproteinase (MMP)-dependent 3D invasiveness of breast tumor cells, in an in vitro tissue culture model (Example 3). Additional examples show that the Pyk2-PRR2 peptide inhibits the interaction between Pyk2 and cortactin (Example 4), as well as inhibiting cortactin tyrosine phosphorylation (Example 5), thereby regulating the maturation and functional activation of invadopodia, and inhibiting invadopodium maturation and function in breast tumor cells.
The interaction of the Pyk2-PRR2 peptide with the SH3 domain of cortactin was mapped by analyzing the three-dimensional structure of this complex using nuclear magnetic resonance (NMR) (Example 6), identifying specific amino acids important for the interaction. Finally, a mutant peptide was prepared by modifying specific positions in the Pyk2-PRR2 peptide based on the 3D analysis, showing that the inhibiting effect is lost (Example 7).
In some embodiments, the present invention provides an isolated Pyk2-derived peptide including a consensus proline-rich region 2 (PRR2) sequence, wherein the consensus PRR2 sequence is PxxPx(R/K)P(K/R)(Y/W/F) in which “x” stands for any amino acid; and the peptide is capable of inhibiting metastasis by binding to a Src homology 3 (SH3) domain of cortactin.
The term “isolated peptide” means that the peptide of the invention does not encompass a natural peptide, if such a peptide even exists, functioning in its natural environment. It is also clarified that the term “isolated” does not mean that the peptide is necessarily obtained by isolating it from a cell (but it could be). Additionally, the term “isolated” does not preclude the peptide from being attached or conjugated to additional elements (including amino acid sequences), as further mentioned below.
Proline-rich tyrosine kinase 2 (Pyk2), encoded by the PTK2B gene, is a cytoplasmic protein tyrosine kinase involved in calcium-induced regulation of ion channels and activation of MAP kinase signaling pathway. It contains three proline-rich domains, two of them, proline-rich region 2 (713PPPKPSRP720, PRR2, positions in UniProt accession No. Q14289), and proline-rich region 3 (855PPQKPPRL862, PRR3) are involved in Src homology domain 3 (SH3)-mediated protein-protein interactions.
The consensus PRR2 sequence of PxxPx(R/K)P(K/R)(Y/W/F), corresponding to the amino acids at positions 714-722 of the human Pyk2 sequence (UniProt Accession No. Q14289), is based on the below examples, which define the amino acids important for interaction with the cortactin SH3 domain both by an analysis of the complex structure and by testing mutant peptides. The cortactin SH3 domain is between amino acids 495-542 of the cortactin protein.
The analysis presented in Example 6 shows the importance of positions P714, P717, P720, K721, and Y722 to binding the cortactin SH3 domain. These amino acids were modified in the super mutant (SEQ ID NO: 3: TAFQEPAPKASQAQYRPPP) and in a mutant carrying a Y722A substitution (SEQ ID NO: 5), and were shown to reduce or abolish the inhibiting effect of the peptide. Additionally, a deletion of amino acids 720-726 (SEQ ID NO: 6) demonstrated a 27-fold loss of affinity (
The amino acids are indicated herein by the amino acid one letter code: Table 1: amino acids one letter code 11 PGP-21.T1.M
In some embodiments, the consensus PRR2 sequence is selected from the group consisting of PxxPxRPKY (SEQ ID NO: 12), PxxPxRPRY (SEQ ID NO: 13), PxxPxKPRY (SEQ ID NO: 14), PxxPxKPKY (SEQ ID NO: 15), PxxPxRPKW (SEQ ID NO: 16), PxxPxRPRW (SEQ ID NO: 17), PxxPxKPRW (SEQ ID NO: 18), PxxPxKPKW (SEQ ID NO: 19), PxxPxRPKF (SEQ ID NO: 20), PxxPxRPRF (SEQ ID NO: 21), PxxPxKPRF (SEQ ID NO: 22), and PxxPxKPKF (SEQ ID NO: 23). In some embodiments, the consensus PRR2 sequence is PxxPxRPKY (SEQ ID NO: 12).
The positions marked with an “x” may be any amino acid. In some embodiments, at least one of the positions marked with “x” is proline. In some embodiments, at least one of the positions marked with “x” is a basic amino acid.
The term “basic amino acid”, as defined herein, relates to the amino acids arginine or lysine.
In some embodiments, the consensus PRR2 sequence is PPKPSRPKY (SEQ ID NO: 24).
In some embodiments, the peptide further includes additional sequence at the amino-terminal side of the consensus PRR2 sequence. In some embodiments, the peptide further includes additional sequence at the carboxy-terminal side of the consensus PRR2 sequence. In some embodiments, the additional sequence comprises sequence derived from the Pyk2 protein.
The term “derived from”, as used herein, means that a first sequence derived from a second sequence has at least a certain level of sequence identity to the second sequence it is said to be derived from. In some embodiments, the certain level of sequence identity is at least about 70%, 75%, 80%, 85%, 90%, 95%, or 99%.
In some embodiments, the peptide includes at least one additional proline outside of the consensus sequence. In some embodiments, the at least one additional proline is N-terminal to the consensus sequence. In some embodiments, the at least one additional proline is C-terminal to the consensus sequence.
In some embodiments, the peptide includes at least one additional basic amino acid outside of the consensus sequence. In some embodiments, the at least one basic amino acid is N-terminal to the consensus sequence. In some embodiments, the at least one basic amino acid is C-terminal to the consensus sequence.
In some embodiments, the peptide includes at least one amino acid selected from the group consisting of glycine, methionine, threonine, alanine, or cysteine outside of the consensus sequence, for stabilizing the protein. In some embodiments, the peptide includes at least one amino acid selected from the group consisting of glycine, methionine, threonine, alanine, or cysteine N-terminal to the consensus sequence.
In some embodiments, the peptide includes at least one tag sequence. In some embodiments, the at least one tag sequence is N-terminal to the consensus sequence. In some embodiments, the at least one tag sequence is C-terminal to the consensus sequence.
The term “tag sequence”, as used herein, relates to a short sequence (typically several amino acids, such as less than 10 amino acids) which is added to a peptide or a protein sequence for a certain purpose such as for easy detection, or for targeting.
In some embodiments, the tag sequence is selected from the group consisting of an HIV-TAT tag, which is typically used for increasing permeability into cells; and a HAIYPRH (Tf tag) used for targeting the peptide to cancer cells.
It is appreciated that the peptide may further include any additional elements or modifications, including protein/peptide fusion, which may be added for any reason, including to increase stability, or help delivery or targeting.
In some embodiments, the peptide is capped at the N-terminal end, the C-terminal end, or both, for protection and stability. The cap may be any suitable cap, including, e.g., an Ac cap at the N-terminal end, or an amino cap at the C-terminal end.
In some embodiments, the peptide includes a sequence at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical to a sequence of Pyk2. In some embodiments, the peptide sequence is at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical to a sequence of Pyk2.
In some embodiments, the peptide includes a sequence at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical to a sequence including the second proline-rich region of Pyk2. In some embodiments, the peptide sequence is at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical to a sequence including the second proline-rich region of Pyk2.
In some embodiments, the Pyk2 is a human Pyk2.
In some embodiments, the peptide does not include a complete Pyk2 protein sequence. In some embodiments, the peptide does not include a proline-rich region 3 (PRR3) sequence of a Pyk2 protein. In some embodiments, the peptide does not include any functional Pyk2 region sequence except for the PRR2 sequence.
In some embodiments, the peptide includes a sequence at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical to the sequence set forth in SEQ ID NO: 1, corresponding to the amino acids at positions 708-726 of the human Pyk2 protein sequence (UniProt accession Q14289). In some embodiments, the peptide sequence is at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical to the sequence set forth in SEQ ID NO: 1. In some embodiments, the peptide sequence includes SEQ ID NO: 1.
In some embodiments, the peptide has a length of about 9-100, 9-50, 10-30, or 15-25 amino acids. In some embodiments, the peptide has a length of about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids.
Peptide therapeutic agents also pose challenges, such as proteolysis, rapid renal clearance, low oral bioavailability, and membrane permeability, these can be effectively overcome using peptidomimetic compounds involving D- or non-proteogenic amino acids, various cyclization approaches, and tailored delivery methods. Other options for stabilization including adding prolines or non-native amino acids.
Since there is a possibility that such modifications may affect the interaction of the peptide with the cortactin SH3 domain, such modifications may be limited to amino acids which are outside of the consensus region. Non-limiting examples for non-conventional or modified amino acids include D-amino acids, and a-substituted amino acids.
In some embodiments, the peptide is a cyclic peptide. In some embodiments, the peptide includes at least one non-conventional and/or at least one modified amino acid. In some embodiments, the peptide includes at least one non-conventional and/or at least one modified amino acid outside of the consensus sequence. In some embodiments, the non-conventional amino acid is a D-amino acid.
In some embodiments, the peptide has a sequence as set forth in SEQ ID NO: 9: TAFQEPPPKPSRPKYRPPP, bold denoting a D-amino acids.
As shown in Example 2, the Pyk2-PRR peptides did not suppress tumor growth, which indicates that the peptides of the invention are most likely specific to metastasis.
Accordingly, in some embodiments, the Pyk2-derived peptide does not inhibit primary tumor growth.
In some embodiments, the present invention provides a nucleic acid including a sequence encoding the Pyk2-derived peptide disclosed herein.
Definitions and embodiments mentioned above and which may be relevant to the nucleic acid embodiments also apply here, and vice versa. Some particularly relevant embodiments may be pointed out or explicitly repeated.
Instead of using the peptide directly, it is conceivable that it may be produced within a cell, by transcription from a nucleic acid template encoding the peptide. To this end, a nucleic acid including a sequence encoding the peptide may be inserted into a suitable expression vector (such as an adenoviral vector, adenovirus-associated vector, or a lentiviral vector) which includes sequences required for expression in mammalian cells (such as promoter, terminator, regulatory elements, etc.), for expression in a desired cell. The vector may be targeted to cancer cells for expressing the peptide.
In some embodiments, the present invention provides a pharmaceutical composition including the isolated Pyk2-derived peptide disclosed herein and a pharmaceutically-acceptable carrier.
Definitions and embodiments mentioned above and which may be relevant to the pharmaceutical composition embodiments also apply here, and vice versa. Some particularly relevant embodiments may be pointed out or explicitly repeated.
The pharmaceutical composition may include any ingredients suitable for delivery of the peptide of the invention, including any suitable form of encapsulation, such as nanoparticles or microparticles, liposomes, fusion proteins, or other lipid or protein or other molecules carriers. The pharmaceutical compositions may further include targeting reagents, for targeting to a desired tissue or cell.
In some embodiments, the pharmaceutically acceptable carrier is a buffer, diluent, adjuvant, excipient, or vehicle suitable for administration with the peptide of the invention. In some embodiments, the pharmaceutically acceptable carrier may be suitable for intravenous infusion. In some embodiments, the pharmaceutically acceptable carrier may be suitable as a cryoprotectant.
In some exemplary embodiments, the carrier may be DMSO (for example, at about 10%). In some embodiments, the pharmaceutically acceptable carrier may include a binder, such as microcrystalline cellulose, polyvinylpyrrolidone (polyvidone or povidone), gum tragacanth, gelatin, starch, lactose or lactose monohydrate; a disintegrating agent, such as alginic acid, maize starch and the like; a lubricant or surfactant, such as magnesium stearate, or sodium lauryl sulphate; and a glidant, such as colloidal silicon dioxide.
In some embodiments, the present invention provides a method of treating a cancer in a subject, the method including administering to the subject a therapeutically-effective dose of the isolated Pyk2-derived peptide disclosed herein.
Definitions and embodiments mentioned above and which may be relevant to the method of treatment embodiments also apply here, and vice versa. Some particularly relevant embodiments may be pointed out or explicitly repeated.
In some embodiments, the present invention provides a method of preventing or inhibiting cancer metastasis in a subject, the method including administering to the subject a therapeutically-effective dose of the isolated Pyk2-derived peptide disclosed herein.
In some embodiments, the present invention provides a method of preventing or inhibiting cancer invasion in a subject, the method including administering to the subject a therapeutically-effective dose of the isolated Pyk2-derived peptide disclosed herein.
The term “treating”, as used herein, refers to means of obtaining a desired physiological effect, in this case, ameliorating the symptoms of the cancer and enhancing recovery by partially or completely inhibiting or blocking metastasis of the cancer. In some embodiments, the treating includes preventing and/or inhibiting cancer invasion and/or metastasis.
The term “cancer invasion”, as used herein, relates to the process in which cancer cells extend beyond the organ or tissue in which the cancer originated and penetrate neighboring tissues or organs. This process generally takes place by migration of cancer cells through the extracellular matrix into surrounding tissues.
The term “cancer metastasis”, as used herein, relates to the process in which the cancer cells spread through the circulatory system or the lymphatic system to more distant locations than neighboring tissues. In solid cancers which are not in the circulatory or the lymphatic systems, metastasis follows invasion.
The term “inhibiting”, as used herein with respect to cancer invasion or metastasis, relates to delaying their appearance or reducing their amount relative to the expected rate or development of invasion or metastasis under similar conditions.
The term “preventing”, as used herein, relates to preventing appearance of cancer invasion or metastasis from the treated cancer completely.
The term “therapeutically effective dose” as used herein means an amount of the peptide that will elicit the desired biological or medical effect. The amount must be effective to achieve the desired therapeutic effect as described above, depending inter alia on the type and severity of the cancer and the treatment regime. The therapeutically effective dose is typically determined in appropriately designed clinical trials (dose range studies) and the person skilled in the art will know how to properly conduct such trials to determine the effective amount.
The administering may be by any method or route suitable for treating the cancer. The administration regimen may be determined by a physician based on the condition of the subject.
The administration may be a systemic or a local administration. Non-limiting examples for administration routes include intravenous, intraarterial, intraperitoneal, intramuscular, subcutaneous, oral, sublingual, enteral, intranasal, buccal, vaginal, rectal, intraocular, intrathecal, topical, transdermal, and intradermal administration.
In some embodiments, the route of administration is selected from the group consisting of intravenous, subcutaneous, intramuscular, intratumoral, intraperitoneal, intranasal, intratumoral, and topical administration.
The added benefit of the peptide of the invention may also be important in view of that cancer treatments have sometimes been shown to have an adverse effect of inducing metastasis.
This further emphasizes the importance of a treatment for preventing or inhibiting invasion and metastasis in addition to any cancer treatment that is directed against the primary tumor. Treating the subject early with a combination of the cancer treatment and the peptide of the invention may have a complementary effect of both treating the primary tumor and preventing metastasis.
Accordingly, the peptide of the invention may be administered to the subject in combination with an anti-cancer treatment, for completely eradicating the cancer. The term “in combination”, as used herein, means as part of the same treatment and not necessarily at the same time. The peptide of the invention may be administered before, after, or at the same time as the anti-cancer treatment, or as part of a regimen which includes both the peptide and the anti-cancer treatment.
Additionally, in some situations the peptide of the invention may be used as part of a palliative treatment, for prolonging life or for enhancing quality of life, without necessarily also administering anti-cancer treatment.
Accordingly, in some embodiments, the method further includes administering to the subject an anti-cancer treatment.
In some embodiments, the present invention provides a method of treating a cancer in a subject, including administering to the subject a combination of the isolated Pyk2-derived peptide disclosed herein and an anti-cancer treatment.
Non-limiting examples for anti-cancer treatments include chemotherapy, immunotherapy, radiotherapy, etc.
As noted above, there may be an advantage to treating with the peptide at an early stage, even before the appearance of invasion or metastasis. However, there is also benefit in treating even after invasion and metastasis have occurred, to limit the level of metastasis and help contain the disease, thereby increasing the efficacy of anti-cancer treatments.
In some embodiments, the cancer is invasive. In some embodiments, the cancer is not invasive. In some embodiments, the cancer is metastatic. In some embodiments, the cancer is not metastatic.
In some embodiments, the cancer does not include metastases.
In some embodiments, the cancer includes metastases.
In some embodiments, the cancer includes invadopodia.
In some embodiments, the cancer is selected from the group consisting of breast cancer, pancreatic cancer, head and neck cancer, prostate cancer, fibrosarcoma, and melanoma.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains.
The term “a” and “an” refers to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.
The term “about” when referring to a measurable value such as an amount, a ratio, and the like, is meant to encompass variations of ±10% of the indicated value, as such variations are also suitable to perform the disclosed invention. Any numerical values appearing in the application are intended to be construed as if preceded by “about”, unless indicated otherwise.
While certain embodiments of the invention have been illustrated and described, it will be clear that the invention is not limited to the embodiments described herein. Numerous modifications, changes, variations, substitutions, and equivalents will be apparent to those skilled in the art without departing from the spirit and scope of the present invention as described by the claims, which follow.
The following examples are presented in order to more fully illustrate some embodiments of the invention. They should in no way be construed, however, as limiting the broad scope of the invention. One skilled in the art can readily devise many variations and modifications of the principles disclosed herein without departing from the scope of the invention.
QAQ
YRPPP
RPK
A
RPPP
HAIYPRHTAFQEP
The positions indicated in Table 2 correspond to UniProt accession No. Q14289. In SEQ ID Nos: 1-6 bold indicates consensus positions, and substitutions are underlined.
Peptides were synthesized by GL Biochem (Shanghai, China). In all cellular biology and in vivo experiments, peptides contained the HIV-TAT sequence at their C-termini (SEQ ID NO: 11) to increase their permeability into cells. For invadopodia, matrix degradation, 2D motility assays, and 3D invasion assays, peptides containing the TAT sequence were conjugated to FITC at their C-termini to track their delivery into cells.
The assays of the present disclosure were conducted using Pyk2-PRR2 (SEQ ID NO: 1), which corresponds to positions 708-726 of UniProt accession No. Q14289 for human Pyk2. Additional assays that were conducted by adding a Tf-tag (HAIYPRH, SEQ ID NO: 10), which targets sequences to transferrin receptors overexpressed in cancer cells, either N-terminal (HAIYPRHTAFQEPPPKPSRPKYRPPP, SEQ ID NO: 7) or C-terminal (TAFQEPPPKPSRPKYRPPPHRPYIAH, SEQ ID NO:8) to the Pyk2-PRR2 sequence.
It is also possible to use sequences wherein D-amino acids are used, such as TAFQEPPPKPSRPKYRPPP (SEQ ID NO: 9, bold denotes a d-amino), as described above.
Some of the sequences used included a cap for protection at the N-terminus (Ac cap, used for the peptide containing a C-terminal Tf-tag and for the sequence containing D-amino acids), the C-terminus (NH2 cap used for the peptide containing an N-terminal Tf-tag and for the sequence containing D-amino acids) or both.
MDA-MB-231 human breast adenocarcinoma cells and 4T1 mouse mammary tumor cells were obtained from the American Type Culture Collection. MDA-MB-231 were cultured in DMEM/10% FBS, and 4T1 cells were cultured in RPMI 1640/10% FBS. MDA-MB-231 cells stably expressing cortactin-TagRFP were previously described (Oser et al. Specific tyrosine phosphorylation sites on cortactin regulate Nckl-dependent actin polymerization in invadopodia. J Cell Sci 2010; 123: 3662-3673).
All experimental procedures were conducted following the Federation of Laboratory Animal Science Association (FELSA) and were approved by the Bar-Ilan University animal use and care committee. Mouse xenograft tumors were generated by injecting 106 4T1 cells resuspended in 20% collagen I (BD Biosciences, Franklin Lakes, NJ, USA) in PBS into the lower-left mammary gland of 8-week-old BALB/c female mice. Twelve days following injection, when tumors reached the size of 100 mm3, mice were treated by intravenous injection with 10 mg/kg of control scrambled, Pyk2-PRR2, Pyk2-PRR3, or Pyk2 super-mutant peptides once a day for eight days.
4T1 cells have intrinsic resistance to 6-thioguanine, which enables precise quantification of metastatic cells, even when they are disseminated at sub-microscopic levels at distant organs, upon excision and digestion of selected organs. The clonogenic assay is based on a principle by which one colony represents one metastatic cell. Briefly, spontaneous lung metastasis was measured in BALB/c mice bearing orthotopic 4T1 tumors of equal size (1-1.2 cm in diameter). Lungs were excised and minced and plated in RPMI/10% FBS in the presence of 6-thioguanine to select for tumor cells only. Cells were plated in a 5% CO2 incubator at 37° C. for 14 days to form 4T1 colonies. On day 14 after plating, the medium was removed from dishes, and colonies were fixed in 4% paraformaldehyde for one hour at room temperature. Colonies were then stained in 2% crystal violet solution and thoroughly washed with PBS. Plates were allowed to air dry, and colonies in each plate were quantified.
Data from two cohorts of 1,080 and 825 breast invasive carcinoma samples was obtained from The Cancer Genome Atlas (TCGA, https://cancergenome.nih.gov/) and analyzed with cBioPortal tools (http://www.cbioportal.org) using MATLAB. The analyzed datasets contain mRNA-seq expression Z-scores (RNA-Seq V2 RSEM) and Agilent microarray mRNA Z-scores computed as the relative expression of an individual gene and tumor to the expression distribution of all samples that are diploid for the gene. Putative copy number alteration (GISTIC) was collected from both cohorts. GISTIC is an algorithm that attempts to identify significantly altered regions of amplification or deletion and uses discrete copy number calls: homozygous deletion; heterozygous loss; neutral; gain; high amplification. Breast cancer samples are annotated with OS and/or DFS time and censorship status. Samples were split into high and low expressing groups based upon median mRNA expression Z-scores. Associations between Z-scores and patient survival (DFS and OS) were assessed by Kaplan-Meier time-event curves and Mantel-Haenszel hazard ratios using an implementation of Kaplan-Meier log rank testing from MATLAB Exchange. All statistical tests were two-sided. Mass-spectrometry based proteomic characterization of 102 breast cancer tumor samples [4] was obtained from the Clinical Proteomic Tumor Analysis Consortium (CPTAC) Data Portal (https://cptac-data-portal.georgetown.edu) with cBioPortal using MATLAB. For each protein target, Z-scores were determined and associated P-values were calculated across all samples.
Microarray datasets with Distant Metastasis-Free Survival (DMFS) annotation were obtained from the NCBI Gene Expression Omnibus (GEO) data repository of high-throughput microarray experimental data. The meta-cohort dataset comprised of 1,650 tumor expression profiles of primary invasive breast cancer based on the Affymetrix U133 GeneChip microarray platform. Queried transcripts included 22,283 probe sets common to all microarrays in all study populations. Assembly of the datasets was performed using MATLAB (The Mathworks, Inc.) and GEO series (GSE) files were extracted via GEO accessions GSE11121, GSE25055, GSE7390, GSE25065, GSE17705, GSE12093, GSE1456, GSE5327 and GSE45255. The datasets have been retrieved with uniform normalization of probe intensities with MAS 5.0 using global scaling with a trimmed mean target intensity of each array arbitrary set to 600. Cross-population batch effects were corrected using Z-score transformation. The tumor profiles represent primary invasive breast tumors sampled at the time of surgical resection, annotated with DMFS time and censorship status Patient samples were split into high and low expressing groups based upon median gene expression. Associations between normalized gene expression and patient survival (DMFS) were assessed by Kaplan-Meier time-event curves and Mantel-Haenszel hazard ratios using an implementation of Kaplan-Meier log rank testing from MATLAB Exchange. All statistical tests were two-sided. The synergy index (SI) was calculated as a means of evaluating additive interaction. The synergy index can be interpreted as the excess risk from overexpression of both genes relative to the risk of overexpression of the genes separately. SI of 1 indicates no synergism, and an SI>1 indicates synergistic interaction between the two genes.
For immunofluorescence, anti-cortactin (ab-33333) and anti-PCNA (ab18197) were obtained from Abcam (Cambridge, UK); anti-Arp2 (N1C3) was obtained from GeneTex (Irvine, CA, USA); anti-Tks5 (SH3 #4; 09-268) was obtained from EMD Millipore (Burlington, MA, USA); anti-pY421-cortactin (C0739) was obtained from Sigma-Aldrich (St. Louis, MO, USA); anti-pY402-Pyk2 (44-618G) was obtained from Thermo Fisher Scientific (Waltham, MA, USA); anti-CD31 (550274) was obtained from BD Biosciences (Franklin Lakes, NJ, USA); anti-cleaved caspase 3 (Asp175; 9961) was obtained from Cell Signaling Technology (Danvers, MA); Rhodamine-labelled phalloidin and Alexa Fluor-conjugated secondary antibodies were obtained from Molecular Probes (Thermo Fisher Scientific). For immunoblotting, anti-cortactin (clone 4F11; 05-180) was obtained from EMD Millipore; anti-β-actin (clone AC-15; A5441) was obtained from Sigma-Aldrich. Secondary antibodies (goat anti-mouse 680LT and goat anti-rabbit 800CW) were obtained from LI-COR Biosciences (Lincoln, NE, USA).
4T1 cells were injected into the mammary fat pad of 8-week-old BALB/c female mice and allowed to grow for 12 days. On day 13 following injection, mice were treated by intraperitoneal injection with scrambled control, Pyk2-PRR2, or Pyk2-PRR3 peptides once a day for eight days. Mice bearing 4T1 mammary tumors were then sacrificed, and tumors or lungs were excised, fixed in 4% paraformaldehyde overnight, washed for 1 hour in cold PBS, and dehydrated overnight in 30% sucrose. Next, tissue was embedded in OCT, and 5 m thick cryostat sections were placed on silane-coated slides and dried at room temperature followed by permeabilization with 0.1% Triton X-100 for 15 min and blocking in 1% BSA and 1% FBS for 1 hour at room temperature. Samples were then incubated overnight at 4° C. with the indicated primary antibodies, washed, and incubated with the appropriate secondary antibodies. Nuclei were counterstained with 4′,6-diamino-2-phenylindole (DAPI). Tissue was imaged using an inverted laser scanning confocal microscope (Zeiss LSM780; 63×, NA 1.4, oil objective, ZEN black edition acquisition software) (Oberkochen, Germany). For all assays results were based on n=7-10 mice/tumors per group.
MDA-MB-231 were plated and allowed to adhere for 4 h. Adherent cells were incubated with 0.1 or 1 μM Pyk2 peptides containing HIV-TAT sequences and conjugated to FITC for 20 min, 8 h, and 24 h. At the end of the incubation time, the cells were fixed and analyzed by fluorescence microscopy.
MDA-MB-231 cells were resuspended to reach a concentration of 5,000 cells/well and were seeded in triplicates in flat bottom 96 well microtiter plates. Following overnight incubation at 37° C. in a tissue culture incubator, Pyk2-PRR2, Pyk2-PRR3, or a scrambled control peptide was added to a final concentration of 0.1, 1, 5, or 10 μM, and cells were followed every 24 h for 96 h in total. Activated XTT solution was prepared according to the manufacturer's instructions (Biological Industries, Beit Haemek, Israel) and added to the cells for 4 h. Two filters were used, the specific absorbance filter at 475 nm and the non-specific absorbance filter at 660 nm. Specific absorbance was calculated using the formula: A 475 nm (test)—A475 nm (blank)—A660 nm (test).
The 2D single-cell random migration assay was performed as previously described in Genna et al. Pyk2 and FAK differentially regulate invadopodia formation and function in breast cancer cells. J Cell Biol 2018; 217: 375-395. In brief, 6-well microtiter plates were coated with 10 μg/ml fibronectin and blocked with 1% denatured BSA. Peptide pre-treated cells were plated at 20,000 cells/well in DMEM/10% FBS containing peptides and allowed to adhere for 12-16 h. Plates were placed in a 37° C. heated chamber, and images were collected using the IncuCyte® Zoom platform (Essen Bioscience, Ann Arbor, MI, USA; 20×, NA 0.60, air objective). Phase images were collected every one hour for a total of 12 h using the IncuCyte® acquisition software. Trajectory plots, accumulated distance (total cell path length), Euclidean distance (the shortest distance between the starting point and endpoint of migration), and velocity were calculated using the chemotaxis and migration tool (ibidi GMBH, Grafelfing, Germany).
The 3D scratch wound assay was performed as described in Meirson et al. Targeting invadopodia-mediated breast cancer metastasis by using ABL kinase inhibitors. Oncotarget 2018; 9: 22158-22183. Briefly, 96 well Image Lock microtiter plates (Essen Bioscience) were coated with 100 μg/ml Matrigel (cat #354234; Corning, New-York, NY, USA). Following overnight incubation at 37° C., MDA-MB-231 cells were plated at a final concentration of 350,000 cells/ml and allowed to adhere for 12-16 h in the presence of Pyk2-PRR or control peptides. Following wounding of the bottom Matrigel-cells layer, a top layer of Matrigel was added to a final concentration of 2 mg/ml and allowed to polymerize for 30 min at 37° C. Pyk2-PRR or control scrambled peptides were then added again in DMEM/10% FBS to a final concentration of 0.1, 1, or 5 μM, with or without the MMP inhibitor GM6001 (25 M). Plates were placed in a 37° C. heated chamber, and images were collected using the IncuCyte® Zoom platform (Essen Bioscience; 20×, NA 0.60, air objective). Phase images were collected every one hour for a total of 12 h using the IncuCyte® acquisition software.
Acceptor photobleaching experiments were performed as described in Genna et al., 2018 (supra). In brief, cortactin-TagRFP MDA-MB-231 cells were plated on Alexa 405 gelatin in a medium containing Pyk2-PRR or scrambled control peptides at 0.1, 1, and 5 μM concentration, fixed, and immunostained with pY402-Pyk2/Alexa Fluor 488-goat anti-rabbit (donor) and cortactin/Alexa Fluor 555-goat anti-mouse (acceptor). As a control, cells were stained with Tks5/Alexa Fluor 488-goat anti-rabbit (donor) and cortactin/Alexa Fluor 555-goat anti-mouse (acceptor). Co-localization of cortactin with degradation holes in Alexa 405-labeled gelatin was used to identify mature degrading invadopodia. For all FRET experiments, cells were imaged in PBS at room temperature on a laser-scanning inverted confocal microscope (Zeiss LSM780; 60×, NA 1.4, oil objective). A region of interest surrounding invadopodia was bleached using 100% 561-nm laser power (acceptor channel), and images were acquired in both the 488 nm and the 561 nm channels before and after bleaching. FRET efficiency was calculated as the E=1−(donor pre/donor post) in background-subtracted images and was corrected for fluctuations in laser power and donor bleaching in ImageJ.
The invadopodium precursor formation assay was performed as described in Genna et al., 2018 (supra). Briefly, gelatin was conjugated to Alexa 405 dye (Thermo Fisher Scientific). MatTek dishes were treated with 1 N HCl and coated with 50 μg/ml poly-L-lysine. A 0.2% gelatin solution was prepared in PBS, and a 1:20 mixture of Alexa 405-labeled gelatin/unlabeled gelatin was warmed to 37° C. before addition to the poly-L-lysine coated plates. Gelatin was cross-linked with 0.01% glutaraldehyde followed by quenching with 5 mg/ml sodium borohydride. 125,000 MDA-MB-231 cells were plated on Alexa 405-labeled gelatin plates in the presence of 0.1, 1, or 5 μM of Pyk2-PRR peptides or scrambled control for 4 h. At the end of incubation, cells were fixed in 3.7% paraformaldehyde, permeabilized with 0.1% Triton X-100, blocked with 1% FBS, 1% BSA in PBS, and labeled with anti-Tks5 and anti-cortactin. Images were acquired using an inverted fluorescent microscope (Leica AF6000; 63×, NA 1.4, oil objective, Leica LAS AF acquisition software) (Wetzlar, Germany) equipped with an ORCA-Flash 4.0 V2 digital CMOS camera (Hamamatsu Photonics, Shizuoka, Japan). Invadopodium precursors were identified as Tks5- and cortactin-rich punctate structures found in the ventral plane of the cell that do not co-localize with degradation areas, whereas mature invadopodia were identified as Tks5 and cortactin-rich puncta that co-localize with degradation areas at the ventral plane of the cell.
The in vitro matrix degradation assay was performed as described in Genna et al., 2018 (supra). Briefly, MatTek dishes were treated with 2.5% gelatin/2.5% sucrose, cross-linked with 0.5% glutaraldehyde, treated with 10 μg/ml of fluorescently labeled fibronectin (Alexa 568; Invitrogen, Thermo Fischer Scientific), and then with 1 mg/ml NaBH4 in PBS. 125,000 cells were plated on the fibronectin/gelatin matrix in Pyk2-PRR or control peptides and allowed to degrade for 24 h. Cells were then fixed in 3.7% paraformaldehyde, and random fields were imaged using an inverted fluorescent microscope (Leica AF6000; 40×, NA 1.3, oil objective). ECM degradation was analyzed by quantifying the average degraded area in pixels per field using ImageJ.
Twenty-four hours before each experiment, cells were plated on fibronectin/gelatin unlabeled matrix in the presence of a Pyk2-PRR or control peptides at 0.1, 1, and 5 μM concentration and allowed to attach for eight hours. Cells were then starved in DMEM/0.5% FBS for 16 h in the presence of peptides and stimulated with 2.5 nM EGF for 3 min or left un-stimulated (0 min EGF). Cells were fixed and stained with mouse phospho-specific cortactin antibody (pY421). To image invadopodia Z-plane, focus was on the ventral surface of the cell where the leading edge containing cortactin is focused. The pixels corresponding to invadopodia dots were then analyzed, not the whole cell. The intensity (mean grey value (mgv) minus background) of cortactin-TagRFP and pY421-cortactin at invadopodium precursors was quantified. The pY421-cortactin/cortactin-TagRFP ratio was calculated as a measure of tyrosine-phosphorylated cortactin at cortactin-rich puncta. Data were normalized to resting cells (0 min EGF) and presented as the relative fold change in cortactin tyrosine phosphorylation.
The barbed end assay was performed using biotin-conjugated actin as described in Genna et al., 2018 (supra). In brief, 24 h before each experiment, cells were plated on fibronectin/gelatin unlabeled matrix in the presence of Pyk2-PRR or control peptides at 0.1, 1, or 5 μM concentration and allowed to attach for 8 h. Cells were starved for 16 h in the presence of the peptides and then stimulated with 2.5 nM EGF and permeabilized with a permeabilization buffer (20 mM Hepes, pH 7.5, 138 mM KCl, 4 mM MgCl2, 3 mM EGTA, 0.2 mg/ml saponin, 1 mM ATP, and 1% BSA) containing 0.4 μM biotin-conjugated muscle actin (AB07) (Cytoskeleton Inc., Denver, CO, USA) for 1 min at 37° C. Cells were fixed in 3.7% paraformaldehyde for 5 min, blocked in PBS containing 1% FBS, 1% BSA, and 3 μM un-labeled phalloidin (Molecular Probes, P3457), then labeled with FITC anti-biotin (200-092-211) (Jackson ImmunoResearch Laboratories, West Grove, PA, USA) to visualize barbed ends, and with rhodamine-phalloidin (R415) (Molecular Probes, Eugene, OR, USA) and Arp2 to identify regions of cells rich in invadopodia. The barbed end intensity at invadopodia-rich regions was quantified by measuring mgv at invadopodia-rich areas minus mgv of the background. Data were normalized to the control condition for each experiment.
The DNA sequence encoding the SH3 domain of the mouse cortactin SH3 domain (GenBank ID U03184; nucleotides1468-1641) was sub-cloned into a pET-28a plasmid (Novagen, Merck) in frame with the cleavable N-terminal 6×His tag. Isotopically labeled cortactin-SH3 for heteronuclear NMR experiments was expressed in transformed E. coli BL21 cells (DE3) grown in the presence of kanamycin (50 mg/1) and chloramphenicol (34 mg/1) at 37° C. in M9 minimal medium [13]. For 15N (13C) labeling the medium contained 1 g/L of 15NH4Cl (4 g/L 13C6-glucose), and 0.25 g/l of appropriately labeled Celtone Base Powder (Cambridge Isotope Laboratories, Andover, MA) was added. For triple-labeled 2H,13C,15N—SH3, expression began in 100 ml of the above medium grown to an OD600 of 0.4-0.5 and gently centrifuged. Pellets were then resuspended in 500 ml filter-sterilized D20-based 15N-M9 supplemented with 2H7-13C6-glucose (2 g/1) and DCN-Isogro (0.25 g/1) to reach a final OD600 of −0.1. In both cases cells were then grown at 37° C. and induced with 0.5 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) upon reaching OD600-0.8, followed by overnight growth at 27° C. To purify the SH3 domain, cells were harvested by centrifugation, subjected to a single freeze-thaw cycle, and resuspended in lysis buffer (50 mM NaPi pH 8.0, 300 mM NaCl, 1 mM dithiothreitol (DTT), 100 ml per liter of culture) supplemented with 1 tablet EDTA-free protease inhibitor cocktail (Merck) and benzamidine (5 mM). Cells were lysed by French press, followed by addition of benzamidine (final concentration 10 mM) and phenylmethylsulfonyl fluoride (PMSF, 1 mM), and the lysate was clarified by centrifugation. The supernatant was loaded on a HisTrap HP column (GE Healthcare, Chicago, IL, USA) equilibrated with lysis buffer and the protein eluted through a gradient (Buffer B: 50 mM NaPi pH 8.0, 100 mM NaCl, 300 mM imidazole, 1 mM DTT). To the fraction eluting at ˜40% B purified tobacco etch virus (TEV) protease (1:30 mol:mol) was added, and the mixture dialyzed overnight at 4° C. against dialysis buffer (50 mM NaPi at pH 8.0, 100 mM NaCl, 1 mM DTT). The dialysate was brought to a final concentration of ˜5 mM imidazole, filtered, and loaded onto a HisTrap column. The collected flowthrough was dialyzed against 20 mM Tris (pH 7.5), 1 mM DTT for 3 h to reach a final concentration of 10 mM NaCl. This fraction was then loaded onto a Q HP Sepharose ion-exchange column (GE Healthcare) and eluted through a gradient at −45% buffer B (20 mM Tris pH 7.5, 1 M NaCl, 1 mM DTT), flash-frozen and stored at −80° C.
Purified SH3 was dialyzed against NMR sample buffer (30 mM NaPi, pH ˜6.5) and concentrated to the desired concentration in an Amicon concentration tube (3 kDa MWCO) (Merck). The PRR2 peptide was dissolved in D20 to a final concentration 14-fold higher than the SH3 domain and combined with the SH3 sample, affording SH3/PRR2 complex in 7% 2H2O solution to which NaN3 (0.02% v/v) was added. Final sample concentrations were 0.7-1.0 mM at a 1:1 molar ratio SH3:PRR2. For quantitative titrations, purchased peptides were dissolved in 100-300 μl NMR sample buffer, and samples at varying (SH3:PRR2 1:0-1:6, SH3 concentration constant at 0.1 mM) ratios were prepared. Fittings were performed with 5-6 measured points.
NMR measurements were conducted on a DRX700 Bruker spectrometer (Bruker BioSpin GmbH, Rheinstetten, Germany) with a cryogenic triple-resonance TCI probe head equipped with z-axis pulsed-field gradients at 16.4 T and 296-298 K. External calibration was performed with DSS dissolved in the same buffer as for the experiments. Acquisition parameters for 2D TOCSY, NOESY and COSY homonuclear experiments, 2D HSQC experiments, the triple resonance HNCACB experiment, and 3D filtered-edited NOESY experiments are detailed below. For two-dimensional homonuclear experiments (COSY, TOCSY, and NOESY) 2048 complex points were acquired in the direct dimension (1H) (acquisition time: 208.9 ms) and 280-480 complex points in the indirect dimension (1H) (acquisition times: 40-57.1 ms), with mixing times of 150 ms (TOCSY) or 200-300 ms (NOESY). Typical experiment times were 3-36 h. Conditions for filtered-edited NOESY were 1024 complex points in the direct dimension (1H), 96-97 complex points in the indirect 1H dimension, and 25 complex points in the 13C dimension, affording acquisition times of 104.4 ms, 4.2-5.3 ms, and 17.1 ms, respectively, and a mixing time of 200-400 ms throughout 59-66 h. 15N,1H-HSQC spectra were acquired at 1024 complex points in the 1H dimension and 188 complex points in the 15N dimension (acquisition times: 91.8 ms and 88.3 ms, respectively). The HNCACB experiment (90 h) was acquired at 1024 complex points in the direct 1H dimension and 40 (52) complex points in the indirect 15N (13C) dimension, affording acquisition times of 104.4, 16.6, and 4.9 ms, respectively. The aromatic-TROSY 13C,1H correlation spectrum (1.5 h) was acquired with 1024 complex points in the direct 1H dimension and 100 complex points in the indirect 13C dimension. Assignment of the unbound SH3 domain was assisted by BMRB entry 10240. Chemical shift perturbation values representing a normalized Euclidean distance Δδ were calculated as ((ΔH)2+(0.14*ΔN)2)1/2, where ΔH and ΔN are the shifts in the hydrogen and nitrogen dimensions, respectively.
The 1H, 15N-HSQC spectrum of a 0.2 mM sample of the SH3 domain was acquired at 0:1 (black), 0.5:1, 1:1, 2:1 and 4:1 (light green) mol:mol PRR2:SH3 and 0:1 (black), 0.75:1, 1.5:1, 3:1 and 6:1 (light green) mol:mol PRR3:SH3. Peptides were unlabeled in all cases. Isotherms were fitted to the equation
NOE crosspeaks were ranked by intensity and binned into 4 groups corresponding to distances of 2.7, 3.3, 4.0-4.5 and 5.0-5.5 A. Dihedral angle constraints were derived from the TALOS+ server [14] from SH3 domain HA, HN, CA and CB chemical shifts. Initial structures were calculated using distance and dihedral angle constraints by simulated annealing and torsional angle dynamics as applied in the program CNS [15]. Final structures were calculated with CNS using distance, dihedral, and H-bond constraints in addition to two electrostatic constraints, SH3(E506)-PRR2(R719) and SH3(D522)-PRR2(K721) suggested by the NOESY data and derived from characteristic salt-bridge patterns in SH3 complexes. Stereochemical quality of the ensemble of final structures was evaluated using with VADAR 1.8 and PROCHECK. For the final ensemble of 20 structures, 100 structures were generated, and structures with lowest-energy and highest stereochemical quality were chosen by analysis with PROCHECK. Final coordinates were deposited at the Protein Data Bank under accession code 7PLL.
Statistical analysis was performed using GraphPad Prism 8.0. For XTT, cortactin phosphorylation, and barbed end assays, statistical significance was calculated using two-way ANOVA followed by Bonferroni post-hoc test. For all other cellular and in vivo assays, one-way ANOVA followed by Tukey's post-hoc test was used. For patient database analysis, statistical significance was calculated using log-rank and Student's t-test implemented in MATLAB. Values were considered statistically significant if the P<0.05. For all figures, *, P<0.05; **, P<0.01; ***, P<0.001. Error bars represent standard errors of the mean (SEM).
The inventors have previously shown that the Pyk2/cortactin interaction is critical for invadopodia-mediated breast tumor cell invasiveness. To study this further, DNA, RNA, and protein expression data of breast cancer tissues from the cancer genome atlas (TCGA) was integrated and stratified by hormone- and HER2-receptor status. A comparison of copy number alterations (CNA), mRNA expression, and protein expression demonstrated similar distribution among all hormone- and HER2-receptor statuses (
Pyk2 peptides derived from the second (Pyk2-PRR2, SEQ ID NO: 1) or third (Pyk2-PRR3, SEQ ID NO: 2) proline-rich regions and a control scrambled peptide (SEQ ID NO: 4) were synthesized and fused to an HIV-TAT sequence to increase their permeability into cells. To examine their effect on breast cancer metastasis in mice, the highly tumorigenic mouse 4T1 cell line was used. This cell line efficiently forms mammary tumors upon injection into the mammary fat pad of syngeneic immune-competent BALB/c mice and spontaneously metastasizes to multiple distant sites in a pattern similar to that of human breast cancer. Tumor-bearing mice were treated with the peptides by intraperitoneal injection for 8 days, followed by careful examination and quantification of lung metastases at the end of the treatment. As demonstrated in
To investigate these further, primary tumors were isolated and examined at the end of the treatment. As demonstrated in
Metastatic dissemination is initiated by the invasion of tumor cells through the tumor basement membrane and extracellular matrix (ECM). To study the effect of Pyk2 peptides on tumor cell invasiveness, the MDA-MB-231 was used, a highly invasive, triple-negative basal-like human breast adenocarcinoma cell line that forms functional invadopodia in culture.
Peptide permeability into MDA-MB-231 cells was validated using an immunofluorescence assay, showing all peptides penetrated the cells and were stable at least 24 h following incubation. An XTT assay on cells incubated with 0.1, 1, 5, and 10 μM concentrations of peptides showed that the three lower concentrations did not affect cell viability and were therefore used in all further cellular studies (data not shown).
The ability of the three peptides to inhibit the invasion of breast cancer cells embedded in a three-dimensional (3D) Matrigel matrix towards a scratch wound was tested over 24 hours. As demonstrated in
Further evidence of the Pyk2-PRR2 effect upon tumor cell invasiveness was provided by a 2D random migration assay. Invasiveness is controlled by coordinating MMP-dependent invadopodia-mediated invasion and focal adhesion-mediated motility, and the inventors have previously shown that Pyk2 localizes to these regions and that knockdown of Pyk2 inhibits both MMP-dependent invasion and two-dimensional (2D) motility. Following accumulated and Euclidean distances as well as velocity over 12 hours, cells treated with Pyk2-PRR2 at all concentrations and with Pyk2-PRR3 at the highest concentration showed reduced 2D motility over an ECM substrate compared to the control peptide (data not shown). Thus, Pyk2-PRR2, and to a lesser extent Pyk2-PRR3, regulate 2D motility and inhibit tumor cell invasiveness.
Breast cancer metastasis has previously been correlated in vivo with invadopodia maturation and function, and in mouse models and human patients with invadopodia assembly and function.
Binding of Pyk2-PRR peptides to purified cortactin-SH3 was determined by following ligand-induced chemical shift changes along the course of a titration isotherm. Pyk2-PRR2 (SEQ ID NO: 1) and Pyk2-PRR3 (SEQ ID NO: 2) ligands exhibited affinities of KD=40±5 and KD=650±10 μM, respectively, and the Pyk2-PRR2 derivatives HAIYPRHTAFQEPPPKPSRPKYRPPP-NH2 (SEQ ID NO: 7+C-term cap), Ac-TAFQEPPPKPSRPKYRPPPHRPYIAH (SEQ ID NO: 8+N-term cap), and Ac-T*A*F*Q*EPPPKPSRPKYR*P*P*P*-NH2 (SEQ ID NO: 9+N- and C-term caps) wherein * denotes d-amino acid exhibited affinities of KD=8±5, KD=2±1 and KD=15±2, while a scrambled control peptide did not display significant affinity to the SH3 domain (KD>1 mM) (
Largest Pyk2-PRR2-induced changes were observed for residues 1521, A539 and the W525 of the cortactin indole cross-peak, consistent with a canonical binding mode. Thus, the Pyk2-PRR2 peptide binds to cortactin SH3 in vitro with a significantly (16-fold) higher affinity than Pyk2-PRR3. The ability of Pyk2-PRR2 to block the access of Pyk2 to cortactin in invadopodia in Förster resonance energy transfer (FRET) acceptor photobleaching experiments was also measured. These demonstrated that the Pyk2-PRR2, but not Pyk2-PRR3 or a scrambled peptide, significantly inhibited the interaction between Pyk2 and cortactin in invadopodia (
To examine whether Pyk2-PRR2 regulates the initial assembly of invadopodium precursors, MDA-MB-231 cells were treated with Pyk2-PRR2, Pyk2-PRR3, or scrambled control peptide, plated on fluorescently labeled gelatin matrix and labeled for Tks5 and cortactin as invadopodium precursor markers. The significant increase in immature invadopodium precursors, along with a significant decrease in mature, matrix-degrading invadopodia observed in Pyk2-PRR2-treated cells implied that this peptide blocks the maturation and activation of pre-assembled invadopodium precursors in breast tumor cells (
At the final maturation stage invadopodia locally degrade the ECM by secretion and activation of MMPs, allowing invasive cancer cells to leave the primary tumor and penetrate blood vessels. Breast tumor cells were plated on fluorescent fibronectin/gelatin matrix and allowed to degrade the matrix for 24 h in the presence of increasing concentrations of control, Pyk2-PRR2, or Pyk2-PRR3 peptides. Quantification of the degradation area revealed a significant decrease in the ability of Pyk2-PRR2 treated cells to degrade the matrix in all concentrations tested (
Tyrosine phosphorylation of invadopodial cortactin mediates invadopodia maturation and activation, and involves the Pyk2 kinase, both directly and indirectly via Src-mediated Arg activation. To see whether Pyk2-PRR peptides can inhibit this process in breast cancer cells, MDA-MB-231 cells stably expressing Tag-RFP-cortactin were treated with Pyk2-PRR or control peptides and further induced to form invadopodia by EGF. Cells were then fixed and labeled with phosphorylation-specific antibodies for cortactin tyrosine Y421. As demonstrated in
With extensive evidence of the Pyk2-PRR2/cortactin-SH3 interaction and its potent role in regulating invadopodia-mediated metastasis, the complex was characterized at the molecular level to identify critical residues which contribute to the interaction affinity. The NMR-based SH3/PRR2 complex structure was determined using distance constraints established through homonuclear and filtered-edited heteronuclear NOESY experiments and dihedral angle constraints derived from analysis of chemical shifts (
Three main features contribute to the affinity of this complex. First, a network of hydrogen bonds stabilizes the complex, including the P714 CO with the Y541 sidechain hydroxyl, the P715 CO with the N540 polar sidechain, and P717/S718 CO groups with the W525 indole NH. In addition, stabilizing electrostatic contacts are found between R719 and the acidic specificity pocket of SH3 residues D505/E506 and between K721 and D522 in surface II (not shown). Second, the P714/P717/P720 pyrrolidine rings adopt an orientation parallel to the SH3 groove-lining aromatic residues W525 and Y541, thus favoring C—H-π interactions between the electron-rich aromatic rings and the partial positive charge of polarized Cα-Hα and Cδ-Hδ bonds in prolines (not shown). Finally, close packing of hydrophobic surfaces is found for the three inserted proline residues and residue Y722 with the methyl groups of 1521 and L536.
The two-surface interaction between SH3 and PRR2 was further confirmed in the NMR chemical shift perturbation (CSP) data, as the strongest effects were observed for both the canonical binding surface (Y497, Q500, D505, W525, A539, N540) as well as residues near the second surface (M520, 1521, D522, W526) (
A super-mutant Pyk2-PRR2 peptide (SEQ ID NO: 3) was synthesized, in which five critical amino acids were mutated (P714/717/720A, R719Q, K721Q). The loss of SH3 affinity of this peptide was verified by NMR, in which no detectable affinity could be observed (estimated KD>1 mM). Similarly, FRET acceptor photobleaching experiments demonstrated that, unlike the Pyk2-PRR2 peptide, this super-mutant peptide does not significantly inhibit the Pyk2-cortactin interaction in invadopodia of breast tumor cells at any of the concentrations tested (
The xenograft model assay was also repeated with the Pyk2 super-mutant peptide, treating tumor-bearing mice with a cell-permeable Pyk2 super-mutant (with a scrambled peptide as control, SEQ ID NO: 4) by intraperitoneal injection for eight days. As above, no significant differences were detected in primary tumor growth in all groups (
This application claims the benefit of U.S. Provisional Application No. 63/499,265 filed on May 1, 2023. Any and all applications for which a foreign or domestic priority claim is identified above and/or in the Application Data Sheet as filed with the present application are hereby incorporated by reference under 37 CFR 1.57. The contents of the electronic sequence listing (BRD-003-US.xml; Size: 25,225 bytes; and Date of Creation: Apr. 27, 2024) is herein incorporated by reference in its entirety.
| Number | Date | Country | |
|---|---|---|---|
| 63499265 | May 2023 | US |
