Patent Publication Number: US-2023133141-A1

Title: Methods for treating multidrug resistant breast cancer

Description:
CROSS-REFERENCE TO RELATED APPLICATION 
     This application claims priority from the U.S. provisional patent application Ser. No. 63/260,736 filed Aug. 31, 2021, and the disclosure of which is incorporated herein by reference in its entirety. 
    
    
     TECHNICAL FIELD 
     The present invention relates to methods for treating multi-drug resistant cancers, in particular, to methods for treating multi-drug resistant breast cancer. 
     BACKGROUND 
     Breast cancer is the most prevalent cancer among women, impacting 1.5 million women each year and 15% of deaths from all cancers. Although various therapeutic strategies are commonly employed, treated patients can suffer from disease relapse and metastasis due to the presence of cancer stem cells (BCSCs). Breast CSCs (BCSCs) are a subset of tumor population with abilities of self-renewal, quiescence and generating heterogenic lineages of cancer cells. BCSCs possess different intrinsic mechanisms of resistance to current chemotherapeutic drugs, like overexpression of ATP-binding cassette (ABC) transporters family, and activation of different signaling pathways such as Wnt/β-catenin signaling, Notch and Akt survival pathways, being one of the biggest hurdles to effective chemotherapy. Current drugs to reverse chemoresistance such as ABC transporter inhibitors, verapamil, possess many side effects, for example, cardiovascular side effects associated with continuous and high-dose intravenous verapamil therapy. 
     Integrins are a family of αβ-heterodimeric cell surface receptors that mediate cell adhesion to the surrounding extracellular matrix (ECM), triggering cellular responses such as cell migration and proliferation, contributing to tumor progression, CSC phenotypes and chemoresistance. They are also markers of certain CSCs and able to mediate cell-cell adhesion which helps corresponding stem cells anchor to their niche, in addition to their adhesion to the surrounding substrata. Some studies proposed that integrins contribute to an attachment-dependent chemoresistance through their membrane proximal conserved KXGFFKR motif, which is critical for receptor functions and activation of β integrins. This highly conserved motif in their α integrins can interact with various proteins including calreticulin (CRT), which is a multifunctional protein involved in various cellular activities such as protein folding, phagocytosis, cell proliferation and metastasis. Its abilities to bind ITGA2 and activate ITGB1 prevent cell surface localization of CRT, in turn mediates phagocytosis. 
     Another function of the highly conserved KXGFFKR motif is to inhibit ITGB1 by interacting with SHARPIN through its ubiquitin-like (UBL) domain, resulting in inactivation of ITGB1. 
     Thus, inhibition of integrin activation through interaction with the UBL domain of SHARPIN to compete with CRT for a highly conserved KXGFFKR motif appears to be a potential mechanism to reserve the chemoresistance in the CSCs arising from interaction between integrin ITGA2 and the highly conserved motif, and the activation of the ITGB1, as the activation of ITGB1 renders cytoplasmic CRT to chemoresistance by degradation of p53 for cell survival ( FIG.  9   ). 
     SUMMARY 
     Accordingly, the present disclosure proposes methods and composition for treating multi-drug resistant cancers including breast cancer in a subject in need thereof. The methods include administering to the subject the composition including a peptide, where the peptide includes a cell penetrating sequence and a sequence complementary to a membrane proximal conserved motif present in substantially all integrins in tumor-initiating cells including those associated with integrin-mediated attachment-independent chemoresistance such that the peptide of the composition is capable of reversing the integrin-mediated attachment-independent chemoresistance in the tumor-initiating cells, in order to restore the function of one or more chemotherapeutic agents against the tumor-initiating cells. 
     In certain embodiments, the peptide of the composition is represented by SEQ ID NO: 4. 
     In certain embodiments, the cell penetrating sequence is a TAT peptide represented by SEQ ID NO: 1. 
     In certain embodiments, the membrane proximal conserved motif is a highly conserved KXGFFKR motif of α integrin. 
     In certain embodiments, the sequence complementary to the highly conserved KXGFFKR motif is derived from a ubiquitin-like domain of an endogenous β1-integrin activation inhibitor. 
     In certain embodiments, the endogenous β1-integrin activation inhibitor is SHARPIN, and the sequence complementary to the highly conserved KXGFFKR motif is a SHARPIN-derived peptide represented by SEQ ID NO: 2. 
     In certain embodiments, the TAT peptide and SHARPIN-derived peptide (SP) are synthesized together into a D-retro-inverso (DRI) form for preventing thereof from protease degradation in vivo. 
     Preferably, the SHARPIN-derived peptide is synthesized based on D-amino acids to make the peptide resistant to enzymatic digestion by endogenous protease when the composition is administered in vivo. 
     In certain embodiments, the DRI form of the TAT-SP peptide (DRI-TAT-SP) is synthesized by modifying N- and C-termini thereof. 
     Preferably, the N-terminus of the TAT-SP peptide is acetylated while the C-terminus thereof is amidated to obtain the DRI-TAT-SP peptide. 
     In certain embodiments, the DRI-TAT-SP peptide is represented by SEQ ID NO: 6. 
     In certain embodiments, the composition is administered in combination with one or more chemotherapeutic agents to the subject. 
     In certain embodiments, the one or more chemotherapeutic agents include an anthracycline or a DNA topoisomerase II inhibitor. 
     In certain embodiments, the one or more chemotherapeutic agents is/are administered as a neoadjuvant therapy prior to the administration of the present composition. 
     In certain embodiments, the one or more chemotherapeutic agents include doxorubicin (DOX), paclitaxel (PTX) and vincristine (VCR). 
     In certain embodiments, the composition is administered to the subject via one or more routes including, but not limited to, intravenous and intraperitoneal injections. 
     In certain embodiments, the subject is human and the multi-drug resistant cancers include human breast cancer. 
     This summary is provided to introduce a selection of concepts in a simplified form that are further described below in the Detailed Description. This Summary is not intended to identify key features or essential features of the claimed subject matter, nor is it intended to be used as an aid in determining the scope of the claimed subject matter. Other aspects of the present invention are disclosed as illustrated by the embodiments hereinafter. 
    
    
     
       BRIEF DESCRIPTION OF DRAWINGS 
       The appended drawings, where like reference numerals refer to identical or functionally similar elements, contain figures of certain embodiments to further illustrate and clarify the above and other aspects, advantages and features of the present invention. It will be appreciated that these drawings depict embodiments of the invention and are not intended to limit its scope. The invention will be described and explained with additional specificity and detail through the use of the accompanying drawings in which: 
         FIG.  1 A  shows mRNA levels of different integrins in MCF-7 and MCF-7 derived cell lines obtained by using RT2 profiler PCR array; CSC: cancer stem cell; 
         FIG.  1 B  shows mRNA levels of different integrins in MCF-7 and MCF-7 derived cell lines measured by quantitative real-time PCR (qRT-PCR); *P&lt;0.05 and **P&lt;0.01 compared to MCF-7 AC; NS: not significant 
         FIG.  1 C  shows ITGA2 protein levels in MCF-7 and MCF-7 derived cell lines detected by Western Blotting (WB); data shown are representative of independently conducted experiments; 
         FIG.  1 D  shows an overall survival Kaplan-Meier estimate generated from the cBioPortal for Cancer Genomic; 
         FIG.  2 A  schematically depicts construction of ITGA2 mutants; 
         FIG.  2 B  shows ITGA2 constructs used to transfect ITGA mutants as in  FIG.  2 A ; A2 denotes full-length ITGA2; A2dm denotes KLGFFKR motif-deficient (dm) ITGA2, with the KLGFFKR motif removed from the cytosolic tail; Tac is an unrelated membrane protein fused to KLGFFKR (Tacδ) or scrambled KLRFGFK (TacδScr); ECD: extracellular domain; TMD: transmembrane domain; 
         FIG.  2 C  shows western blot results of ITGA2 and Tac protein levels (using anti-ILR2 antibody) in different ITGA2 mutants as in  FIG.  2 A ; 
         FIG.  2 D  shows half maximal inhibitory concentration (IC 50 ) values of DOX, PTX and VCR in MCF-7 AC, CSC and different ITGA2 mutants as in  FIG.  2 A ; 
         FIG.  2 E  shows flow cytometry analysis results of CSC population in MCF-7 AC, CSC and different ITGA2 mutants as in  FIG.  2 A ; 
         FIG.  3 A  shows normalized phosphorylated-AKT (p-AKT) protein levels in MCF-7 AC, CSC and different ITGA2 mutants treated with 1 μM DOX for 24 hours at 37° C.; 
         FIG.  3 B  shows western blot results of MRP1 and P-gp protein expressions in MCF-7 AC, CSC and different mutants with the same treatment as in  FIG.  3 A ; 
         FIG.  3 C  shows relative DOX accumulation in CSC and different ITGA2 mutants with respect to MCF-7 AC treated with 5 μM DOX for 2 hours at 37° C.; the DOX accumulation is indicated by fluorescence signal from their cell-free supernatants; data shown are representative of at least 3 independently conducted experiments (mean±S.E.M.); *P&lt;0.05 and **P&lt;0.01 compared to MCF-7 AC; NS: not significant; 
         FIG.  3 D  shows percentages of apoptosis by quantifying annexin V expression level in MCF-7 AC, CSC and different ITGA2 mutants via flow cytometry analysis of annexin V-FITC and propidium iodide positive staining of the cells; 
         FIG.  4 A  shows western blot results of CRT and total p53 protein levels in MCF-7 AC, CSC and different ITGA2 mutants with respect to KXGFFKR motif (motif) interaction with the ITGA2 in the cells; 
         FIG.  4 B  shows relative cell surface CRT expression in CSC and different ITGA2 mutants with respect to MCF-7 AC with or without DOX treatment in the presence of the motif as in  FIG.  4 A , which is analyzed by flow cytometry; 
         FIG.  4 C  shows CRT expression level in cell lysate from different ITGA2 mutants by co-immunoprecipitation of different mutants with anti-CRT antibody and analyzed by blotting; data shown are representative of at least 3 independently conducted experiments (mean±S.E.M.); 
         FIG.  5 A  shows an alignment of different SHARPIN-derived peptides according to certain embodiments of the present invention; 
         FIG.  5 B  shows blotting results of from CSC lysate immunoprecipitated with anti-CRT antibody in the presence of different concentrations of SP and SPScr peptides, respectively (left panel) and from CSC lysate immunoprecipitated with anti-CRT in the presence of 1 μM TAT-SP and TAT-SPScr peptides, respectively (right panel) for 24 hours; 
         FIG.  5 C  shows half maximal effective concentration (EC 50 ) values of DOX in CSCs treated with TAT-SP and TAT-SPScr peptides, respectively; 
         FIG.  5 D  shows half maximal inhibitory concentration (IC 50 ) values of DOX, PTX and VCR in CSCs treated with TAT-SP and TAT-SPScr peptides, respectively; data shown are representative of at least 3 independently conducted experiments (mean±S.E.M.); *P&lt;0.05 and **P&lt;0.01 compared to CSC; NS: not significant. IP: immunoprecipitation; 
         FIG.  6 A  shows quantification of p-Akt (T308) protein expression in CSCs treated with or without 1 μM TAT-SP or TAT-SPScr peptide for 24 hours; 
         FIG.  6 B  shows blotting results of ITGA2, MRP1, CRT and p53 protein levels in CSC lysate from cells treated with 1 μM TAT-SP and TAT-SPScr peptides, respectively, for 24 hours; 
         FIG.  6 C  shows relative percentage of DOX accumulation in different CSC groups treated with 5 μM DOX in the presence of 1 μM TAT-SP and TAT-SPScr peptides, respectively, for 24 hours, with respect to the untreated CSC control group and MCF-7 AC; data shown are representative of at least 3 independently conducted experiments (mean±S.E.M.). *P&lt;0.05 and **P&lt;0.01 compared to CSC; NS: not significant; 
         FIG.  6 D  shows percentages of apoptosis by quantifying annexin V expression level in untreated CSC control group and CSC groups treated with 1 μM TAT-SP and TAT-SPScr peptides, respectively, via flow cytometry analysis of annexin V-FITC and propidium iodide positive staining of the cells; 
         FIG.  7 A  shows half maximal inhibitory concentration (IC 50 ) values of DOX, PTX and VCR in MCF-7 CSC at various concentration in the absence or presence of 3 μM of D-retro-inverso form of TAT-SP peptide (DRI-TAT-SP) according to certain embodiments, and half maximal effective concentration (EC 50 ) of the DRI-TAT-SP in reversing chemoresistance to DOX, PTX and VCR in MCF-7 CSC, respectively; 
         FIG.  7 B  shows plasma concentration of the DRI-TAT-SP administered at 25 mg/kg by intravenous (i.v.) injection to a breast cancer animal model; AUC (0-obs) : Area under the plasma concentration-time curve from time 0 up to 7 hours; C max : maximum plasma concentration; T max : time to maximum plasma concentration; AUC (0-obs) /DOSE: dose normalized area under the plasma concentration-time curve from time 0 up to 7 hours; the values are presented as mean±SD. n=1-3; 
         FIG.  7 C  shows body weight change in the animal model administered with the DRI-TAT-SP as in  FIG.  7 B , 1.2 mg/kg DOX alone, or both once every two days for 10 injections; 
         FIG.  7 D  shows histological examination results of different organs from the animal model after being administered with the DRI-TAT-SP, DOX, or both once every 2 days for 10 injections as in  FIG.  7 C ; the values are presented as mean±SD. n=3-4; 
         FIG.  8 A  shows tumor volume change (%) in different treatment groups of animal model with (1) PBS, (2) 1.2 mg/kg DOX, (3) 25 mg/kg DRI-TAT-SP and (4) 1.2 mg/kg DOX+25 mg/kg DRI-TAT-SP, at a frequency of once every 2 days for 10 times via i.v. injection (upper panel); tumor volume is measured using digital caliper once every 4 days (lower panel), where an estimated tumor volume (mm 3 ) is calculated as ½×length×width 2  in mm and normalized to the tumor volume on the first day of treatment; the values are presented as mean±SD; n=8-10; 
         FIG.  8 B  shows body weight change in different treatment groups of the animal model treated and sampled according to administration scheme as in  FIG.  8 A ; 
         FIG.  8 C  shows an in vivo DRI-TAT-SP accumulation in tumors over time after i.v. injection; the values are presented as mean±SD; n=2-4; 
         FIG.  9    schematically depicts a proposed mechanism of ITGA2-mediated attachment-independent chemoresistance in CSC, and how it can be inhibited or reversed by the SHARPIN or SHARPIN-derived peptide according to certain embodiments of the present invention. 
     
    
    
     Skilled artisans will appreciate that elements in the figures are illustrated for simplicity and clarity and have not necessarily been depicted to scale. 
     DETAILED DESCRIPTION OF THE INVENTION 
     It will be apparent to those skilled in the art that modifications, including additions and/or substitutions, may be made without departing from the scope and spirit of the invention. Specific details may be omitted so as not to obscure the invention; however, the disclosure is written to enable one skilled in the art to practice the teachings herein without undue experimentation. 
     (1) Role of ITGA2 in Multidrug Resistance in CSCs 
     The present disclosure provides methods and related composition for targeting an integrin-mediated attachment-dependent chemoresistance mechanism in cancer stem cells (CSCs) including breast cancer stem cells (BCSCs). ITGA2, a type of integrin, shows the greatest upregulation among 84 genes studied RT 2  qPCR primer assay ( FIG.  1 A ) in CSCs sorted from MCF-7 adherent cells (AC), which is confirmed by quantitative PCR (qRT-PCR) and Western Blotting (WB) ( FIGS.  1 B and  1 C ). An overall survival Kaplan-Meier estimate shown in  FIG.  1 D  indicates a poorer prognosis from overexpression of ITGA2 in MCF-7 AC-derived CSCs. 
     In order to evaluate the role of ITGA2 in chemoresistance of BCSCs, a knock-out (KO) ITGA2 in vitro model is created in MCF-7 AC, named as A2KO AC, by CRISPR and its mammospheres (A2KO MS) are produced ( FIGS.  2 A- 2 C ). Compared to its corresponding MCF-7 AC and MS ( FIG.  2 B ), in the absence of ITGA2, A2KO MS remains drug sensitive with a significantly lower drug IC 50  to DOX, PTX and VCR ( FIG.  2 C ). Reintroduction of full-length ITGA2 to A2KO MS rescues A2KO-A2OX MS from anticancer drugs, confirming the role of ITGA2 in chemoresistance in MS. Such ITGA2-mediated chemoresistance is not due to manipulation of CSC population ( FIG.  2 E ) but other mechanisms. 
     (2) Role of Conserved Membrane Proximal KXGFFKR Motif in DOX-Resistant CSCs 
     A highly conserved motif, KXGFFKR, in ITGA2 has been found in all α-integrins. To study the correlation between this conserved membrane proximal motif and drug resistance to DOX, a commonly used chemotherapeutic agent of breast cancer, ITGA2 is overexpressed in A2KO AC by transfection of different ITGA2 plasmids as shown in  FIG.  2 B  into the A2KO AC cells, followed by selection by hygromycin B to derive into several ITGA2 mutants, namely A2KO-ASOX MS, A2KO-ASdmOX MS, A2KO-TacδOX MS, and A2KO-TacδScrOX MS, where A2KO-ASOX MS denotes the MS derived from A2KO AC with an overexpression of full-length ITGA2; whereas A2KO-ASdmOX MS denotes the MS derived from A2KO AC with an overexpression of KXGFFKR motif-deficient ITGA2 (by removing KLGFFKR motif from its cytosolic tail), while A2KO-TacδOX MS denotes the MS derived from ASKO AC with an overexpression of the KXGFFKR motif alone in Tacδ protein (where Tac is an unrelated membrane protein fused to KLGFFKR motif) and A2KO-TacδScrOX MS denotes MS derived from ASKO AC with an overexpression of a scrambled KLGFFKR in Tacδ protein. It is observed that the overexpression of full-length ITGA2 (A2KO-A2OX MS), but not the motif-deficient ITGA2 (A2KO-A2dmOX MS), could successfully rescue the KO clone from DOX. However, overexpression of KXGFFKR motif alone (A2KO-TacδOX MS) does not fully rescue the KO clone and restore DOX IC 50 , implying that KXGFFKR motif is required but not sufficient to induce attachment-independent DOX-resistance in MCF-7 CSCs. 
     (3) Role of Activation of ITGB1 in DOX Resistance of CSCs via Stimulation of Akt Pathway by KXGFFKR Motif 
     ITGA2, through its KXGFFKR motif, is responsible for activation of ITGB1, which in turn stimulates different downstream pathways including Akt signaling pathway.  FIG.  3 A  shows that ITGA2 mutants with overexpression of full length of ITGA2 (A2KO-A2OX MS) and overexpression of KXGFFKR motif (A2KO-TacδOX MS) have elevated DOX-induced Akt phosphorylation (elevated T308-phosphorylated Akt level), whereas S473-phosphorylated Akt level remains unchanged upon DOX incubation, excluding its participation in KXGFFKR-mediated DOX-resistance. 
     Stimulation of Akt pathway leads to upregulation of ABC transporters and enhanced drug efflux activity to mediate drug resistance in CSCs.  FIG.  3 B  shows that overexpression of full-length ITGA2 (A2KO-A2OX MS) upregulates MRP1 and P-gp, but not motif-deficient ITGA2 (A2KO-A2dmOX MS) or KXGFFKR motif alone (A2KO-TacδOX MS). With upregulation of MRP1 and P-gp, CSC, MCF-7 MS and A2KO-A2OX MS exhibit enhanced DOX efflux, resulting in low DOX accumulation ( FIG.  3 C ). In the absence of KXGFFKR motif, Akt pathway could not be activated in A2KO-A2dmOX MS for enhanced drug efflux activity and accumulated high level of DOX, even though the MRP1 and P-gp levels are relatively higher when compared to A2KO MS. On the other hand, KXGFFKR motif alone in A2KO-TacδOX MS is sufficient to activate Akt pathway, allowing it to efflux DOX more efficiently and in turn accumulates relatively less DOX. 
     Activation of Akt pathway by ITGA2 is also susceptible to DOX resistance via inhibition of DOX-induced apoptosis. To verify this potential mechanism, an annexin V apoptosis assay is performed on different ITGA2 mutants. Consistent with the results obtained from DOX-induced Akt phosphorylation in  FIG.  3 C , KXGFFKR motif of ITGA2 is sufficient to activate Akt pathway to promote cell survival and inhibit DOX-induced apoptosis ( FIG.  3 D ). 
     (4) Role of Interaction Between KXGFFKR Motif and Calreticulin (CRT) in ITGB1 Activation and DOX Resistance 
     CRT is a multifunctional protein involved in various cellular activities like protein folding, phagocytosis, cell proliferation and metastasis. CRT binds to ITGA2 and helps activate ITGB1, which in turn prevents cell surface localization of CRT and mediates phagocytosis.  FIG.  4 A  shows that the total CRT expression elevates in CSCs and MS with overexpression of full-length ITGA2 but not the KXGFFKR motif alone, which is consistent with some other studies. 
     To assess whether CRT is involved in activation of ITGB1 and ITGA2-mediated DOX-resistance, cell surface CRT localization is measured by indirect immunofluorescence staining with flow cytometry.  FIG.  4 B  shows that the KXGFFKR motif at ITGA2 (CSC, MS and A2KO-A2OX MS) or Tacδ protein (A2KO-TacδOX MS) interacts with CRT and prevents its surface localization, regardless of total CRT expression level as shown in  FIG.  4 A .  FIG.  4 C  shows that CRT is co-immunoprecipitated (co-IP) with full-length ITGA2 but not the motif-deficient ITGA2. Similarly, intact KXGFFKR motif allows co-immunoprecipitation of Tacδ protein with CRT, but not the scrambled motif, confirming the physical interaction between KXGFFKR motif and CRT. These results indicate that CRT interacts with KXGFFKR motif of full-length ITGA2, helps activate ITGB1 to simulate the downstream Akt pathway as evident by  FIG.  3 A , and prevents subsequent cell surface localization ( FIG.  4 B ). 
     Cytoplasmic CRT is known to promote cell survival and inhibit metastasis through activation of MAPK pathway and degradation of p53.  FIGS.  4 A and  4 B  show respectively reduced p53 level and reduced cell surface CRT localization in MCF-7 derived cell lines overexpressed with intact KXGFFKR motif, suggesting that ITGA2 mediates DOX-resistance by interacting with CRT using its KXGFFKR motif for activation of ITGB1, which in turn (1) stimulates subsequent Akt pathway for reduced DOX-induced apoptosis and enhanced DOX-efflux, and (2) prevents cell surface localization of CRT by capturing them in cytoplasm for cell survival. 
     (5) Use of SHARPIN-Derived TAT-Peptide to Reverse Chemoresistance in DOX-Resistant CSCs by Competition with CRT for KXGFFKR Motif 
     SHARPIN, an inhibitor of ITGB1, can interact with the highly conserved KXGFFKR motif of α-integrin. More specifically, a ubiquitin-like (UBL) domain of the SHARPIN interacts with that motif to inactivate ITGB1. In this regard, a SHARPIN-derived (SP) peptide (SEQ ID NO: 2) derived from the UBL domain of the SHARPIN (residues 259-281) is synthesized ( FIG.  5 A ).  FIG.  5 B  demonstrates that both SP peptide and TAT-SP peptide (SEQ ID NO: 4), but not SPScr (SEQ ID NO: 3) or TAT-SPScr (SEQ ID NO: 5), compete with CRT for the KXGFFKR motif and inhibit the immunoprecipitation of ITGA2, respectively, confirming the interaction between the UBL domain of SHARPIN and KXGFFKR motif of ITGA2. TAT-SP peptide, but not TAT-SPScr, is able to modulate CSC chemoresistance with respect to DOX, PTX and VCR ( FIG.  5 D ) in the presence of DOX with EC 50  of 0.4 μM ( FIG.  5 C ). 
       FIG.  6 A  shows that incubation of CSCs with TAT-SP peptide inhibits the activation of Akt pathway by ITGA2, but neither the protein expression of ITGA2 nor MRP1 ( FIG.  6 B ). In the absence of enhanced drug efflux by Akt pathway, MRP1 could not efficiently conduct DOX efflux, resulting in high level of DOX accumulation ( FIG.  6 C ). At the same time, inhibition of Akt pathway by TAT-SP leaves CSCs unprotected from DOX-induced apoptosis ( FIG.  6 D ), reversing DOX-resistance in CSC. 
     (6) Pharmacokinetics of DRI-TAT-SP 
     To achieve a better peptide stability, a D-retro inverso (DRI) peptide of TAT-SP, namely DRI-TAT-SP, is synthesized, which is shown to be effective in reversing chemoresistance in BCSCs in the presence of DOX with EC 50  of 2.3±0.4 μM ( FIG.  7 A ). 
     Six- to eight-week-old female Balb/c mice are administered with DRI-TAT-SP intravenously (i.v.) at 25 mg/kg, and the peptide plasma concentration is monitored from 0 to 7 hrs.  FIG.  7 B  shows that DRI-TAT-SP reaches a C max  at 870 ng/mL 5 min post administration, and exhibits a rapid distribution rate at t 1/2α  of 15.9 min. The elimination phase is observed 60 min post i.v. administration at t 1/2β  of 442 mins. 
     (7) In Vivo Toxicity of DRI-TAT-SP 
     Healthy Balb/c mice are administered intravenously with: (1) DOX 1.2 mg/kg, (2) DRI-TAT-SP 25 mg/kg or (3) DOX 1.2 mg/kg plus DRI-TAT-SP 25 mg/kg once every two days for a total of 10 injections. DRI-TAT-SP alone causes a small drop in body weight by 2.7±4.4% after 10 injections. However, when combined with DOX, the mice suffer from a more significant body weight loss by 10.5±3.7%, given that DOX alone does not cause significant body weight loss (+0.6±2.4%) ( FIG.  7 C ). The organs including heart, liver, spleen, lung, kidneys, and brain are collected after sacrifice, which do not show significance difference in wet weight between the three groups. 
     (8) DRI-TAT-SP Combined with DOX Significantly Inhibits Tumor Growth in MCF7 Breast Cancer Xenograft 
     The MCF7 human breast cancer xenograft model is established in female Balb/c nude mice supplemented with 17β-estradiol to support the tumor growth. The MCF7 tumor could reach 3.3-fold increase in volume (325±84 mm 3 ) in 20 days with a tumor doubling time of 11.8 days if only PBS was given. DRI-TAT-SP 25 mg/kg alone showed 10.1% tumor inhibition (325±135 mm 3 ) with no statistical significances (P&gt;0.1) while DOX 1.2 mg/kg alone could only inhibit the tumor growth by 21.5% (270±73 mm 3 , P&lt;0.1) ( FIG.  8 A  &amp; Table 1). The T d  for DRI-TAT-SP and DOX alone groups are nearly identical (DOX alone: 14.8 days; DRI-TAT-SP alone: 12.9 days). However, the DOX/DRI-TAT-SP combination could significantly reduce the tumor volume by 49.2% (202±63 mm 3 , P&lt;0.001), delaying the tumor growth at T d  of 27.7 days which is 2.3-fold slower compared to the PBS control group (Table 1). The rapid distribution of DRI-TAT-SP to tumor at around 10,000 ng/g tumor tissues 5 minutes administration further supported its effectiveness in DOX sensitization to kill the breast cancer cells in vivo ( FIG.  8 C ). the DOX/DRI-TAT-SP combination causes 13.7±6.6% body weight drop throughout the study ( FIG.  8 B ). No treatment-induced deaths are observed in all the four groups. However, because of estrogen support, 7 out of 36 mice died of urolithiasis, showing symptoms of depression and dehydration (Table 1). 
                     TABLE 1                  Summary of in vivo efficacy and toxicity of doxorubicin       combined with DRI-TAT-SP in treating MCF7 breast cancer.                                         Tumor   Tumor   No. of           Animals   doubling   inhibition   toxicity       Treatment group   (n)   time (Days)   (%)   death                                         PBS   10   11.8   0   0/8       Doxorubicin (1.2 mg/kg)   8   14.8   21.5   0/8       DRI-TAT-SP (25 mg/kg)   8   12.9   10.1   0/6       Doxorubicin (1.2 mg/kg) +   10   27.7   49.2   0/7       DRI-TAT-SP (25 mg/kg)                    
Data obtained are analyzed to evaluate treatment outcomes. Tumor doubling time is calculated using tumor volumes obtained from the end of experiment versus the tumor volumes on the first day of treatment. Tumor inhibition (%) is calculated using the tumor volumes obtained from the end of experiment. Corresponding equations of determining tumor inhibition (T/C) and tumor doubling time (Td) are provided as follows:
 
     
       
         
           
             
               Tumor 
               ⁢ 
                   
               
                 inhibition 
                 ⁢ 
                 
                      
                     
                 
                 ( 
                 
                   T 
                   / 
                   C 
                 
                 ) 
               
             
             = 
               
             
               
                 [ 
                 
                   1 
                   - 
                   
                     
                       % 
                       ⁢ 
                           
                       Tumor 
                       ⁢ 
                           
                       
                         volume 
                         ⁢ 
                         
                             
                              
                         
                         ( 
                         
                           Treatment 
                           ⁢ 
                               
                           group 
                         
                         ) 
                       
                     
                     
                       % 
                       ⁢ 
                           
                       Tumor 
                       ⁢ 
                           
                       volume 
                       ⁢ 
                           
                       
                         ( 
                         
                           PBS 
                           ⁢ 
                               
                           group 
                         
                         ) 
                       
                     
                   
                 
                 ] 
               
               × 
               100 
               ⁢ 
               % 
             
           
         
       
       
         
           
             
               Tumor 
               ⁢ 
                   
               doubling 
               ⁢ 
                   
               time 
               ⁢ 
                   
               
                 ( 
                 Td 
                 ) 
               
             
             = 
             
               
                 ( 
                 
                   
                     t 
                     2 
                   
                   - 
                   
                     t 
                     1 
                   
                 
                 ) 
               
               ⁢ 
               
                 
                   log 
                   ⁡ 
                   ( 
                   2 
                   ) 
                 
                 
                   
                     log 
                     ⁡ 
                     ( 
                     
                       % 
                       ⁢ 
                           
                       tumor 
                       ⁢ 
                           
                       volume 
                       ⁢ 
                       
                            
                           
                       
                       ⁢ 
                       2 
                     
                     ) 
                   
                   - 
                   
                     log 
                     ⁢ 
                         
                     % 
                     ⁢ 
                         
                     
                       ( 
                       
                         tumor 
                         ⁢ 
                             
                         volume 
                         ⁢ 
                             
                         1 
                       
                       ) 
                     
                   
                 
               
             
           
         
       
       
         
           
             where 
             , 
               
             
               
                 
                   t 
                   2 
                 
                 = 
                     
                 
                   Final 
                   ⁢ 
                       
                   day 
                 
               
               ; 
               
                 
                   t 
                   1 
                 
                 = 
                 
                   Initial 
                   ⁢ 
                       
                   day 
                 
               
               ; 
             
           
         
       
       
         
           
             
               
                 % 
                 ⁢ 
                     
                 tumor 
                 ⁢ 
                     
                 
                   volume 
                   2 
                 
               
               = 
               
                 Percentage 
                 ⁢ 
                     
                 change 
                 ⁢ 
                     
                 in 
                 ⁢ 
                     
                 tumor 
                 ⁢ 
                     
                 volume 
                 ⁢ 
                     
                 at 
                 ⁢ 
                     
                 
                   t 
                   2 
                 
               
             
             ; 
           
         
       
       
         
           
             
               % 
               ⁢ 
                   
               tumor 
               ⁢ 
                   
               
                 volume 
                 1 
               
             
             = 
             
               Percentage 
               ⁢ 
                   
               change 
               ⁢ 
                   
               in 
               ⁢ 
                   
               tumor 
               ⁢ 
                   
               volume 
               ⁢ 
                   
               at 
               ⁢ 
                   
               
                 t 
                 1 
               
             
           
         
       
     
     In summary, the present disclosure proposes that an attachment-independent, ITGA2 KXGFFKR motif-mediated chemoresistance mechanism in BCSCs is a potential target to be reversed. Integrin ITGA2 coupled with ITGB1 to serve as a high affinity receptor for collagen type I and its significant upregulation in CSC and MCF-7 MS are crucial to chemoresistance in CSC and MS derived from MCF-7 AC. Silencing ITGA2 significantly reduces anticancer drug IC 50  in A2KO MS, confirming the role of ITGA2 in chemoresistance. The pronounced effect of ITGA2 is mostly due to the downregulation of other integrins after ITGA2 KO. Reintroduction of full-length ITGA2, but not the motif-deficient ITGA2 or KXGFFKR motif alone, successfully rescues corresponding mutants, suggesting that KXGFFKR motif is required but not sufficient to induce attachment-independent chemoresistance. Upon binding to CRT at its KXGFFKR motif, ITGA2 induces chemoresistance by activation of ITGB1, stimulating subsequent Akt pathway for (1) upregulation of MRP1/P-gp and promotion of their drug efflux activities for enhanced drug efflux; and (2) inhibition of anticancer drug-induced apoptosis. Activated ITGB1 (3) prevents cell surface localization of CRT, confining them at the cytoplasm to promote cell survival. The proposed SHARPIN-derived peptides, TAT-SP and DRI-TAT-SP, compete with CRT for KXGFFKR motif via their UBL domain of SHRAPIN to disrupt ITGA2/CRT interaction as well as ITGB1 inactivation. 
     The proposed SHARPIN-derived peptides, TAT-SP and DRI-TAT-SP, reverse effectively the multidrug resistance in MCF-7 breast CSCs at DOX-EC 50  of 0.4±0.1 μM and 2.3±0.4 μM, respectively. When combined with DOX at 1.2 mg/kg in vivo, DRI-TAT-SP could suppress the tumor progression in MCF-7 xenograft model by 49.2% after 10 injections in 20 days, showing its potential to treat breast cancers. Although i.v. administration of DRI-TAT-SP at 25 mg/kg does not give a prolonged residence in systemic circulation, a rapid peptide distribution to tumor at around 10,000 ng/g tissues supports its effectiveness in DOX sensitization to kill the breast cancer cells in vivo. Despite body weight drop observed, DOX/DRI-TAT-SP combination does not cause severe organ alteration. Data provided in the present disclosure support ITGA2 as a new therapeutic target to reverse chemoresistance in breast cancer by disrupting ITGA2/CRT interaction. 
     The following examples are intended to aid the understanding and enablement of certain embodiments in the present disclosure, which should not be considered as limiting the scope of the present invention. 
     EXAMPLES 
     (A) General DNA Amplification Methods: 
     DNA amplification was conducted by PCR using Phusion polymerase (ThermoFisher Scientific). All sgRNA and cDNA expression plasmids were transformed and stored as glycerol stocks at −80 C in  E coli  cells. All vectors and plasmids used in mammalian cell assays were purified using endotoxin-free DNA plasmid maxiprep kit (Bioteke). Hygromycin B used for either plasmid maintenance or selection were purchased from Invitrogen. 
     (B) General Mammalian Cell Culture Conditions: 
     MCF-7 and derived cells were cultured in Minimal Eagles Media (MEM) containing 10% (v/v) fetal bovine serum (FBS), 100 U/mL penicillin, 100 μg/mL streptomycin and 0.01 mg/mL human recombinant insulin. CSCs were produced by sorting a CD24 low /CD44 high  subpopulation. CSCs or MCF-7-derived MS were cultured in MAMMOCULT™ Human Basal media (STEMCELL TECHNOLOGIES) containing 10% (v/v) MAMMOCULT™ Proliferation Supplement (STEMCELL TECHNOLOGIES), 4 μg/mL Heparin Solution (STEMCELL technologies) and 0.48 μg/mL Hydrocortisone (STEMCELL TECHNOLOGIES). All cell types were incubated, maintained and cultured at 37° C. with 5% CO 2 . 
     (C) Generation of ITGA2 KO Clone: 
     Construction of an expression plasmid for sgRNAs was done as previously reported. Two oligo pairs encoding the 20-nt guide sequences of ITGA2 (Table 2) were annealed and ligated into pSpCas9(BB)-2A-GFP (PX458) plasmid. pSpCas9(BB)-2A-GFP (PX458) was a gift from Feng Zhang (Addgene plasmid #48138). Correct cloning and sequences were confirmed by Sanger sequencing. MCF-7 AC were seeded on 6-well plates and transfected at approximately 70% confluency with 2.5 μg with sgRNAs and 10 ng GFP plasmid expression (generous gift from Dr. Ben Ko), 5 μL Lipofectamine P3000 and 7.5 μL Lipofectamine 3000 (Life Technologies) as per the manufacturer&#39;s instructions. Cells were cultured for 48 hours and sorted for GFP fluorescence using FACSAria III Flow Cytometer (Becton Dickenson Bioscience). GFP-positive cells were collected and cultured. 
     
       
         
           
               
             
               
                 TABLE 2 
               
             
            
               
                   
               
               
                 Primers used for generating sgRNA plasmids and mammalian cell genomic 
               
               
                 DNA amplification: 
               
            
           
           
               
               
               
            
               
                   
                   
                 SEQ ID 
               
               
                 Primer 
                 Sequence 
                 NO. 
               
               
                   
               
               
                 ITGA2 KO sgRNA-A 
                 5’-CACCGTGTGTTTCTAGGTTACTGGT-3’ 
                  7 
               
               
                 forward 
                   
                   
               
               
                 ITGA2 KO SgRNA-A 
                 5’-AAACACCAGTAACCTAGAAACACAC-3’ 
                  8 
               
               
                 reverse 
                   
                   
               
               
                   
               
               
                 ITGA2 KO sgRNA-B 
                 5’-CACCGATCAAGCCGAGGCTCATGT-3’ 
                  9 
               
               
                 forward 
                   
                   
               
               
                 ITGA2 KO SgRNA-B 
                 5’-AAACACATGAGCCTCGGCTTGATC-3’ 
                 10 
               
               
                 reverse 
                   
                   
               
               
                   
               
               
                 ITGA2 sequencing 
                 5’-AGAAAGGCAGCAGGTCAAATC-3’ 
                 11 
               
               
                 forward 
                   
                   
               
               
                 ITGA2 sequencing 
                 5’-CCTCAGATTGGGAGGGATGGT-3’ 
                 12 
               
               
                 reverse 
                   
                   
               
               
                   
               
               
                 ITGA2 dm 
                 5’-CTCTggTTgCAATTTTATggAAATATgAAAAgATgA-3’ 
                 13 
               
               
                 forward 
                   
                   
               
               
                 ITGA2 dm  
                 5’-TTggTCATCTTTTCATATTTCCATAAAATTgCAACC-3’ 
                 14 
               
               
                 reverse 
               
               
                   
               
            
           
         
       
     
     (D) Generation of Overexpression Clones: 
     pCMV-ITGA2-CH cDNA plasmid was purchased from Sino Biological Inc. for overexpression full-length ITGA2. KLGFFKR motif was removed by PCR-mediated deletion as previously described with ITGA2 dm primers and pCMV-ITGA2-CH (Sino Biological Inc.) plasmid as template listed in above Table 2. pcDNA 3.1 (+) Tacδ and pcDNA 3.1 (+) TacδScr plasmids were a gift from Chinten James Lim (Addgene plasmid #80019). MCF-7 and A2KO AC were seeded on 6-well plates and transfected at approximately 70% confluency with 2.5 cDNA plasmid, 5 μL Lipofectamine P3000 and 7.5 μL Lipofectamine 3000 (Life Technologies) as per the manufacturer&#39;s instructions. Cells were selected with 50 μg/mL hygromycin B for 3-5 days and maintained in medium with 10 μg/mL hygromycin B (Invitrogen) for maintenance of cDNA expression. 
     (E) Chemotherapeutic Drugs and Culture Media: 
     Doxorubicin (DOX), Paclitaxel (PTX) and Vincristine (VCR), 3-(4,5-Dimethylthiazol-2-yl)-5-[3-(carboxymethoxy)phenyl]-2-(4-sulfo-phenyl)-2H-tetrazolium (MTS) and phenazine methosulfate (PMS) was purchased from Promega. Minimal essential Media (MEM) was purchased from Life Technologies. MAMMOCULT™ Human Basal Media and its supplements were purchased from STEMCELL Technologies. 17β-estradiol pellets (0.72 mg, 60-day release) was purchased from Innovative Research of America (Sarasota, Fla.). 
     (F) Flow Cytometry Analysis: 
     Cells were detached by Phosphate buffered saline (PBS) with 2.5 mM EDTA and washed with PBS. For determining CSC population, a total of 1×10 6  cells were incubated with APC-CD44 (BD Biosciences) and PE-CD24 (BD Biosciences) in FACS solution (PBS with 1% BSA and 1 mM EDTA) for 30 minutes on ice. APC-IgG2 (BD Biosciences) and PE-IgG2 (BD Biosciences) were used as isotype controls. For determining apoptotic population, a total of 1×10 6  cells were first treated with or without 1 μM DOX for 24 hours, in the absence or presence of 1 μM TAT-SP or TAT-SPScr, followed by incubation with FITC-Annexin V (BD Biosciences) and propidium iodide (PI) in binding buffer (10 mM Hepes, pH 7.4, 140 mM NaCl and 2.5 mM CaCl 2 ) for 30 minutes on ice as per the manufacturer&#39;s instructions. For determining CRT surface localization, a total of 1×10 6  cells, treated with or without 1 μM DOX for 24 hours, were incubated with anti-CRT antibody (1:800, CST) in PBS with 3% BSA for 1 hour on ice. After washing with PBS, cells were further incubated with 2 μg/mL goat anti-rabbit Alexa Fluor 488 (Invitrogen) for 45 minutes on ice. Fluorescence signals were analyzed by BD Accuri C6 flow cytometer (BD Biosciences). 
     (G) Semi-Quantitative Reverse Transcription-Polymerase Chain Reaction (RT-PCR) and Quantitative Real-Time PCR (qRT-PCR) and RT 2  qPCR Primer Assay: 
     The total RNA was extracted from MCF-7 AC and derived cells using TRIzol reagent (Invitrogen) according to the manufacturer&#39;s instructions and reverse-transcribed into cDNA using High Capacity cDNA Reverse Transcription Kits (Applied Biosystems). The primer sequences were designed using Primer Blast and listed as Table 3. qRT-PCR amplification was performed using GOTAQ® qPCR Master Mix (PROMEGA) according to the manufacturer&#39;s instructions. The relative mRNA level was normalized to β-actin and the difference in mRNA levels was estimated using the 2 −ΔΔCt  method. RT 2  qPCR Primer Assay was performed using RT 2  PROFILER™ PCR array (Qiagen) containing a set of 84 human cancer stem cell-focus genes. 
     
       
         
           
               
             
               
                 TABLE 3 
               
             
            
               
                   
               
               
                 Primers used for qRT-PCR: 
               
            
           
           
               
               
               
            
               
                 Primer 
                 Sequence 
                 SEQ ID NO 
               
               
                   
               
            
           
           
               
               
               
               
            
               
                 ITGA1 
                 forward 
                 5’-GGTGCTTATTGGTTCTCCGTTAG-3’ 
                 15 
               
               
                   
                 reverse 
                 5’-TTCTCCTTTACTTCTGTGACATTGG-3’ 
                 16 
               
               
                   
               
               
                 ITGA2 
                 forward 
                 5’-GGAACGGGACTTTCGCAT-3’ 
                 17 
               
               
                   
                 reverse 
                 5’-GGTACTTCGGCTTTCTCATCA-3’ 
                 18 
               
               
                   
               
               
                 ITGA10 
                 forward 
                 5’-AGTAAAGGCAGTTGGATTCTCATAGAC-3’ 
                 19 
               
               
                   
                 reverse 
                 5’-GAGCTGCACTCTGGAGACCAAT-3’ 
                 20 
               
               
                   
               
               
                 ITGB1 
                 forward 
                 5’-AATGAATGCCAAATGGGACACGGG-3’ 
                 21 
               
               
                   
                 reverse 
                 5’-TTCAGTGTTGTGGGATTTGCACGG-3’ 
                 22 
               
               
                   
               
               
                 MRP1 
                 forward 
                 5’-ATGTCACGTGGAATACCAGC-3’ 
                 23 
               
               
                   
                 reverse 
                 5’-GAAGACTGAACTCCCTTCCT-3’ 
                 24 
               
               
                   
               
               
                 β-actin 
                 forward 
                 5’-CTCTTCCAGCCTTCCTTCCT-3’ 
                 25 
               
               
                   
                 reverse 
                 5’-AGCACTGTGTTGGCGTACAG-3’ 
                 26 
               
               
                   
               
            
           
         
       
     
     (H) Peptide Synthesis: 
     The peptides were composed of a ubiquitin-like (UBL) domain, SELGFPPAVQRWVIGRCLCVPER, with or without a cell penetrating peptide sequence (TAT), RKKRRQRRR (SEQ ID NO: 1). The DRI-TAT-SP was produced in a reversed sequence using D-amino acids and was protected by acetylation at N-terminus and amidation at C-terminus to make it more resistant to enzymatic degradation. 
     In certain exemplary embodiments, the following peptides are used to prepare the present composition:
         (i) TAT-peptide (SEQ ID NO: 1):   (ii) SHARPIN-derived peptide (SP): SELGFPPAVQRWVIGRCLCVPER (SEQ ID NO: 2);   (iii) TAT-SP peptide: RKKRRQRRRSELGFPPAVQRWVIGRCLCVPER (SEQ ID NO: 4);   (iv) DRI-TAT-SP peptide: Ac-repvc1crgivwrqvappfglesarqrrkkr-NH 2  (SEQ ID NO: 6),       

     where one-letter amino acid code in upper case denotes L-amino acids; one-letter amino acid code in lower case denotes D-amino acids. 
     Other Peptides such as SPScr (SEQ ID NO: 3) and TAT-SPScr (SEQ ID NO: 5) are Used to Synthesize Comparative Compositions in Certain Assays with the Afore-Mentioned Exemplary Compositions, in Particular, to Compare with the Compositions Comprising TAT-SP Peptide (SEQ ID NO: 4) and DRI-TAT-SP Peptide (SEQ ID NO: 6). 
     (I) DOX Accumulation Assay: 
     To assess MRP1 activity, 2×105 MCF-7 AC and derived cells, with or without 24-hour incubation of 1 μM TAT-SP or TAT-SPScr, were treated with 5 μM DOX at 37° C. for 2 hours. After removal of extracellular drugs, cells were divided into two equal halves. One half was lysed with lysis buffer (isopropanol with 0.75 M HCl and 0.2% Triton X-100) at room temperature for 30 minutes; the other half was incubated with 5 μM CMFDA (Invitrogen) in darkness at room temperature for 30 minutes. The fluorescent intensities of drugs and dyes were detected with CLARIOstar (BMG LABTECH) and relative drug concentrations were normalized to CMFDA level. 
     (J) Immunoprecipitation: 
     Calreticulin (CRT) from MCF-7-derived cells was immunoprecipitated by anti-CRT (CST) antibody. In brief, MCF-7-derived cells, with or without 24-hour incubation of 1 μM TAT-SP or TAT-SPScr, were collected and subsequently lysed with RIPA buffer on ice for 30 minutes. The lysate was diluted 5-fold with PBS and incubated with anti-CRT antibody overnight at 4° C. with simultaneous mixing on a rotating platform, followed by further incubation of Protein-G PLUS agarose (Santa Cruz Biotechnology) at 4° C. for at least 2 hours. After incubation, agarose beads were centrifuged and washed by PBS. Immunoprecipitated proteins were collected by treating the beads with 0.2 M glycine and subjected to Western Blotting (WB) analysis. 
     (K) Protein Preparation and Western Blotting: 
     Total protein was extracted with RIPA buffer supplied with protease inhibitor (Roche), resolved in 10% SDS-PAGE and transferred onto Immobilon-P PVDF membrane (Millipore). Membranes were blocked with 5% non-fat dry milk in Tris-buffered saline with 0.1% Tween (TBST) for 2 hours, then were incubated overnight with corresponding antibodies at 4° C., followed by incubation with horseradish peroxidase-conjugated goat anti-mouse IgG (Santa Cruz Biotechnology) or goat anti-rabbit IgG (Santa Cruz Biotechnology) as appropriate, for 2 hours at room temperature in TBST. Antibody binding was detected with Pierce ECL Western Blotting Substrate (Thermo Scientific) and images were visualized with Azure C600 imaging system (Azure Biosystems). The antibodies used were anti-ITGA2 (Abcam), anti-CRT (CST), anti-ILR2 (CST), anti-MRP1/ABCC1 (CST), anti-Mdr-1 (Santa Cruz Biotechnology), anti-Akt (pan) (CST), anti-pAkt (S473) (CST), anti-pAkt (T308) (CST), p53 (Santa Cruz Biotechnology) and anti-β-actin (Santa Cruz Biotechnology). 
     (L) Determination of 50% Inhibitory Concentration (IC 50 ) by MTS Assay: 
     MCF-7 AC and derived cells were seeded onto 96-well plates at a density of 1×10 4  cells per well and treatments of anticancer drugs or peptides were administered accordingly. Following 72-hour incubation at 37° C. with 5% CO 2 , cell viability was assessed using CellTiter 96 AQueous Non-Radioactive Cell Proliferation Assay (Promega). The IC 50  value was estimated from the optical density at 492 nm using GraphPad. 
     (M) Pharmacokinetic Study of SHARPIN-Derived Peptides: 
     SHARPIN-derived peptides powder was dissolved in 1× PBS and administered intravenously at 25 mg/kg to overnight-starved Balb/c mice (n=3). At different time points (5, 15, 30, 60, 120, 240, 420 min), blood was collected via cardiac puncture and placed in a heparinized Eppendorf tube followed by centrifuged at 14,000 rpm for 5 min to obtain blood plasma. Plasma was stored at −80° C. until analysis. Plasma concentration data were analyzed by non-compartmental model. Area under plasma concentration-time curve (AUC), distribution half-life (t 1/2α ) and elimination half-life (t 1/2β ) were calculated by PK solution2.0 (Summit Research Service, Ashland, USA). 
     (N) DRI-TAT-SP Accumulation Assay in Tumors: 
     DRI-TAT-SP 25 mg/kg were administered intravenously to female Balb/c nude mice bearing MCF7 xenograft. At different time points (5, 30, 60, 120 and 240 min), tumors were collected after the sacrifice of the mice. Tumors were weighed and added with 3 volumes of 1× PBS for tissue homogenization. The samples were stored at −80° C. before analysis. 
     (O) UPLC/QQQ-MS Analysis Protocol: 
     Agilent 6460 Ultra Performance Liquid Chromatography-Electrospray Ionization Triple Quadrupole mass spectrometer (UPLC-QQQ-MS) was used in sample separation, peptide detection and quantification. Waters Acquity UPLC BEH Protein C4 column (1.7 μm, 2.1×100 mm) was used for separation. SHARPIN-derived peptides were separated and eluted by a gradient of water (0.1% FA, v/v) and acetonitrile (ACN) (0.1% FA, v/v) at a constant flow rate of 0.3 mL/min. During separation, an elution protocol was used as follows. Equilibration: 0 to 1 min, 5% ACN. Elution gradient: 1 to 10 min, ACN from 5 to 99%. Regeneration: 10 to 11 min, 99% ACN, then 11 to 11.5 min, ACN from 99% to 5%. Re-equilibration: 11.5 to 13 min: 5% acetonitrile. A hundred microlitres of plasma/tumor samples were added with 300 μL acetonitrile (1% formic acid (FA)) for protein and peptide precipitation. Supernatants were discarded after centrifugation at 14,000 rpm for 10 minutes. Then the samples were reconstituted in 50% methanol (0.1% FA, v/v), filtered through a 0.22 μm nylon filter, and kept in the autosampler at 4° C. for analysis. Twenty microliters were injected into the MS. The MS was operated in positive electrospray ionization (ESI+) mode with supply of 300° C. gas temperature and sheath gas temperature, 8 L/min drying gas, and 11 L/min sheath gas flow and 3.5 kV capillary voltage. The signals of TAT-SP and DRI-TAT-SP were recorded at m/z 563 to 661 at collision energy (CE) of 15 eV and m/z 569 to 637 at CE of 20 eV, respectively. TAT-SP and DRI-TAT-SP were eluted at 4.05 and 3.86 minutes, respectively. 
     (P) In Vivo Toxicity Test in Balb/c Mice: 
     Healthy female Balb/c mice (6-8 weeks old) were randomized into 3 groups and were administered with (1) DOX 1.2 mg/kg i.v., (2) DRI-TAT-SP 25 mg/kg i.v, and (3) DOX 1.2 mg/kg+DRI-TAT-SP 25 mg/kg i.v. once every two days (q2d) for 10 injections via tail vein injection. The body weight of the mice was measured once every 2 days. Any toxicity symptoms including body weight change and behavioral changes will be monitored. Body weight drop more than 15% suggested treatment induced toxicity. 
     (Q) In Vivo Efficacy Study of DRI-TAT-SP Combined with DOX in Treating MCF7 Breast Cancer Xenograft Model: 
     Six- to eight-week-old female Balb/c nude mice were maintained under specific pathogen-free conditions prior to in vivo efficacy study. Each animal was implanted with a piece of 17β-estradiol pellet 0.72 mg, 60-day release. (Cat No. SE-121, Innovative Research of America, Sarasota, Fla.). Four days later, the nude mice were inserted at the both flanks with a sesame sized (around 10 mm 3 ) MCF-7 tumor piece originated from a tumor bulk. When the tumor reached approximately 100 mm 3  (21-28 days), the mice were randomized and administered intravenously with (1) 1× PBS, (2) DOX 1.2 mg/kg, (3) DRI-TAT-SP 25 mg/kg or (4) DOX 1.2 mg/kg+DRI-TAT-SP 25 mg/kg once every 2 days for a total of 10 injections. Tumor volume was measured using digital caliper once every 4 days. Estimated tumor volume (mm 3 ) was calculated as ½×length(mm)×width(mm) 2 . The mouse body weights were recorded once every two days. The tumor doubling time (Td) was calculated as t×log 2/log(V f /V 0 ), where t, V f  and V 0  stand for interval time, percentage change of tumor volume on final day and percentage change of tumor volume on initial day. 
     Flow cytometry data were analyzed with BD Accuri C6 software (BD Biosciences). Densitometric analysis was carried out by ImageJ software. Statistical analysis was performed using GraphPad Prism 5 and SPSS. Student t-test was employed to determine significant differences. 
     Although the invention has been described in terms of certain embodiments, other embodiments apparent to those of ordinary skill in the art are also within the scope of this invention. Accordingly, the scope of the invention is intended to be defined only by the claims which follow. 
     INDUSTRIAL APPLICABILITY 
     The proposed disruption of ITGA2/CRT interaction by two SHARPIN-derived peptides, TAT-SP and DRI-TAT-SP, is a new therapeutic target to reverse chemoresistance in breast CSCs ( FIG.  9   ). The proven DRI-TAT-SP efficacies in DOX sensitization in breast cancer cells of in vivo human breast cancer xenograft suggest a novel peptide therapeutic candidate to treat breast cancers. Despite body weight drop observed, the data in the present disclosure supports that DRI-TAT-SP is safe for in vivo application and provides a synergistic effect when being administered in combination with DOX, which in turn lowers the dosage of DOX, ameliorating detrimental side effects from continuous and high dose of DOX administration.