CN112168820B - Application of SRCAP ATPase inhibitor in colorectal cancer treatment - Google Patents

Abstract

The invention provides the use of a reagent for detecting the expression level of SRCAP ATPase enzyme activity or a downstream transcription factor regulated by the SRCAP ATPase enzyme activity in the preparation of a diagnostic agent/kit for diagnosing or prognosing colorectal cancer diseases in a mammal. The invention also provides the use of an inhibitor/antagonist of SRCAP ATPase enzyme activity or a regulated downstream transcription factor thereof in the manufacture of a medicament/kit for the prevention or treatment of colorectal cancer disease in a mammal.

Description

Application of SRCAP ATPase inhibitors in the treatment of colorectal cancer

Technical field

The present invention provides the use of SRCAP ATPase inhibitors to inhibit colorectal cancer stem cells.

Background technique

Intestinal epithelial cells cover the entire surface of the intestine and perform many important functions in the body such as nutrient absorption, mucosal barrier, secretion and immune regulation, maintaining the body's dynamic balance. The intestinal epithelium consists of rapidly proliferating intestinal crypts and differentiated villi, which constitute the basic tissue structure. Crypts composed of intestinal stem cells (ISCs), Paneth cells and proliferating cells are distributed in the intestinal epithelium. It also includes absorptive epithelial cells, cup cells, endocrine cells and Tuft cells. The self-renewal and pluripotent differentiation of ISCs maintain intestinal epithelial homeostasis under physiological conditions. Pathological conditions can lead to severe disorders of epithelial function. ISCs are located at the bottom of intestinal crypts and are in close contact with Paneth cells that serve as the adjacent microenvironment. It is now known that the microenvironment can provide Wnt/β-catenin, Notch, BMP, ErbB and Hedgehog signals to support the self-renewal and differentiation of ISCs. Lgr5 ⁺ cells at the bottom of the crypt are activated ISCs, and their activity is mainly regulated by signals such as Wnt to maintain intestinal homeostasis under physiological conditions.

Colorectal cancer is a common tumor in Western countries. With economic and social development and lifestyle changes, new cases and deaths in my country each year account for 20% of colorectal cancer cases in the world during the same period. Colorectal cancer is the most common malignant tumor of the digestive tract in Beijing. The incidence rate increased from 27.42/100,000 to 39.93/100,000 in 10 years. The existence of cancer stem cells has been confirmed in leukemia and various solid tumors such as breast cancer and colon cancer. Cancer stem cells have the ability of self-renewal and multi-directional differentiation. They account for a very small proportion of tumor cells, but they are closely related to tumor recurrence, metastasis, drug resistance and treatment failure. The origin of intestinal cancer stem cells is still a focus of debate in the field. Some studies believe that mutations in normal stem cells can lead to abnormal structure and function of intestinal epithelial tissue and instability of the internal environment, thereby having the potential to develop colon cancer. Lgr5 is a G protein-coupled receptor family protein. It can be used as an intestinal stem cell marker to participate in cell differentiation, and is also an important intestinal cancer stem cell marker. At the same time, colon cancer stem cells involve Wnt signaling, PI3K/AKT signaling, and the receptor tyrosine kinase Ras-MAPK pathway. These pathways are also related to the proliferation and differentiation of normal stem cells.

The chromatin remodeling complex is a type of DNA-dependent helicase-like protein complex with ATPase activity and is an important component of epigenetic regulation. It plays an important regulatory role in stem cell self-renewal, cell fate determination and reprogramming. Chromatin remodeling complexes mainly include four protein families, including SWI/SNF, CHD, ISWI and INO80. The SRCAP complex, composed of 11 subunits including chromatin remodeling enzymes SRCAP, Ruvbl1, YL-1, and Arp6, also belongs to the INO80 family. Its chromatin remodeling activity mainly catalyzes the replacement of classic histones by histone variant H2A.Z. H2A forms nucleosomes and regulates gene transcription. SRCAP gene mutations cause the genetic disease Floating-Harbor syndrome. The full-length protein consists of HSA, ATPase, CBP binding, SANT and other domains. The histone variant H2A.Z regulated by SRCAP acts on Oct4/MLL/PRC2 to regulate the expression of stemness genes during the self-renewal process of embryonic stem cells, and participates in the regulation of key genes such as muscle cell differentiation and hematopoietic stem cell differentiation during adult development. expression. The role of regulation of SRCAP complex activity in Lgr5 ⁺ intestinal cancer stem cells has not yet been determined.

Amtizole, Chinese name: 1,2,4-Thiadiazole-3,5-diamine, CAS#:34283-30-2, English chemical name: 1,2,4-Thiadiazole-3,5-diamine, molecular formula: C ₂ H ₄ N ₄ S. Amtizole is a known antioxidant and Na+/K+ATPase inhibitor. The inhibitory effect of this compound on other ATPase activities and its regulatory effect on the self-renewal of Lgr5 ⁺ intestinal cancer stem cells and colorectal cancer have not been reported.

Contents of the invention

The present invention attempts to explore the relationship between the self-renewal of Lgr5 ⁺ intestinal cancer stem cells and SRCAP ATPase activity, and attempts to explore the therapeutic effect of the small molecule compound Amtizole and each of its possible active modification products on colorectal cancer, thereby providing information for disease treatment. More convenient method. Although great progress has been made in understanding the mechanisms of Lgr5 ⁺ intestinal cancer stem cell self-renewal and colorectal cancer, there is still a need to identify specific pathways and proteins that activate Lgr5 ⁺ intestinal cancer stem cell self-renewal as druggable targets to provide Provides new avenues for treatment of colorectal cancer mediated by it. The present invention fulfills this and related needs.

Accordingly, the present invention provides the following:

1. Application of inhibitors/antagonists of SRCAP ATPase enzyme activity or downstream transcription factors regulated by it in the preparation of drugs/kits for preventing or treating colorectal cancer in mammals.

2. The application according to 1 above, wherein the downstream transcription factor is a PPARδ molecule.

3. The application according to 1 or 2 above, wherein the inhibitor/antagonist is a monoclonal/polyclonal antibody, antisense RNA or small molecule compound inhibitor.

4. The application according to any one of 1-3 above, wherein the inhibitor/antagonist is Amtizole or a precursor or derivative thereof, wherein the precursor or derivative retains the SRCAP ATPase activity of Amtizole. Inhibitor/antagonist activity.

5. The application according to the above 4, wherein the Amtizole or its precursor or derivative has the structure of the following formula (I),

in:

Optional modifying groups on formula (I) are independently selected from: C ₁ -C ₁₂ alkyl (such as C ₁ -C ₆ alkyl), C ₁ -C ₆ alkylene, C ₂ -C ₁₂ alkenyl (such as C ₃ -C ₆ alkenyl), C ₃ -C ₁₀ cycloalkyl, phenyl, substituted phenyl, optionally substituted five- or six-membered heteroaryl, or C ₆ -C ₁₀ aralkyl, Halogen, -OH, -OR ^a , C ₄ -C ₁₂ dienyl, C ₆ -C ₁₂ trienyl, C ₈ -C ₁₂ tetraenyl, C ₆ -C ₁₀ aryl (optionally independently One, two or three substituents selected from C ₁ -C ₆ alkyl, halogen, C ₁ -C ₆ alkoxy, or hydroxyl), C ₁ -C ₁₀ alkoxy, carboxyl, cyano, C ₁ -C ₁₀ alkanoyloxy, C ₁ -C ₁₀ alkylthio, C ₁ -C ₁₀ alkylsulfonyl, C ₁ -C ₁₀ alkoxycarbonyl, C ₁ -C ₁₀ alkanoyl amino, -SR ^b , -SO ₂ R ^c , -NHSO ₂ R ^d and -NHCO ₂ ^Re ; wherein R ^a , R ^b , R ^c , R ^d , and ^Re are independently selected from C ₁ -C ₆ alkyl, C ₁ -C ₆ alkyl, C ₂ -C ₆ alkenyl, C ₃ substituted with _one to three groups of C 6 -C 10 aryl, C ₁ -C ₆ alkoxy _, halogen, and hydroxyheteroaryl -C ₁₀ cycloalkyl, phenyl, or naphthyl, the hydroxyheteroaryl containing one to five heteroatoms independently selected from sulfur, nitrogen, and oxygen.

6. The application according to any one of 1-5 above, wherein the mammal is a human, a mouse or a domestic animal, such as a cat, a dog, a pig, a cow, a sheep, a goat, a horse and a rabbit.

7. Use according to any one of 1-6 above, wherein the colorectal cancer disease is colon cancer or rectal cancer.

8. Application of reagents for detecting SRCAP ATPase enzyme activity or the expression level of downstream transcription factors regulated by it in the preparation of diagnostic agents/kits for diagnosing or prognosticating colorectal cancer in mammals.

9. The application according to 8 above, wherein the mammal is a human, a mouse or a domestic animal, such as a cat, a dog, a pig, a cow, a sheep, a goat, a horse and a rabbit.

10. Use according to 8 or 9 above, wherein the colorectal cancer disease is colon cancer or rectal cancer.

Description of the drawings

The results shown from A to K in Figure 1 demonstrate that SRCAP can promote colorectal carcinogenesis and intestinal cancer stem cell self-renewal. Specifically, as shown in Figure 1, A, it was observed on the human colorectal cancer tissue chip that SRCAP is lowly expressed in human normal colorectal epithelial cells, but highly expressed in colorectal cancer tumors, and the expression continues to increase with the progression of the tumor. Figure 1, B shows the expression statistics of SRCAP in different stages and grades of colorectal cancer. Figure 1, C shows the analysis of SRCAP expression and survival, showing that patients with high SRCAP expression of colorectal cancer have a poor prognosis. Figure 1, D shows that on SRCAP knockdown HCT116, the ability of tumor stem cells to form spheres in vitro is inhibited. Figure 1, E shows that SRCAP knockdown HCT116 cells significantly reduced the subcutaneous tumor-forming ability in vivo. Figure 1, F shows the strategy for constructing Srcap-specific gene knockout mice in intestinal Lgr5 ⁺ stem cells. Figure 1, G shows the PCR identification results of the genotype of Srcap-specific gene knockout mice in intestinal Lgr5 ⁺ stem cells. Figure 1, H shows the application of anti-SRCAP protein antibody in Western blotting experiments to prove that SRCAP protein was specifically knocked out in mouse intestinal Lgr5 ⁺ stem cells. Figure 1. I shows the tissue-specific changes in the intestine in the mouse colorectal cancer model induced by dextran sulfate sodium (DSS)/chemical mutagen (azoxymethane, AOM). The number of tumors formed in sexual Srcap knockout mice was significantly reduced. Figure 1, J shows the improved survival rate of colorectal cancer induced by Srcap knockout mice. Figure 1, K shows that the number of tumors formed in mice with tissue-specific knockout of Srcap in the intestine was significantly reduced.

The results shown from A to J in Figure 2 demonstrate that the SRCAP ATPase inhibitor Amtizole has an inhibitory effect on colorectal cancer caused by the self-renewal of intestinal cancer stem cells. Specifically, Figure 2, A shows that the small molecule compound Amtizole significantly inhibits SRCAP ATPase activity in the in vitro SRCAP ATPase enzyme activity detection system. Figure 2, B shows the proportion of CCO that inhibits the formation of organoids from intestinal cancer stem cells in the culture system. Figure 2, C shows that Amtizole has a significant inhibitory effect on spontaneous colorectal cancer in Apc ^min mice. Figure 2, D shows that Amtizole alleviates the weight loss of mice. Figure 2, E shows that Amtizole reduces mouse mortality. Figure 2, F shows the reduction in the number of colorectal cancer tumors in mice treated with Amtizole. Figure 2, G shows the reduction in colorectal cancer tumor volume in mice treated with Amtizole. Figure 2, H shows the staining of pathological sections showing a reduction in the number of colorectal cancer tumors in mice treated with Amtizole. Figure 2, I shows cells derived from clinical patient tumor samples injected subcutaneously into NSG mice to induce a PDC model. It was found that the use of Amtizole in tumors can also effectively inhibit tumor growth. Figure 2, J shows further immunohistochemical staining analysis of PDC tumor specimens. It was found that Amtizole inhibited the expression of the proliferation-related marker molecule PCNA and the signaling pathway molecule PPARδ.

Detailed ways

The inventor of the present invention found in the related research on the self-renewal of Lgr5 ⁺ intestinal cancer stem cells and the occurrence of colorectal cancer tumors: SRCAP is lowly expressed in normal human colorectal epithelial cells, but highly expressed in colorectal cancer tumors, and it evolves with the tumor. Process expression continues to rise. Colorectal cancer patients with high SRCAP expression have poor prognosis. The ability of SRCAP knockdown HCT116 cells to form spheres in vitro and subcutaneous tumor formation in vivo was significantly reduced. In a mouse colorectal cancer model induced by dextran sulfate sodium (DSS)/chemical mutagen azomethane (AOM), tissue-specific knockout of Srcap in the intestine formed The number of tumors was significantly reduced and the survival rate of the mice was improved. The small molecule compound Amtizole significantly inhibits SRCAP ATPase activity in the in vitro SRCAPATPase enzyme activity detection system. Inhibit the proportion of intestinal cancer stem cells forming organoids CCO in the culture system. Amtizole also has a significant inhibitory effect on spontaneous colorectal cancer in Apc ^min mice. Effectively alleviate the weight loss of mice and reduce mouse mortality. The number and size of tumors decrease. Cells derived from clinical patient tumor samples were injected subcutaneously into NSG mice to induce a PDC model, and it was found that intratumoral use of Amtizole can also effectively inhibit tumor growth. Further immunohistochemical staining analysis of PDC tumor specimens found that Amtizole inhibited the expression of the proliferation-related marker molecule PCNA and the signaling pathway molecule PPARδ.

The first aspect of the present invention provides the role of SRCAP in promoting the evolution of intestinal cancer and the self-renewal of intestinal cancer stem cells. It is characterized in that SRCAP is highly expressed in late-stage colorectal cancer specimens and is related to prognosis, and SRCAP can promote the occurrence of colorectal cancer. and intestinal cancer stem cell self-renewal.

In some embodiments of the present invention, the expression is low in normal human colorectal epithelial cells, but highly expressed in colorectal cancer tumors, and the expression continues to increase with tumor progression. Colorectal cancer patients with high SRCAP expression have poor prognosis.

In some embodiments of the invention, the ability of SRCAP knockdown HCT116 cells to form spheroids in vitro and subcutaneous tumor formation in vivo is significantly reduced.

In other embodiments of the invention, in a mouse colorectal cancer model induced by dextran sulfate sodium (DSS)/chemical mutagen (azoxymethane, AOM), Srcap The number of tumors formed in mice with tissue-specific knockout of tract was significantly reduced and the survival rate of mice was improved.

A second aspect of the invention provides a method for inhibiting SRCAP ATPase in intestinal cancer stem cells, the method comprising contacting the cells with an effective amount of Amtizole, an inhibitor of SRCAP ATPase, and each of its possible active modification products. touch. and provides a method for treating colorectal cancer caused by intestinal cancer stem cell self-renewal in a patient in need thereof, the method comprising administering to the patient a therapeutically effective amount of an inhibitor of SRCAP ATPase, Amtizole, and its possible An active modification product.

In some embodiments of the present invention, the small molecule compound Amtizole was verified to inhibit SRCAP ATPase in vitro and inhibit the formation of intestinal cancer CCO organoids by establishing an in vitro enzymatic activity screening system for SRCAP ATPase.

In some embodiments of the invention, Amtizole inhibits spontaneous colorectal cancer in Apc ^min mice. It can effectively alleviate the weight loss of mice and reduce mouse mortality. Reduce the number and size of tumors.

In other embodiments of the present invention, intratumoral injection of Amtizole inhibits the growth of intestinal cancer PDC model tumors and inhibits the expression of the proliferation-related marker molecule PCNA and the signaling pathway molecule PPARδ.

In a third aspect, the method of any of the above first and second aspects is that the inhibitor of SRCAP ATPase is a compound of formula (I) and each of its possible active modification products:

In a fourth aspect, the present method relates to compounds that inhibit each of the SRCAP ATPase enzymes or pharmaceutically acceptable salts thereof for inhibiting intestinal cancer stem cell self-renewal.

In a fifth aspect, the method relates to compounds that inhibit each of the SRCAP ATPase enzymes or pharmaceutically acceptable salts thereof for the treatment of colorectal cancer caused by self-renewal of intestinal cancer stem cells.

In any of the preceding aspects, the inhibitor of each of the SRCAP ATPase enzymes may be the same compound or a different compound.

In the present invention, the term "SRCAP (NCBI database gene ID: 10847)" refers to SRCAP with the original sequence shown in the SRCAP gene in the current international shared nucleic acid database GeneBank, which includes natural or synthetic SRCAP and its analogs. SRCAP analogues refer to derivatives or variants that are still biologically active after substitution, deletion or addition of one or several nucleotides, or biochemical modification.

In the present invention, the term "PCNA (NCBI database gene ID: 5111)" refers to PCNA with the original sequence shown in the PCNA gene in the current international shared nucleic acid database GeneBank, which includes natural or synthetic PCNA and its analogs. PCNA analogues refer to derivatives or variants that are still biologically active after substitution, deletion or addition of one or several nucleotides, or biochemical modification.

In the present invention, the term "PPARD (NCBI database gene ID: 5467)" refers to PPARD with the original sequence shown in the PPARD gene in the current international shared nucleic acid database GeneBank, which includes natural or synthetic PPARD and its analogs. PPARD analogues refer to derivatives or variants that are still biologically active after being substituted, deleted or added with one or several nucleotides, or biochemically modified.

The effective dose of Amtizole of the present invention can be adjusted accordingly according to the mode of administration and the severity of the disease to be treated. The preferred effective amount can be determined by those of ordinary skill in the art considering various factors. The factors include but are not limited to: pharmacokinetic parameters of Amtizole or its analogues, health status, body weight, route of administration, etc. of the patient being treated.

The following examples are used to illustrate the invention but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.

The following are the materials and general methods used in the embodiments of the present invention:

Antibodies and reagents

Anti-mouse and human SRCAP antibodies (ESL104) were purchased from Kerafast (Boston, USA). Anti-PPAR-δ (10156-2-AP) antibodies were purchased from Proteintech (Chicago, USA). Anti-mouse PCNA (ab29) antibodies were purchased from Abcam (Cambridge, USA). ATPase/GTPase activity assay kit, EZ prep nucleic acid isolation kit were purchased from Sigma-Aldrich (St. Louis, USA). Matrigel (356234) w was purchased from Corning (Corning, USA).

Statistical Analysis

Unpaired Student's t-test was used as statistical analysis in this invention. Statistical calculations were performed using Microsoft Excel or SPSS13. When P<0.05, the P value is significant.

Example

Example 1: Prove that SRCAP can promote colorectal carcinogenesis and intestinal cancer stem cell self-renewal.

Low expression of SRCAP in human normal colorectal epithelial cells was observed on the human colorectal cancer tissue chip (purchased from Shanghai Xinchao Biotechnology Co., Ltd., colon cancer 180-point survival tissue chip, product number HColA180Su15), while in colorectal cancer It is highly expressed in tumors and continues to increase as the tumor evolves, as shown in Figure 1, A. The expression statistics of SRCAP in different stages and grades of colorectal cancer are shown in Figure 1, B. Analysis of SRCAP expression and survival showed that patients with colorectal cancer with high SRCAP expression had a poor prognosis, as shown in Figure 1, C. On the SRCAP knockdown human colon cancer cell line HCT116 (HCT116 cells were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences, catalog number TCHu 99. The SRCAP knockdown cell line was screened by the laboratory itself, shSRCAP:5'-GCTTTCCATGGAGGAGCTATT-3'), The ability of tumor stem cells to form spheres in vitro was inhibited, as shown in Figure 1, D. The subcutaneous tumor-forming ability of SRCAP knockdown HCT116 cells in vivo was significantly reduced, as shown in Figure 1, E. Srcap conditional knockout mice were constructed through the CRISPR-Cas9 method, and Lgr5-Cre mice (B6.129P2-Lgr5 ^{tm1(cre /ERT2)Cle} /J, which specifically expresses intestinal stem cells) were purchased from Jackson Laboratory, USA, Stock No.008875) to obtain knockout mice (Srcap ^fl/fl ; Lgr5-Cre) and control mice (Srcap ^fl/fl ) that specifically knock out the Srcap gene in mouse intestinal stem cells, as shown in Figure 1, F shown. The results of genotype PCR identification of Srcap-specific gene knockout mice in intestinal Lgr5 ⁺ stem cells are shown in Figure 1, G. SRCAP protein was specifically knocked out in mouse intestinal Lgr5 ⁺ stem cells, as shown in Figure 1, H. In the mouse colorectal cancer model induced by dextran sulfate sodium (Dextran Sulfate, sodium, DSS)/chemical mutagen (azoxymethane, AOM) (see reference [1] for model construction methods), in the intestine The number of tumors formed in the tissue-specific Srcap knockout mice of the tract (see Figures 1F-1H for specific knockout methods and experimental verification results) was significantly reduced, as shown in Figure 1, I. The survival rate of colorectal cancer induced by Srcap knockout mice is improved, as shown in Figure 1, J. The number of tumors formed in mice with tissue-specific knockout of Srcap in the intestine was significantly reduced. As shown in Figure 1, K.

Example 2: Prove that the SRCAP ATPase inhibitor Amtizole has an inhibitory effect on colorectal cancer caused by the self-renewal of intestinal cancer stem cells.

The small molecule compound Amtizole significantly inhibited SRCAP ATPase activity in the in vitro SRCAP ATPase enzyme activity detection system (reference [2]), as shown in Figure 2, A (colorimetric reading at OD ₆₂₀ , using IgG as a negative control, positive The difference between the reading value and IgG is used as the relative enzyme activity value, DMSO=0.38±0.04, Amitizole=0.09±0.02). The ratio of inhibiting the formation of CCO (colorectal cancer organoid, human colorectal cancer organoid) organoids from intestinal cancer stem cells in the culture system is shown in Figure 2, B. Amtizole (1.5 mg/kg body weight) has a significant inhibitory effect on spontaneous colorectal cancer in Apc ^min mice (see [1] for references on animal models/methods), as shown in Figure 2, C. Amtizole (1.5 mg/kg body weight) alleviated the weight loss of mice, as shown in Figure 2, D. Amtizole (1.5 mg/kg body weight) reduced mouse mortality, as shown in Figure 2, E. The number of colorectal cancer tumors in mice in the Amtizole-treated group (1.5 mg/kg body weight) was reduced, as shown in Figure 2, F. The colorectal cancer tumor volume of mice in the Amtizole-treated group (1.5 mg/kg body weight) decreased, as shown in Figure 2, G. Pathological section staining showed that the number of colorectal cancer tumors in the Amtizole-treated mice was reduced, as shown in Figure 2, H. Cells derived from clinical patient tumor samples (ascending colon cancer surgical samples were obtained from the 301 Hospital of the People's Liberation Army) were injected into NSG (NOD-Prkdcscid Il2rgtm1/Bcgen, purchased from Biocytogen) to subcutaneously induce PDC (patient-derived cells) models in mice (animals For references of models/methods, see [1]). It was found that intratumoral use of Amtizole (10 μg intratumoral injection, once every 3 days) can also effectively inhibit tumor growth, as shown in Figure 2, I. Further immunohistochemical staining analysis of PDC tumor specimens showed that compared with control DMSO, Amtizole inhibited the expression of the proliferation-related marker molecule PCNA (Proliferating Cell Nuclear Antigen, Proliferating Cell Nuclear Antigen) and the signaling pathway molecule PPARδ, as shown in Figure 2 , shown in J.

Those skilled in the art should understand that although the present invention has been specifically described with reference to the above embodiments, the present invention is not limited to these specific embodiments. Based on the methods and technical solutions taught in the present invention, those skilled in the art can make appropriate modifications or improvements without departing from the spirit of the present invention, and equivalent embodiments obtained thereby are within the scope of the present invention. references

1. Zhu, P. et al. LncGata6maintains stemness of intestinal stem cells and promotes intestinal tumorigenesis. Nat Cell Biol 20, 1134-1144 (2018).

2. Ye, B. et al. Suppression of SRCAP chromatin remodelling complex and restriction of lymphoid lineage commitment by Pcid2. Nat Commun 8, 017-01788 (2017).

Claims (4)

1. The use of Amtizole in the preparation of drugs or kits for preventing or treating colorectal cancer in mammals, the Amtizole having the structure of the following formula (I) 2. Use according to claim 1, wherein the mammal is a human, a mouse or a domestic animal. 3. Use according to claim 1, wherein the mammal is a cat, dog, pig, cow, sheep, goat, horse or rabbit. 4. Use according to any one of claims 1-3, wherein the colorectal cancer disease is colon cancer or rectal cancer.