A predictive biomarker for human colorectal cancer or human colorectal cancer metastasis and its application

Abstract

The present invention discloses the application of cytochrome c oxidase subunit 4 isoform 2 (COX4I2) in the preparation of a predictive biomarker for the prognosis and immunotherapy responsiveness of human colorectal cancer patients. The present invention evaluated the correlation between COX4I2 levels and clinical characteristics and their biological effects in vitro and in vivo, and found that elevated COX4I2 levels were associated with poor prognosis in colorectal cancer patients. Experimental studies also observed that COX4I2 could participate in the epithelial-mesenchymal transition (EMT) of colorectal cancer cells, promote the expression of fibroblast growth factor 1 (FGF1), activate and recruit tumor-associated fibroblasts, and promote tumor angiogenesis. Therefore, COX4I2 can be used as a biomarker for the prognosis of colorectal cancer patients and may also be a new target for therapeutic development.

Description

Technical Field

[0001] The present invention relates to a cytochrome C oxidase and a predictive biomarker COX4I2 for the prognosis of human colorectal cancer, belonging to the technical field of biomedicine. Background Art

[0002] Colorectal cancer (CRC) is a common digestive tract tumor, ranking third in incidence among malignant tumors and second in lethality among cancers. Its pathogenesis is very complex, involving numerous susceptibility factors and related to genetics, diet, and lifestyle habits. Statistics show that 30%-50% of CRC tumors recur and metastasize distantly after surgery, especially to the liver, lungs, bones, and ovaries, and the five-year survival rate often does not exceed 10%.

[0003] The growth and metastasis of tumors require the nourishment of blood vessels, and the neovascularization of tumors is a characteristic of the tumor microenvironment. The hypoxic microenvironment disrupts the balance of vascular-related factors, activates endothelial cells, increases vascular permeability, promotes the delivery of nutrients to cancer cells, enhances the migration and proliferation of cancer cells, and leads to tumor development. In recent years, anti-angiogenic therapies, such as bevacizumab and regorafenib, have been widely used in CRC. However, the formation of tumor-related blood vessels involves the action of multiple genes and signaling pathways, and its regulatory mechanism is extensive and complex. Relatively speaking, only a few patients can benefit from anti-angiogenic therapy. Therefore, it is particularly important to screen suitable target populations and prognostic markers related to anti-angiogenic therapy.

[0004] Cytochrome C oxidase (COX) is the last enzyme in the respiratory chain, responsible for catalyzing the transfer of electrons and oxygen (O2). The catalytic subunits encoded by mitochondrial COX genes are combined with the structural subunits encoded by nuclear genes. Subunit 4 is the largest of them, participating in the mitochondrial respiratory chain and optimizing respiratory efficiency under different oxygen concentrations. Therefore, mammalian cells respond to the hypoxic environment by changing the composition of COX subunits. The levels of both COX4I1 and COX4I2 are regulated by oxygen concentration: at normal concentration, the level of COX4I1 rises while the level of COX4I2 drops. Under hypoxic conditions, hypoxia-inducible factor (HIF) induces the expression of COX4I2, thereby increasing the level of COX4I2, changing the mitochondrial membrane potential, and increasing the levels of ATP and reactive oxygen species (ROS). The level of ROS is related to angiogenesis, including stimulating the growth and migration of endothelial cells and regulating the levels of vascular endothelial growth factor (VEGF), its receptor, nuclear factor κB (NF-κB), mitogen-activated protein kinase (MAPK), and matrix metalloproteinases (MMPs). It is well known that hypoxia is related to tumor progression and poor prognosis. However, the role of COX4I2 upregulated under hypoxic conditions in tumor progression is not yet clear. Summary of the Invention

[0005] Object of the Invention: The technical problem to be solved by the present invention is to overcome the low accuracy of biomarkers for predicting the prognosis of colorectal cancer patients and the insufficient ability to predict the metastasis of colorectal cancer patients in existing clinical diagnoses. Through in-depth research on COX4I2, it is shown that COX4I2 is involved in regulating the epithelial-mesenchymal transition (EMT) of colorectal cancer cells, and COX4I2 can promote the recruitment and angiogenesis of vascular endothelial cells and the recruitment and activation of tumor-associated fibroblasts by increasing the expression level of fibroblast growth factor 1 (FGF1).

[0006] Through a large number of transcriptome data screening and clinical specimen verification, the present invention shows that COX4I2 can be used as a predictive biomarker for human colorectal cancer metastasis or as a component of a kit for predicting or screening human colorectal cancer or human colorectal cancer metastasis.

[0007] Technical Solution: To achieve the above object, the technical solution adopted by the present invention is as follows:

[0008] A predictive biomarker for human colorectal cancer or human colorectal cancer metastasis, which belongs to the cytochrome C oxidase family Gene Cards Gene Symbol: COX4I2.

[0009] A predictive biomarker for the immunotherapeutic response of human colorectal cancer, which belongs to the cytochrome C oxidase family Gene Cards Gene Symbol: COX4I2.

[0010] A kit for predicting or screening the prognosis of human colorectal cancer or immunotherapy, which comprises COX4I2 protein.

[0011] The present invention provides a kit for predicting or screening human colorectal cancer metastasis, and the amino acid sequence of the COX4I2 protein is as follows:

[0012] MLPRAAWSLVLRKGGGGRRGMHSSEGTTRGGGKMSPYTNCYAQRYYPMPEEPFCTELNAEEQALKEKEKGSWTQLTHAEKVALYRLQFNETFAEMNRRSNEWKTVMGCVFFFIGFAALVIWWQRVYVFPPKPITLTDERKAQQLQRMLDMKVNPVQGLASRWDYEKKQWKK. That is:

[0013] Met-Leu-Pro-Arg-Ala-Ala-Trp-Ser-Leu-Val-Leu-Arg-Lys-Gly-Gly-Gly-Gly-Arg-Arg-Gly-Met-His-Ser-Ser-Glu-Gly-Thr-Thr-Arg-Gly-Gly-Gly-Lys-Met-Ser-Pro-Tyr-Thr-Asn-Cys-Tyr-Ala-Gln-Arg-Tyr-Tyr-Pro-Met-Pro-Glu-Glu-Pro-Phe-Cys-Thr-Glu-Leu-Asn-Ala-Glu-Glu-Gln-Ala-Leu-Lys-Glu-Lys-Glu-Lys-Gly-Ser-Trp-Thr-Gln-Leu-Thr-His-Ala-Glu-Lys-Val-Ala-Leu-Tyr-Arg-Leu-Gln-Phe-Asn-Glu-Thr-Phe-Ala-Glu-Met-Asn-Arg-Arg-Ser-Asn-Glu-Trp-Lys-Thr-Val-Met-Gly-Cys-Val-Phe-Phe-Phe-Ile-Gly-Phe-Ala-Ala-Leu-Val-Ile-Trp-Trp-Gln-Arg-Val-Tyr-Val-Phe-Pro-Pro-Lys-Pro-Ile-Thr-Leu-Thr-D-Glu-Arg-Lys-Ala-Gln-Gln-Leu-Gln-Arg-Met-Leu-D-Met-Lys-Val-Asn-Pro-Val-Gln-Gly-Leu-Ala-Ser-Arg-Trp-D-Tyr-Glu-Lys-Lys-Gln-Trp-Lys-Lys。

[0014] Beneficial effects:

[0015] Through a large number of experimental screenings and in-depth studies on the COX4I2 protein, the present invention shows that the activation of COX4I2 can significantly induce the expression of fibroblast growth factor 1 (FGF1), thereby promoting tumor-related angiogenesis and the activation of cancer-associated fibroblasts (CAFs), and promoting CRC progression. Therefore, the COX4I2 protein can be used as a marker for predicting rectal cancer prognosis or screening for immunotherapy, and can also be used as a therapeutic target, with important application value. Description of the drawings

[0016] Figure 1For the LASSO and Cox regression models. In the figure, (A) shows the fitting of the LASSO (Least Absolute Shrinkage and Selection Operator) Cox model. Each curve represents a gene. The curve of the coefficient against log(λ) is plotted. The vertical line indicates the positions of 7 genes with coefficients greater than 0 determined by 10-fold cross-validation. (B) λ is determined by 10-fold cross-validation. The x-axis represents log(λ); the y-axis represents the binomial deviance. The optimal value calculated by the minimum criterion and one standard error of the criterion is indicated by the vertical dashed line. The univariate forest plot (C) and the multivariate forest plot (D) show the associations between COX expression and clinical features related to OS in CRC. The univariate forest plot (E) and the multivariate forest plot (F) show the associations between COX expression and clinical features related to DSS in CRC. The univariate forest plot (G) and the multivariate forest plot (H) show the associations between COX expression and clinical features related to PFI in CRC.

[0017] Figure 2 The figure shows the results that overexpression of COX4I2 promoted the malignant development of CRC. In the figure, (A) analyzes the IHC results and COX4I2 levels in CRC and control tissues (n = 30). (B) shows the COX4I2 levels in tumors (T) and paired normal tissues (N) by Western blotting (n = 30). (NS: not significant, *P < 0.05, **P < 0.01, and ***P < 0.001). (C) shows the COX4I2 protein levels in normal human colon epithelial cells and CRC cell lines. (D) shows the transfection efficiency (%) by GFP expression and Western blotting. (E) shows the colony formation of CRC cells transfected with NC, sh-COX4I2, and oe-COX4I2 constructs. (F) shows mouse xenograft tumors (6 mice per group). (G) measures the volume of xenograft tumors twice a week. (H) shows the weight of xenograft tumors at the end of the study. (I-J) shows the correlation between the IHC staining intensities of COX4I2 and Ki-67 in xenograft tumors (n = 24) and CRC tissues (n = 30). Measurements are taken from different samples. (K) shows the FGF1 protein level in the culture supernatant of CRC cells 48 hours after transfection, measured by enzyme-linked immunosorbent assay (ELISA). (L) shows the FGF1 level in mouse serum, measured by ELISA. Measurements are taken from different samples (n = 6). (M) shows the IHC staining of FGF1 protein in mouse xenograft tumor tissues. (N) shows the COX4I2 and FGF1 protein levels in CRC and normal tissues.

[0018] (O) Correlation between COX4I2 and FGF1 in CRC. Statistical analysis was performed by χ2 test. R, Pearson correlation coefficient. Data are expressed as mean ± SEM, *p < 0.05; **p < 0.01; ***p < 0.001.

[0019] Figure 3 For the results that overexpression of COX4I2 promoted the invasion and metastasis of CRC cells by affecting epithelial-mesenchymal transition (EMT), in the figure, (A) cell invasion (Transwell assay). (B) Cell migration (scratch assay). (C) Correlation between COX4I2 and EMT-related genes. (D) EMT-related proteins were shown by western blotting. Data are expressed as mean ± SEM, *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

[0020] Figure 4 For the results that overexpression of COX4I2 promoted angiogenesis of CRC and activation of CAF. In the figure, (A) Schematic diagram of HUVEC migration assay. (B-C) Migration analysis of human umbilical vein endothelial cells (HUVEC) (Transwell assay). (D) Schematic diagram of tube formation assay. (E-F) Tube formation of HUVEC and a chart showing the tube formation index of different groups. (G) Non-contact co-culture of MSCs and CRC cells. Cells were used at a ratio of 1:1. (H-I) Western blot showing CAFs-related markers after 4 days of co-culture. (J-L) Immunofluorescence (magnification, ×400) when MSCs were co-cultured with CRC cells (control cells and CRC cells transfected with NC, sh-COX4I2, and oe-COX4I2 constructs) with or without 5 μM PD-166866 treatment. (M) CAFs migration assay. (N-O) Recruitment of CRC cells to CAF (Transwell) (magnification ×200). Data are expressed as mean ± SEM, *P < 0.05; **P < 0.01; ***P < 0.001. Detailed implementation mode

[0021] The following combines specific embodiments to further clarify the present invention. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. After reading the present invention, various equivalent forms of modification of the present invention by those skilled in the art all fall within the scope defined by the appended claims of this application.

[0022] Example 1 Determine the valuable genes in cytochrome c oxidase

[0023] In this invention, all 42 cytochrome c oxidase-related genes were subjected to LASSO regression to identify the target genes for subsequent research. The cross-validation was performed in 10 rounds to prevent overfitting ( Figure 1 A and B). Successive univariate and multivariate Cox regression analyses were conducted, showing that both COX4I2 and COX19 were associated with CRC prognosis, identifying these genes as the research targets ( Figure 1 C-H).

[0024] Example 2 COX4I2 promotes the malignant phenotypes of colorectal cancer

[0025] In this invention, Western blotting and IHC (immunohistochemistry) were used to explore the expression of COX4I2 in CRC tissues, indicating that the levels of COX4I2 were high both in CRC cells and tissues. In CRC and paired control tissues, the average H-SCOREs of COX4I2 expression were 95.53±15.96 and 19.14±6.65, respectively ( Figure 2 A-C). The levels of COX4I2 were the highest in the RKO and SW480 cell lines, so these cell lines were used for further research. The transfection efficiency was confirmed by GFP expression and Western blotting ( Figure 2 D). Silencing of COX4I2 reduced clone formation ( Figure 2 E). In addition, stable overexpression of COX4I2 led to the successful formation of mouse xenograft tumors ( Figure 2 F-H). Ki-67 (a nuclear proliferation marker, which is an index for detecting the proliferation activity of tumor cells) is a known factor related to CRC prognosis, and its strong expression is associated with invasive tumors and poor outcomes, and it is also a proliferation marker. In this invention, it was observed that in CRC cases and tumors of nude mice, high expression of COX4I2 also showed strong Ki-67 staining signals ( Figure 2 I-J). These results suggest that COX4I2 may be associated with Ki-67. According to the results of enrichment analysis and the observed correlation between COX4I2 and FGF1 (fibroblast growth factor 1) in the sera of CRC patients, this invention speculated that fibroblast growth factor 1 may be involved in some cancer-related behaviors regulated by COX4I2. Subsequently, this invention used culture medium ( Figure 2 K), mouse serum ( Figure 2 L), mouse tumor ( Figure 2 M) and CRC tissue ( Figure 2 N-O) to further verify the correlation between COX4I2 and FGF1. It was found that in all cases, the expression of COX4I2 was closely related to the expression of FGF1.

[0026] Example 3 Overexpression of COX4I2 promotes the EMT process

[0027] GSEA and single-cell RNA-Seq analysis indicated that the expression of COX4I2 was closely associated with the epithelial-mesenchymal transition (EMT) of cells. An increase in EMT-related phenotypes was found to be associated with COX4I2 overexpression. After treatment with the integrin FGF1-specific inhibitor PD-166866, this effect was reduced ( Figure 3 A-B). TIMER analysis also found that the expression of COX4I2 was positively correlated with the expression of N-cadherin (CDH2) (R = 0.320, P < 0.001), matrix metalloproteinase-2 (MMP2) (R = 0.450, P < 0.001), matrix metalloproteinase-9 (MMP9) (R = 0.400, P < 0.001), SNAI2 (human homologue of snail 2, a member of the SNAIL superfamily of zinc finger transcription factors and one of the epithelial-mesenchymal transition transcription factors) (R = 0.380, P < 0.001) and SNAI1 (human homologue of snail 1) (R = 0.450, P < 0.001), and negatively correlated with the expression of CDH1 (E-cadherin) (R = 0.070). These results were confirmed by western blotting ( Figure 3 D).

[0028] Example 4 COX4I2 Promotes Angiogenesis and CAF Activation

[0029] To verify the GSEA analysis and single-cell RNA-Seq analysis of angiogenesis and fibroblast activation, the present invention established several in vitro co-culture models. First, it was found that overexpression of COX4I2 in CRC promoted the migration of human umbilical vein endothelial cells ( Figure 4 A-C), as well as their ability to form blood vessels ( Figure 4 D-F). The FGF1 inhibitor could rescue this malignant phenotype. Cancer-associated fibroblasts (CAFs) are derived from circulating mesenchymal stem cells (MSCs) and are well-known for their properties in forming the tumor microenvironment. The non-contact co-culture model ( Figure 4 G) showed that overexpression of COX4I2 promoted the differentiation of MSCs into CAFs, which was mainly reflected in a significant increase in CAF activation markers in the co-culture system ( Figure 4(H-L). Fibroblast activation protein (FAP), fibroblast-specific protein-1 (S100A4), and α-smooth muscle actin (α-SMA) are all commonly used to label activated CAFs. These observations also showed that pretreatment with an FGF1 inhibitor reduced the differentiation of MSCs into CAFs in the co-culture unit. Cancer cell invasion refers not only to the tumor cells themselves but also to the recruitment of endothelial cells and fibroblasts in the surrounding stromal microenvironment. Migration assays confirmed that COX4I2 overexpression increased the recruitment of cancer cells to CAFs ( Figure 4 M-O).

[0030] The above experimental results indicate that COX4I2 promotes CRC progression by stimulating angiogenesis and fibroblast activation. Therefore, the COX4I2 protein can be used as a biomarker for predicting the prognosis of rectal cancer or screening for immunotherapy, and can also be used as a therapeutic target.

[0031] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made, and these improvements and modifications should also be regarded as the protection scope of the present invention. Sequence Listing <110> Jiangsu Provincial Hospital of Traditional Chinese Medicine <120> A Predictive Biomarker for Human Colorectal Cancer or Human Colorectal Cancer Metastasis and Its Application <160> 1 <170> SIPOSequenceListing 1.0 <210> 1 <211> 171 <212> PRT <213> Artificial Sequence (2 Ambystoma laterale x Ambystoma jeffersonianum) <400> 1 Met Leu Pro Arg Ala Ala Trp Ser Leu Val Leu Arg Lys Gly Gly Gly 1 5 10 15 Gly Arg Arg Gly Met His Ser Ser Glu Gly Thr Thr Arg Gly Gly Gly 20 25 30 Lys Met Ser Pro Tyr Thr Asn Cys Tyr Ala Gln Arg Tyr Tyr Pro Met 35 40 45 Pro Glu Glu Pro Phe Cys Thr Glu Leu Asn Ala Glu Glu Gln Ala Leu 50 55 60 Lys Glu Lys Glu Lys Gly Ser Trp Thr Gln Leu Thr His Ala Glu Lys 65 70 75 80 Val Ala Leu Tyr Arg Leu Gln Phe Asn Glu Thr Phe Ala Glu Met Asn 85 90 95 Arg Arg Ser Asn Glu Trp Lys Thr Val Met Gly Cys Val Phe Phe Phe 100 105 110 Ile Gly Phe Ala Ala Leu Val Ile Trp Trp Gln Arg Val Tyr Val Phe 115 120 125 Pro Pro Lys Pro Ile Thr Leu Thr Asp Glu Arg Lys Ala Gln Gln Leu 130 135 140 Gln Arg Met Leu Asp Met Lys Val Asn Pro Val Gln Gly Leu Ala Ser 145 150 155 160 Arg Trp Asp Tyr Glu Lys Lys Gln Trp Lys Lys 165 170

Claims

1. Use of a reagent for detecting COX4I2 protein in the preparation of a predictive kit for the prognosis and immunotherapy responsiveness of human colorectal cancer patients.

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