Method for improving sensitivity of esophageal squamous carcinoma patient to neoadjuvant chemoradiotherapy

Abstract

The invention provides a method for improving the sensitivity of an esophageal squamous carcinoma (ESCC) patient to neoadjuvant chemoradiotherapy. Specifically, by reducing the levels of CEBPB, AREG and / or EREG proteins, mitochondrial oxidative phosphorylation (OXPHOS) is inhibited, TCA circulation is inhibited, and / or ERBB signal pathways are inhibited, so that the sensitivity of ESCC patients to neoadjuvant radiotherapy and chemotherapy is improved. In addition, the invention also provides a method for predicting the sensitivity of an ESCC patient to neoadjuvant chemoradiotherapy, and a biomarker for predicting the sensitivity of the ESCC patient.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, and more specifically, this invention relates to a method for improving the sensitivity of esophageal squamous cell carcinoma patients to neoadjuvant chemoradiotherapy. Background Technology

[0002] Esophageal cancer (EC) is the ninth most common malignant tumor worldwide and the sixth leading cause of cancer-related death. In East Asia, 80% of esophageal cancer cases are esophageal squamous cell carcinoma (ESCC). For locally advanced ESCC, neoadjuvant chemoradiotherapy (nCRT) followed by surgery is the standard treatment. However, the efficacy of nCRT for ESCC varies from person to person. Approximately 30-50% of nCRT patients achieve pathological complete remission (pCR) and long-term survival, but a significant number experience short-term recurrence or disease progression. Recent studies have shown that patients who achieve clinical complete remission but refuse surgery have similar overall survival rates to those who undergo standard surgical resection after nCRT, suggesting that nCRT may be a viable method for organ preservation in these patients. Given the crucial role of radiotherapy (IR) in improving the efficacy of nCRT, researchers are increasingly focusing on elucidating the molecular mechanisms and predictive biomarkers of radiosensitivity in ESCC patients.

[0003] Metabolic reprogramming is a prominent feature of cancer, enabling esophageal squamous cell carcinoma (ESCC) cells to develop radioresistance through adaptive plasticity. The glycolytic pathway plays a particularly important role in the response to radiotherapy. Radiotherapy-induced upregulation of glycolytic enzymes (such as GLUT1, HK2, and PDK1) enhances the glycolytic capacity and radioresistance of ESCC cells (the role of metabolism in cancer cell radioresistance and radiosensitization). Therefore, lactate accumulation, as a major substrate of glycolysis, is associated with radioresistance. High expression of the TCA cycle enzyme IDH2 (isocitrate dehydrogenase 2) confers radioresistance in ESCC cells. Furthermore, several metabolites have been identified as biomarkers for predicting nCRT response; however, inconsistencies exist in the predictive results of these biomarkers across different studies, thus the key metabolic mechanisms underlying radioresistance in esophageal squamous cell carcinoma remain unclear.

[0004] The metabolic pathways of radiation response are often regulated by oncogenic signaling pathways. However, how oncogenic signals regulate metabolism to enhance radioresistance in ESCC remains unclear. Summary of the Invention

[0005] The purpose of this invention is to provide a biomarker for predicting the sensitivity of esophageal squamous cell carcinoma to neoadjuvant chemoradiotherapy.

[0006] In a first aspect of the invention, a method is provided for predicting the sensitivity of esophageal squamous cell carcinoma (ESCC) patients to neoadjuvant chemoradiotherapy, the method comprising:

[0007] A. Provide test samples from ESCC patients;

[0008] B. Detect the levels of AREG and / or EREG proteins in the sample to be tested, and obtain the AREG level detection value C1 and the EREG level detection value C2; ​​and

[0009] C. Compare C1 and C2 with the cutoff values ​​of AREG and / or EREG proteins to determine whether the ESCC patients are sensitive to neoadjuvant chemoradiotherapy;

[0010] The serum cutoff value of the AREG protein was 103.6 pg / ml.

[0011] The serum cutoff value of the EREG protein was 150.1 pg / ml.

[0012] In another preferred embodiment, the sample to be tested is selected from serum samples, cancer tissue samples, or combinations thereof.

[0013] In another preferred embodiment, if C1 > 103.6 pg / ml and / or C2 > 150.1 pg / ml, the patient is deemed insensitive to neoadjuvant chemoradiotherapy.

[0014] In another preferred embodiment, if C1 < 103.6 pg / ml and / or C2 < 150.1 pg / ml, the patient is deemed sensitive to neoadjuvant chemoradiotherapy.

[0015] In another preferred embodiment, the cutoff value of the AREG and / or EREG proteins also includes the cutoff value of the tissue immunohistochemical score.

[0016] In another preferred example, the cutoff values ​​for the tissue immunohistochemical scores of both AREG and EREG proteins were 10.5.

[0017] In another preferred embodiment, if the tissue immunohistochemical score of the AREG and / or EREG proteins in the sample to be tested is greater than 10.5, the patient is determined to be insensitive to neoadjuvant chemoradiotherapy.

[0018] In another preferred embodiment, if the tissue immunohistochemical score of the AREG and / or EREG proteins in the sample to be tested is less than 10.5, the patient is deemed to be sensitive to neoadjuvant chemoradiotherapy.

[0019] In a second aspect of the invention, a method is provided to improve the sensitivity of patients with esophageal squamous cell carcinoma (ESCC) to neoadjuvant chemoradiotherapy (nCRT), the method comprising:

[0020] 1) Reduce the levels of CEBPB, AREG, and / or EREG proteins;

[0021] 2) Inhibit mitochondrial oxidative phosphorylation (OXPHOS) and / or the TCA cycle; and / or

[0022] 3) Inhibit the ERBB signaling pathway.

[0023] In another preferred embodiment, the CEBPB regulates the levels of AREG and / or EREG proteins.

[0024] In another preferred embodiment, the CEBPB protein level is positively correlated with the AREG and / or EREG protein levels.

[0025] In another preferred embodiment, the AREG and / or EREG activate OXPHOS, increasing the generation of OCR and ATP.

[0026] In another preferred embodiment, the levels of the AREG and / or EREG proteins are positively correlated with nCRT resistance.

[0027] In another preferred embodiment, the higher the level of the AREG and / or EREG proteins, the less sensitive the ESCC patient is to nCRT.

[0028] In another preferred embodiment, the activity of OXPHOS is positively correlated with nCRT resistance.

[0029] In another preferred embodiment, the higher the activity of the OXPHOS, the less sensitive the ESCC patient is to nCRT.

[0030] In another preferred embodiment, the AREG and / or EREG activate the ERBB pathway.

[0031] In another preferred embodiment, the more active the ERBB pathway, the less sensitive the ESCC patient is to nCRT.

[0032] In another preferred embodiment, the ERBB pathway is inhibited, thereby reducing the activity of OXPHOS.

[0033] In another preferred embodiment, the levels of CEBPB, AREG, and / or EREG proteins are reduced to decrease the activity of OXPHOS.

[0034] In another preferred embodiment, reducing the levels of CEBPB, AREG, and / or EREG proteins thereby reducing OXPHOS activity, thus increasing the sensitivity of ESCC patients to nCRT.

[0035] In another preferred embodiment, the levels of CEBPB, AREG, and / or EREG proteins are reduced, thereby inhibiting the proliferation, colony-forming ability, wound healing ability, and / or migration ability of ESCC cells.

[0036] In another preferred embodiment, the drug that inhibits the TCA cycle is selected from the group consisting of CPI-613, FH-IN-1, 3-NP, or combinations thereof.

[0037] In another preferred embodiment, the drug that inhibits mitochondrial oxidative phosphorylation is selected from the group consisting of: IACS-010759, rotenone, Gboxin, antimycin A, or combinations thereof.

[0038] In another preferred embodiment, the drug that inhibits the ERBB signaling pathway is selected from the group consisting of varlitinib or dacomitinib.

[0039] In a third aspect of the invention, an ESCC cell line highly sensitive to neoadjuvant chemoradiotherapy is provided, said ESCC cell line being an ESCC cell line treated using the method described in the second aspect of the invention.

[0040] In another preferred embodiment, the ESCC cell line is selected from the group consisting of KYSE30, KYSE410, or TE-1.

[0041] In a fourth aspect of the invention, there is provided the use of CEBPB, AREG, and / or EREG for predicting the sensitivity of esophageal squamous cell carcinoma (ESCC) patients to neoadjuvant chemoradiotherapy as described in the first aspect of the invention.

[0042] In a fifth aspect of the invention, a kit is provided for use in the method as described in the second aspect of the invention, the kit comprising:

[0043] 1) Inhibitors that reduce the levels of CEBPB, AREG, and / or EREG proteins;

[0044] 2) Inhibitors of mitochondrial oxidative phosphorylation (OXPHOS) and / or the TCA cycle; and / or

[0045] 3) Inhibitors that suppress the ERBB signaling pathway.

[0046] In another preferred embodiment, the kit is used to improve the sensitivity of patients with esophageal squamous cell carcinoma (ESCC) to neoadjuvant chemoradiotherapy.

[0047] In another preferred embodiment, the inhibitor includes nucleic acid inhibitors, small molecule inhibitors, or inhibitors that specifically promote protein degradation for reducing the levels of CEBPB, AREG, and / or EREG proteins, inhibiting mitochondrial oxidative phosphorylation (OXPHOS), the TCA cycle, and / or inhibiting the ERBB signaling pathway.

[0048] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0049] Figure 1 The alterations in TCA circulating intermediate metabolites characterize the nCRT response in ESCC patients: a. Compared to non-pCR patients, pCR patients exhibited stronger nCRT-induced perturbations in their metabolic profiles. a. Serum metabolic profiles were compared across four experimental groups using a PLS-DA model: pre-pCR (serum from pCR patients before nCRT, n=23); post-pCR (serum from pCR patients after nCRT, n=23); pre-non-pCR (serum from non-pCR patients before nCRT, n=36); and post-non-pCR (serum from non-pCR patients after nCRT, n=36). b. Metabolic profiles before and after nCRT were analyzed using a stratified PLS-DA model in both pCR and non-pCR patient populations. c. The PLS-DA model in (b) was validated by a displacement assay. d. Heatmaps of differentially expressed metabolites (DEMs) before and after nCRT in both pCR and non-pCR ESCC patients. e. KEGG pathway enrichment analysis of DEMs in pCR and non-pCR ESCC patients before and after nCRT. The Y-axis represents the ln (p-value) of the pathway analysis. The X-axis represents the influencing factor (number of enriched DEMs in the pathway / number of all metabolites in the pathway). f. After nCRT, the abundance of circulating TCA metabolites increased in the pCR group, but not in the non-pCR group. Statistical analysis was performed using Student's t-test. Data are expressed as mean ± SD. *p<0.05, **p<0.01.

[0050] Figure 2This study demonstrates that inhibition of OXPHOS enhances the radiosensitivity of ESCC cells: a. Colony formation assay survival fractions of parental KYSE410 (410) and radiation-resistant KYSE410R (410R) cells after irradiation. b. Oxygen consumption rate (OCR) was measured using a Seahorse XF24 analyzer. Oligo: oligomycin; FCCP: carbonyl cyanide-4-phenylhydrazone; Rot / Ant: rotenone / antimycin A. c. ATP production was analyzed using a Seahorse analyzer. d. Schematic diagram of inhibitors targeting the TCA cycle (CIP-613, 3-NP, FH-IN-1) and OXPHOS (IACS-010759, rotenone, Gboxin, antimycin A). e. Evaluation of the radiosensitizing effect of TCA cycle or OXPHOS inhibitors on KYSE30 cells by colony formation assay survival fractions. f. The relative abundance of circulating TCA metabolites in KYSE30 cells with or without OXPHOS inhibitors was analyzed using UHPLC-QTRPMS (n=6).

[0051] Figure 3This study demonstrates how AREG and EREG regulate the radiation sensitivity of ESCCs via OXPHOS: a. Venn diagrams of differentially expressed genes (DEGs) identified by RNA-seq in KYSE410R or KYSE410 cells before and after irradiation (IR) (410R vs. 410, 410_IR vs. 410, 410R_IR vs. 410R, 410R_IR vs. 410_IR). b. KEGG pathway enrichment of 55 overlapping DEGs in the four comparison groups. c. Correlation between ERBB signaling pathway characteristic gene expression and OXPHOS characteristic gene expression in the TCGA ESCA dataset. de. Validation of irradiation-induced upregulation of AREG and EREG mRNA levels by RNA-seq (d) and qRT-pCR (e). fj. ERBB signaling pathway regulation of OXPHOS activity. Validation of AREG or EREG knockout effects by qRT-pCR (f). Oxygen consumption ratio (OCR) (g) and ATP production (h) of AREG or EREG knockout ESCC cells were analyzed using a Seahorse XF24 analyzer. AREG (Amphiregulin) and EREG (Eipregulin) treatments increased OCR (i) and ATP production (j), but ERBB inhibitor (Dacomitinib or Varlitinib) treatments decreased OCR (i) and ATP production (j) in ESCC cells. k. Radiation resistance and IR treatment increased the expression of mitochondrial complex proteins through the AKT / mTOR signaling pathway. l. ERBB inhibitor (Dacomitinib or Varlitinib) treatment increased the radiosensitivity of ESCC cells. m. Knockdown of AREG or EREG increased the radiosensitivity of ESCC cells. n. Immunohistochemical staining showed that AREG and EREG expression was higher in non-pCR tumor tissues than in pCR. o. Quantitative ELISA confirmed elevated baseline and nCRT-induced serum AREG / EREG levels in non-pCR ESCC patients.

[0052] Figure 4 The study demonstrated that AREG and EREG promote tumorigenesis in ESCC cells: a. Immunohistochemical staining showed upregulated AREG and EREG levels in ESCC tumor tissue compared to normal tissue. b. High levels of AREG or EREG were associated with poorer overall survival in ESCC patients. cf. Functional validation of AREG and EREG in tumor progression. In ESCC cells, knockdown of AREG or EREG reduced cell proliferation (c), colony formation (d), migration (e), and transwell invasion (f).

[0053] Figure 5The following studies demonstrate CEBPB's role as a co-transcriptional factor for AREG and EREG in irradiation responses: a. Immunohistochemical staining showed significant co-expression of AREG and EREG in ESCC tumor samples (n=46). b. Venn diagrams show the common transcriptional regulators of AREG and EREG predicted by the CISTROME, ChEA3, and GTRD databases. c. Immunohistochemical staining showed a positive correlation between CEBPB and AREG or EREG protein levels in ESCC tumor samples (n=46). dg. CEBPB mediates irradiation-induced AREG and EREG expression. CEBPB knockdown of AREG and EREG mRNA levels (d). CEBPB overexpression increased AREG and EREG transcription (e). Irradiation (IR) induces CEBPB transcription (f). Knockdown of CEBPB resulted in the disappearance of IR-induced AREG and EREG expression (g).

[0054] Figure 6 This study demonstrates that CEBPB drives tumor progression and OXPHOS-mediated radioresistance in ESCC: a. CEBPB expression was elevated in ESCC tumor samples compared to normal tissue samples (GEPIA). b. CEBPB promotes tumorigenesis in ESCC. CEBPB knockdown in KYSE30 cells reduced cell proliferation (b), colony formation ability (c), wound healing (d), and transwell migration (e). f. CEBPB regulates OXPHOS activity. CEBPB knockdown reduced oxygen consumption rate (OCR) (f) and ATP production (g), while CEBPB overexpression increased OCR (h) and ATP production (i). j. CEBPB knockdown increased radiosensitivity in KYSE30 cells. k. Immunohistochemistry showed higher CEBPB protein levels in nCRT-non-pCR ESCC tumors compared to pCR specimens.

[0055] Figure 7Targeting OXPHOS or ERBB enhances the radiosensitivity of ESCC tumors: ad. IACS-010759 (an OXPHOS complex I inhibitor) enhances the irradiation effect on ESCC xenograft tumors. Mice with KYSE30-derived xenograft tumors were divided into four groups: control (PBS), IACS-010759 (7.5 mg / kg), irradiation (10 Gy), and a combination of IACS-010759 and irradiation. (a) Xenograft tumors. Tumor volume (b) and tumor weight (c) were measured at specified time points. Xenograft tumor cell apoptosis was detected by TUNEL assay (d). eh. Dacomitinib (a pan-ERBB inhibitor) enhances the irradiation efficacy of ESCC xenograft tumors. Mice with KYSE30 cell line xenograft tumors were divided into four groups: control (PBS), dacomitinib group (7 mg / kg), irradiation group (10 Gy), and combination group. (e) Xenograft tumors. Tumor volume (f) and tumor weight (g) of xenograft tumors were measured at specified time points. Xenograft tumor cell apoptosis (h) was detected by TUNEL assay. A single 10 Gy irradiation was administered. Two days later, control PBS, IACS-010759 (7.5 mg / kg), or dacomitinib (7 mg / kg) were administered orally by gavage three times a week.

[0056] Figure 8 The study revealed significant abnormalities in the levels of tricarboxylic acid cycle (TCA) intermediate metabolites in esophageal squamous cell carcinoma (ESCC) patients who responded to nCRT: a. Comparison of serum metabolite profile principal component analysis (PCA) scores before and after neoadjuvant chemoradiotherapy (pCR group, n=23; non-PCR group, n=36). b. Figure 1 a) Permutation test results of the OPLS-DA model in Figure S1c. c) Partial least squares discriminant analysis (PLS-DA) models of serum metabolic profiles in patients with esophageal squamous cell carcinoma, showing the differences in pCR and non-pCR metabolic characteristics before (pre-nCRT) and after (post-nCRT). d) Permutation test results of the PLS-DA model in Figure S1c.

[0057] Figure 9The study showed that inhibition of OXPHOS enhanced the radiosensitivity of ESCC cells: a) Colony-forming survival fraction of irradiated (IR) KYSE410 cells treated with a TCA cycle inhibitor (CPI-613) or an OXPHOS inhibitor (IACS-010759, antimycin A). b) Relative abundance of TCA cycle intermediates in KYSE30 control cells and 3-NP treated cells was determined by UHPLC-QTRPMS (n=6). c) Relative abundance of TCA cycle intermediates in KYSE410 (410) and radioresistant KYSE410R (410R) cells with and without irradiation was determined by UHPLC-QTRPMS (n=6). Student t-test. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; ns, no significant difference.

[0058] Figure 10 The results showed that both AREG and EREG are oncogenes: a. In various malignant tumors, both AREG and EREG are highly expressed in tumor tissues compared to normal tissues (TIMER2.0). b. High AREG expression is associated with poorer overall survival in cancer patients (GEPIA). c. High EREG expression is associated with poorer overall survival in cancer patients (GEPIA).

[0059] Figure 11 The study revealed that AREG and EREG are co-expressed in various malignant tumors: a. In the TCGA database, the expression levels of AREG and EREG showed a high correlation among different malignant tumors. b. In the TCGA ESCA dataset, the expression level of CEBPB was positively correlated with the expression levels of AREG or EREG.

[0060] Figure 12 The ROC curves for predicting pCR by tissue and serum AREG and EREG levels are shown: a. ROC curves for predicting pCR by tissue AREG and EREG levels; b. ROC curves for predicting pCR by serum AREG and EREG levels. Detailed Implementation

[0061] Through extensive and in-depth research, the inventors have developed a method for predicting the sensitivity of esophageal squamous cell carcinoma (ESCC) to neoadjuvant chemoradiotherapy. Experiments have demonstrated that radioresistant ESCC cells (KYSE410R) exhibit elevated OXPHOS activity. Knocking down AREG / EREG to inhibit the OXPHOS or ERBB signaling pathways can make ESCC cells more sensitive to radiotherapy. Furthermore, this invention has verified that CEBPB can act as a transcriptional regulator of AREG and EREG, regulating the radiosensitivity of ESCC cells through oxidative phosphorylation. This invention is based on these findings.

[0062] the term

[0063] To facilitate understanding of this invention, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined herein, all other technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains. Before describing this invention, it should be understood that it is not limited to the specific methods and experimental conditions described, as such methods and conditions can be varied.

[0064] As used herein, the terms “comprising,” “including,” and “containing” are used interchangeably and include not only closed definitions but also semi-closed and open definitions. In other words, the terms include “consisting of” and “substantially consisting of”.

[0065] AREG

[0066] Amphiregulin (AREG) is an epidermal growth factor (EGF)-like molecule belonging to the EGF family. It is an autocrine growth factor and a mitogen for astrocytes, Schwann cells, and fibroblasts. AREG gene expression and release are triggered by various stimuli, including inflammatory lipids, cytokines, hormones, growth factors, and exogenous drugs. By binding to EGFR, AREG activates intracellular signaling, regulating proliferation, apoptosis, and migration of different cell types, including epithelial cells, fibroblasts, and immune cells (dendritic cells, neutrophils, mast cells, and lymphocytes). Physiologically, AREG plays a crucial role in lung morphogenesis and the development and maturation of mammary glands, bone tissue, and oocytes. AREG also plays a key role in the restoration of tissue integrity after infection or injury.

[0067] EREG

[0068] Epiregulin (EREG) is a member of the epidermal growth factor family, also known as ER or EPR. This protein binds to cell surface receptors, triggering intracellular cascade reactions and participating extensively in tissue repair, embryonic development, and tumor microenvironment regulation. Studies have found that EREG is abnormally highly expressed in various solid tumors, such as colorectal cancer, breast cancer, and non-small cell lung cancer. Excessive secretion accelerates malignant cell proliferation and invasion, while also inducing angiogenesis, providing conditions for tumor metastasis. Furthermore, this protein is also active in chronic inflammatory diseases such as inflammatory bowel disease and psoriasis, possibly related to abnormal activation of immune cells.

[0069] In this invention, AREG and EREG, as activators of the ERBB signaling pathway, affect the sensitivity of ESCC patients to nCRT by regulating the activity of oxidative phosphorylation. Experiments have shown that the levels of AREG and EREG differ significantly between pCR and non-pCR patients. AREG and EREG are expressed at low levels in pCR patients and at high levels in non-pCR patients, both before and after nCRT. Therefore, AREG and EREG can serve as biomarkers for predicting the sensitivity of ESCC patients to nCRT, thereby determining the sensitivity of ESCC patients to nCRT and enabling measures to be taken to improve the treatment effect of nCRT in ESCC patients who are insensitive to nCRT.

[0070] CEBPB

[0071] CEBPB, or CCAAT enhancer-binding protein β, belongs to the CCAAT / enhancer-binding protein family. It is a transcription factor that binds to the regulatory regions of specific genes' DNA (usually the CCAAT box or its associated sequence in the promoter or enhancer), thereby regulating the expression of these genes (turning them on, off, or modulating their expression levels). It plays a pivotal role in regulating key biological processes such as inflammation, immune responses, metabolism, cell differentiation, and stress responses. Dysregulation of CEBPB expression or activity is a significant factor in the development and progression of various inflammatory diseases, cancers, and metabolic disorders, and therefore also a potential therapeutic target.

[0072] In this invention, CEBPB, as a transcriptional regulator of AREG and EREG, is positively correlated with the expression levels of AREG and EREG. Therefore, it can also serve as a biomarker for nCRT sensitivity in ESCC patients.

[0073] Neoadjuvant therapy (nCRT)

[0074] Neoadjuvant therapy (nCRT) is a cancer treatment method that combines radiotherapy and chemotherapy. The administration method is as follows: the total radiotherapy dose is 41.4 Gy, divided into 23 fractions, 5 weeks per week. The chemotherapy regimen includes paclitaxel (45 mg / m²). 2 ) and cisplatin (25mg / m 2 ), administered weekly on days 1, 8, 15, 22, and 29. Surgery is performed 4 to 6 weeks after the completion of the neoadjuvant chemoradiotherapy course.

[0075] Pathological complete response (pCR) and non-pathological complete response (non-pCR)

[0076] After neoadjuvant therapy, pathological examination of surgically removed primary tumor lesions and regional lymph node specimens reveals no residual cancer cells in the primary lesion and no metastatic cancer cells in the regional lymph nodes; this is termed pathological complete response (pCR). Achieving pCR is a key indicator for assessing the sensitivity and prognosis of neoadjuvant therapy.

[0077] ERBB signaling pathway

[0078] The ERBB signaling pathway is closely related to a variety of biological processes, including cell proliferation, differentiation, survival, and metabolism. This pathway consists of multiple receptors and their corresponding ligands, including ERBB1 (also known as EGFR), ERBB2, ERBB3, and ERBB4. Upon binding to their respective ligands, ERBB receptors activate a series of downstream signaling molecules, including the PI3K / Akt, RAS / MAPK, and STAT signaling pathways, thereby regulating cellular biological effects. In this invention, the ERBB signaling pathway affects the nCRT sensitivity of ESCC patients by regulating oxidative phosphorylation.

[0079] Methods for predicting the sensitivity of esophageal squamous cell carcinoma (ESCC) patients to neoadjuvant chemoradiotherapy

[0080] This invention also provides a method for predicting the sensitivity of esophageal squamous cell carcinoma (ESCC) patients to neoadjuvant chemoradiotherapy. Since the levels of AREG and / or EREG proteins differ significantly between ESCC patients sensitive to neoadjuvant chemoradiotherapy and those insensitive to it, this invention also calculates serum cut-off values ​​and immunohistochemical cut-off values ​​of cancer tissue to determine the levels of AREG and / or EREG proteins in patients sensitive to neoadjuvant chemoradiotherapy. Therefore, the serum cut-off values ​​and immunohistochemical cut-off values ​​of cancer tissue of this invention can be used to predict the sensitivity of ESCC patients to neoadjuvant chemoradiotherapy.

[0081] In a preferred embodiment, the method specifically includes:

[0082] A. Provide test samples from ESCC patients;

[0083] B. Detect the levels of AREG and / or EREG proteins in the sample to be tested, and obtain the AREG level detection value C1 and the EREG level detection value C2; ​​and

[0084] C. Compare C1 and C2 with the cutoff values ​​of AREG and / or EREG proteins;

[0085] The serum cutoff value of the AREG protein was 103.6 pg / ml.

[0086] The serum cutoff value of the EREG protein was 150.1 pg / ml.

[0087] In another preferred embodiment, the sample to be tested is selected from serum samples, cancer tissue samples, or combinations thereof.

[0088] In another preferred embodiment, if C1 > 103.6 pg / ml and / or C2 > 150.1 pg / ml, the patient is deemed insensitive to neoadjuvant chemoradiotherapy; if C1 < 103.6 pg / ml and / or C2 < 150.1 pg / ml, the patient is deemed sensitive to neoadjuvant chemoradiotherapy.

[0089] In another preferred embodiment, the cutoff value of the AREG and / or EREG proteins also includes the cutoff value of the tissue immunohistochemical score.

[0090] In another preferred example, the cutoff values ​​for the tissue immunohistochemical scores of both AREG and EREG proteins were 10.5.

[0091] In another preferred embodiment, if the immunohistochemical score of AREG and / or EREG proteins in the sample to be tested is greater than 10.5, the patient is determined to be insensitive to neoadjuvant chemoradiotherapy; if the immunohistochemical score of AREG and / or EREG proteins in the sample to be tested is less than 10.5, the patient is determined to be sensitive to neoadjuvant chemoradiotherapy.

[0092] Methods to improve the sensitivity of nCRT in patients with esophageal squamous cell carcinoma (ESCC)

[0093] This invention provides a method for improving the radiosensitivity of esophageal squamous cell carcinoma (ESCC) patients to nCRT. This invention demonstrates through experiments that oxidative phosphorylation (OXPHOS) activity is crucial for the radiosensitivity of ESCC. The higher the OXPHOS activity, the lower the sensitivity of ESCC patients to nCRT and the higher their resistance. In addition, the ligands AREG and EREG of the ERBB receptor enhance the radioresistance of ESCC by regulating OXPHOS.

[0094] Therefore, treatment that inhibits the OXPHOS or ERBB pathway can improve the radiosensitivity of ESCC, while AREG and EREG, as biomarkers, can be used to predict the nCRT sensitivity of ESCC patients.

[0095] In another preferred embodiment, the method specifically includes:

[0096] 1) Reduce the levels of CEBPB, AREG, and / or EREG proteins;

[0097] 2) Inhibit mitochondrial oxidative phosphorylation (OXPHOS) and / or the TCA cycle; and / or

[0098] 3) Inhibit the ERBB signaling pathway.

[0099] Reagent test kit

[0100] The present invention also provides a kit for improving the sensitivity of nCRT in patients with esophageal squamous cell carcinoma (ESCC), the kit comprising:

[0101] 1) Inhibitors that reduce the levels of CEBPB, AREG, and / or EREG proteins;

[0102] 2) Inhibitors of mitochondrial oxidative phosphorylation (OXPHOS) and / or the TCA cycle; and / or

[0103] 3) Inhibitors that suppress the ERBB signaling pathway.

[0104] The inhibitors include nucleic acid inhibitors, small molecule inhibitors, or inhibitors that specifically promote protein degradation, used to reduce the levels of CEBPB, AREG, and / or EREG proteins, inhibit mitochondrial oxidative phosphorylation (OXPHOS), the TCA cycle, and / or inhibit the ERBB signaling pathway.

[0105] Nucleic acid inhibitors, such as siRNA and shRNA, can inhibit the expression of target proteins by degrading their mRNA. Small molecule inhibitors can regulate the function of target proteins (such as enzymes and kinases) by specifically binding to their active sites. Their mechanisms of action include competing with substrates for binding sites, altering protein structure, or inhibiting protein conformational transitions, thereby reducing protein activity. Inhibitors that specifically promote protein degradation can specifically recognize and target target proteins through coupling with targeting elements, thereby inducing protein degradation. For example, ubiquitinating enzymes or domains with ubiquitination activity that are specific to the target protein can specifically recognize and target the target protein through targeting elements. The ubiquitinating enzyme or the domain with ubiquitination activity then ubiquitinates the target protein, and the proteasome degrades the target protein by recognizing the ubiquitination modification.

[0106] Compared with the prior art, the main advantages of the present invention include:

[0107] 1. This invention validates the individual differences in the sensitivity of ESCC patients to neoadjuvant chemotherapy and validates that AREG and EREG can serve as biomarkers to predict the sensitivity of ESCC patients to neoadjuvant chemotherapy.

[0108] 2. This invention develops a method to improve the sensitivity of ESCC patients to neoadjuvant chemotherapy, which can improve the therapeutic effect of neoadjuvant chemotherapy on ESCC patients.

[0109] 3. According to the present invention, biomarkers can be detected in ESCC patients first, thereby enabling individualized treatment of ESCC patients based on the prediction results.

[0110] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0111] Materials and methods

[0112] 1. Human tissue collection

[0113] This study was approved by the Ethics Committee of Renji Hospital, affiliated with Shanghai Jiao Tong University School of Medicine. All participants signed informed consent forms. Eligible patients were diagnosed with locally advanced resectable thoracic esophageal squamous cell carcinoma (ESCC), staged as T1-4aN1-3M0 or T3-4aN0M0 (stages II to IVA) according to the American Joint Committee on Cancer Staging Manual, 8th edition. From 2020 to 2022, all patients received neoadjuvant chemoradiotherapy (nCRT) at Renji Hospital. The total dose was 41.4 Gy, divided into 23 fractions, 5 weeks per week. The chemotherapy regimen included paclitaxel (45 mg / m²). 2 ) and cisplatin (25mg / m 2Administered weekly on days 1, 8, 15, 22, and 29. Surgery was performed 4 to 6 weeks after the completion of the neoadjuvant chemoradiotherapy cycle. Response to neoadjuvant chemoradiotherapy was assessed based on the pathological results of the surgical specimens, and patients were divided into two groups: pathological complete response (pCR) and non-pathological complete response (non-pCR). Table 1 lists the baseline clinical characteristics of patients in the pCR group (n=23) and the non-pCR group (n=36). Serum samples were collected in the morning after fasting before the start of neoadjuvant chemoradiotherapy and on the last day after the last day of neoadjuvant chemoradiotherapy. All samples were stored at -80°C for subsequent metabolomics analysis, including non-targeted and targeted assays.

[0114] Table 1. Comparison of baseline clinical characteristics between the sensitive and insensitive groups.

[0115]

[0116] 2. Cell Culture

[0117] ESCC cell lines, including KYSE30, KYSE410, and TE-1, were purchased from ATCC. KYSE410 and TE-1 were cultured in RPMI 1640 medium (Sigma), while KYSE30 was cultured in medium containing 50% RPMI 1640, 50% Ham's F-12, and 2 mM L-glutamine. All cell lines were cultured at 37°C and 5% CO2, with the medium supplemented with 10% fetal bovine serum (FBS, Yeasen) and 1% penicillin-streptomycin (Gibco). To ensure mycoplasma-free cell culture, the cell culture medium was periodically tested for mycoplasma (Yeasen).

[0118] 3. Reagents

[0119] Methanol, acetonitrile, and formic acid of high performance liquid chromatography grade were purchased from Thermo Fisher Scientific (Waltham, Massachusetts, USA). 2-Chloro-L-phenylalanine was purchased from Sigma-Aldrich (Missouri, USA). Ultrapure water was produced by a Mill-Q reference system (Milliborough, Massachusetts, USA) equipped with an LC-MS Pak filter.

[0120] Rotenone was purchased from Sigma-Aldrich in St. Louis, USA. Bismuth subtilis, dacomitinib, epithelial regulatory protein, varlitinib, FH-IN-1, IACS-010759, and MDH1-IN-1 were purchased from Medicare in Shanghai, China. CPI613, Gboxin, and 3-nitropropionic acid (3-NPH) were purchased from Selek in Shanghai, China. Antibiotic A was purchased from Glebio in California, USA. In in vivo studies, IASC-010759 was purchased from CSN Pharmaceuticals in Shanghai, China. Dacomitinib was purchased from Macklin in Shanghai, China.

[0121] 4. Non-targeted metabolomics analysis

[0122] Serum samples from patients with esophageal squamous cell carcinoma (ESCC) were thawed at 4°C for 30 minutes. 300 μL of pre-chilled methanol (containing 2 μg / mL 2-chloro-L-phenylalanine as an internal standard) was added to each sample, mixed with 100 μL of serum, and vortexed for 15 seconds. The samples were then centrifuged at 12,000 rpm for 15 minutes at 4°C to remove protein precipitates. After centrifugation, the supernatant was dried under nitrogen. The dried samples were redissolved in 80 μL of 80% methanol. They were then centrifuged again at 12,000 rpm at 4°C for 15 minutes, and 50 μL of the supernatant was transferred to a new vial for non-target and target detection using UHPLC-QTOFMS (AB Sciex) and UHPLC-QTRAPMS (AB Sciex), respectively. Non-target detection was performed using an Agilent Technologies 1290 Infinity Series UHPLC system equipped with a Waters ACQUITY UPLC BEH Amide column (1.7 μm, 2.1 mm × 100 mm) for LC separation. Mobile phase A was 0.1% formic acid aqueous solution, and mobile phase B was acetonitrile. The following elution gradients were used for analysis: 0–0.5 min, 95% B; 0.5–7.0 min, 95%–65% B; 7.0–8.0 min, 65%–40% B; 8.0–9.0 min, 40% B; 9.0–9.1 min, 40%–95% B; 9.1–12.0 min, 95% B. The column temperature was set to 25 °C, the autosampler temperature to 4 °C, and the injection volume to 2 μL.

[0123] MS / MS spectra were acquired using a triple quadrupole time-of-flight mass spectrometer (TripleTOF 6500, AB Sciex) in information-dependent acquisition (IDA) mode. Sample detection was performed using an electrospray ionization (ESI) source in negative ion mode. In each cycle, the top 12 most intense precursor ions with intensities exceeding 100 were selected for MS / MS analysis at a collision energy (CE) of 30 eV. The cycle time was 0.56 seconds. ESI source conditions were set as follows: gas 1 and gas 2 at 60 psi, curtain gas at 35 psi, source temperature at 550 °C, and declustering potential at 60 V. After data acquisition, all raw MS data (.wiff) files were converted to mzXML format using ProteoWizard and processed by XCMSplus (AB Sciex), including peak deconvolution, alignment, and integration. The minimum fraction and cutoff values ​​were set to 0.5 and 0.3, respectively. Metabolite identification was based on Accurate Mass Metabolite Spectral Library Search in The MasterView. TM1.1 The local library in the software (AB Sciex) is used.

[0124] 5. Targeted metabolomics analysis

[0125] For cell samples, 3 × 10^6 cells were seeded in 10 cm culture dishes. When cell confluence reached 90%, the cells were washed twice with PBS. Subsequently, the cells were quenched with pre-chilled methanol solution containing 2-chloro-L-phenylalanine (2 μg / mL) as an internal standard, and metabolites were extracted by sonication. After removing debris by centrifugation at 12,000 rpm for 15 min, the supernatant was transferred to new vials for target detection using an Agilent 1290 ultra-high performance liquid chromatography system with an AB QTRAP 6500 mass spectrometer (AB Sciex, USA). Metabolites were separated using a Waters ACQUITY UPLC BEH amide column (1.7 μm, 2.1 mm × 100 mm). Multiple reaction monitoring (MRM) mode was used for detection. MRM transitions (m / z), dynamic range (V), and correction error (V) of target metabolites in cancer metabolism were referenced from previous literature (Yuan M, Breitkopf SB, Yang X, Asara JM: A positive / negative ion–switching, targeted mass spectrometry–based metabolomics platform for bodily fluids, cells, and fresh and fixed tissue. Nature Protocols 2012, 7(5): 872-881). MRM data were acquired using Analyst 1.6.1 software (ABSciex). Chromatography review and peak area integration were performed using OS software (version 1.5.0.23389, AB Sciex). The abundance of each metabolite was presented as the peak area ratio of the target metabolite to the internal standard 2-chloro-L-phenylalanine.

[0126] 6. Establishment of the radiation-resistant cell line KYSE410R

[0127] KYSE410 cells were seeded in 25 cm² culture flasks. Cells were irradiated with 2 Gy of X-rays using a high-energy linear accelerator (ElektaSynergy, 6 MV X-rays, 600 MU / min). The culture medium was replaced immediately after irradiation, and the cells were returned to the incubator. When the cells reached approximately 90% confluence, they were treated with trypsin and then transferred to new culture flasks for further culture. When the cells again reached approximately 50% confluence, they were irradiated with X-rays again. This process was repeated twice weekly for a total of 18 weeks, with each dose of 36 Gy, for 2 months until a radioresistant cell population was established. The resistance level of these clones was assessed using a clonogenic assay. Parental cells were treated with trypsin, counted, and passaged under the same conditions (without X-ray irradiation).

[0128] 7. Irradiation of cells and tumors

[0129] Cells were irradiated using a medical linear accelerator (Elekta Synergy, 6MV X-rays, 600MU / min) with a 180° irradiation field of 40 cm × 40 cm. For in vivo experiments, mice were irradiated with a total dose of 10 Gy (6MV X-rays, 600 MU / min) using the same medical linear accelerator (Elekta Synergy).

[0130] 8. Cell transfection

[0131] To knock down AREG / EREG in esophageal squamous cell carcinoma (ESCC) cells, two independent shRNA sequences were designed. Another shRNA without the target sequence served as a negative control (NC). Cells were seeded in 6-well plates and transfected with lentivirus. Stable cells were selected using puromycin (4 μg / mL for KYSE30 and 1.0 μg / mL for TE-1) for two weeks. The knockdown effect of AREG / EREG was validated by quantitative real-time pCR.

[0132] 9. Westernbolt

[0133] Cells were washed three times with PBS, followed by extraction of total cellular protein using RIPA lysis buffer (Qiagen, Germany) containing a mixture of protease inhibitors and phosphatase inhibitors. Lysates were incubated on ice for 30 min and then centrifuged at 12,000 rpm for 30 min at 4 °C. Protein concentration was determined using a Bicinchoninic Acid Kit (Pierce, Rockford, IL). 40 μg of total protein was separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a 0.22 μm polyvinylidene fluoride membrane (PDVF; Millipore). The membrane was blocked with 5% skim milk powder at room temperature for one hour and then incubated overnight with primary antibody at 4 °C. Protein bands were visualized using an enhanced chemiluminescence (ECL) detection kit (Tanon, China). Quantitative analysis of Western blots was performed using ImageJ software.

[0134] 10. Immunohistochemical staining

[0135] Paraffin-embedded mouse or human tissue samples were completely dewaxed in xylene and the antigen was restored by boiling for 20 minutes (Beyotime Biotechnology). Samples were incubated overnight with primary antibody at 4°C, followed by immunohistochemistry application solutions kit (Cellsun Technologies) according to the manufacturer's instructions. IHC scoring was based on staining intensity and the number of positive cells. The scoring criteria for positive staining rate were: less than 5% = 0 points, 5-25% = 1 point, 26-50% = 2 points, 51-75% = 3 points, and more than 75% = 4 points. The scoring criteria for staining intensity were: no staining = 0 points, pale yellow = 1 point, brownish-yellow = 2 points, and brown = 3 points. The final score was the product of the two scores, then divided into four grades: negative (0 points), weakly positive (1-4 points), positive (5-8 points), and strongly positive (9-12 points).

[0136] 11. Quantitative Real-Time PCR

[0137] Total RNA was extracted from cell lines using the RNAfast200 kit (Fasted, Shanghai, China), following the manufacturer's instructions. The quality of the isolated RNA was assessed using Nanodrop (Thermo Scientific, Massachusetts, USA). Reverse transcription (RT) was performed using PrimeScript RT Master Mix (TaKaRa, Kurume, Japan). cDNA was used as a template for PCR. Real-time PCR was performed using ChamQ™. Color qPCR Master Mix (Vazyme, Nanjing, China) was used. ATCB (β-actin) was used for gene expression normalization.

[0138] 12. Proliferation and Migration Analysis

[0139] The CCK8 assay was used to assess the proliferation capacity of ESCC cells. Specifically, 1 × 10^3 cells were seeded into each well of a 96-well plate. To assess cell proliferation at 0, 24, 48, and 72 hours, 10 μL of CCK8 reagent was added to each well, and the cells were co-incubated for 2 hours. The absorbance of the ESCC cells in each well was measured using a microplate reader. To investigate cell migration, ESCC cells were resuspended in 200 μL of serum-free RPMI 1640 medium and added to the upper chamber of a transwell transfer apparatus at a density of 1 × 10^4 cells per well. Subsequently, 800 μL of complete medium was added to the lower chamber as a chemical inducer. After incubation at 37°C for 24 hours, cells on the uncoated lower surface of the membrane were fixed with 4% paraformaldehyde and then stained with crystal violet. Five representative fields of view for each membrane were photographed using a 10x microscope. The number of migrating cells was counted, and the relative migration rate was calculated.

[0140] 13. Scratch test

[0141] KYSE30 and TE1 cells were seeded into 6-well plates. When the cell density reached 80%, a straight line was drawn on the cell monolayer using a sterile 200 μL pipette tip. Cell migration was observed under a 10x optical microscope at 0, 24, and 48 hours after the drawing. The closure area was calculated using ImageJ software.

[0142] 14. Clonogenesis assay

[0143] 500 to 3000 cells were seeded into 6-well plates and cultured at 37°C and 5% CO2. The cells were then irradiated with 6 Mv X-rays at different doses of 0, 2, 4, and 6 Gy. After 14 days of culture, the cells were washed with PBS and then stained with 0.5% crystal violet. Finally, the number of colonies formed was counted under a microscope.

[0144] 15. Oxygen Consumption Rate (OCR) Analysis

[0145] OCR (oxidative reduction capacity) measurements were performed using a Seahorse XF24 analyzer (Seahorse Biosciences). Specifically, KYSE30 and TE1 cells were seeded into XF 96-well plates (10,000 cells per well) and cultured for 24 hours using an Agilent Technologies (Santa Clara) instrument. For OCR measurement, cells were covered with 80 μL of assay medium (Seahorse XF basal medium (102353), containing 100 mmol sodium pyruvate, 200 mmol L-glutamine, and 1 mol glucose). 1.5 μmol oligomycin, 1 μmol FCCP, 0.5 μmol rotenone, and antimycin A were injected via a Port syringe. OCR measurements were performed in real-time using a Seahorse XF24 analyzer (Agilent Technologies) and glycolysis stress and mitochondrial stress assay kits, respectively, according to the manufacturer's instructions.

[0146] 16. RNA sequencing and data analysis

[0147] Both KYSE410 and KYSE410R cells were irradiated with 4 Gy. After 24 hours, these irradiated cells (KYSE410_IR and KYSE410R_IR) were collected together with the unirradiated cells (KYSE410 and KYSE410R) and sent to BenQ Biosciences in Shanghai, China for RNA sequencing analysis.

[0148] 17. Mouse xenograft tumor model

[0149] To assess tumor growth, a subcutaneous xenograft mouse model was used. The animal experiments were approved by the Ethics Committee of Renji Hospital affiliated with Shanghai Jiao Tong University. In the experiment, 4-6 week old female nude mice, after one week of acclimatization, were injected subcutaneously into the right thigh with 3 × 10^6 KYSE30 cells. When the average tumor volume reached 100 mm²... 3 Mice were randomly assigned to four groups: a control group, a radiotherapy group, an inhibitor group, or a combination of radiotherapy and inhibitors. The radiotherapy group subsequently received 10 Gy X-ray irradiation targeting the tumor region. Starting the day after irradiation, the IACS-010759 inhibitor was administered orally at a dose of 7.5 mg / kg every two days, while dacomitinib was administered orally at a dose of 7 mg / kg every two days. Tumor volume was measured three times weekly and calculated using the formula (length × width²) / ².

[0150] 18. Statistical Analysis

[0151] Most data are presented as mean ± standard deviation, while growth curves for xenograft tumors are presented as mean ± standard error. Student's t-test was used for comparisons between the two groups, and two-way ANOVA was performed on tumor growth and cell growth curves using GraphPad Prism 5.0 software. A p-value less than 0.05 was considered statistically significant.

[0152] Example 1. ESCC patients with good response to nCRT have disrupted TCA circulation metabolism.

[0153] To identify metabolic differences between nCRT pCR and non-pCR ESCC patients, researchers collected serum samples from 59 ESCC patients before and after nCRT treatment. Based on the pathological results of the surgical specimens, these patients were assessed and assigned to either the pCR or non-pCR group (Table 1). The research team used non-targeted (UPLC-QTOF MS) and targeted (UPLC-QTRAP MS) metabolomics methods to perform metabolomics analysis on serum samples from the four groups (pCR and non-pCR groups before and after nCRT treatment), analyzing approximately 300 metabolites. Unsupervised principal component analysis (PCA) score plots were used to analyze the metabolites. Figure 8 a) The metabolic profiles of the four groups did not show significant separation. However, the supervised PLS-DA model showed no significant differences between the pCR and non-pCR groups, either before or after nCRT treatment. Figure 1 a). However, nCRT treatment appears to help differentiate the metabolic profiles of the pCR and non-pCR groups ( Figure 1 a, 8b).

[0154] Subsequently, a pairwise analysis of metabolic differences among the four groups was performed using the PLS-DA model. The results showed that, both before and after nCRT treatment, no significant separation was observed in the metabolite profiles between the pCR and non-pCR groups. Figure 8 c and 8d). However, for both the pCR and non-pCR groups, the metabolite profiles showed good separation before and after nCRT treatment (c and 8d). Figure 1 b), the permutation test shows that the PLS-DA model fits well ( Figure 1 c). Most importantly, the pCR group showed greater separation in metabolic differences before and after nCRT compared to the non-pCR group. Figure 1 b, 1c) indicate that nCRT has a significant impact on the metabolome of ESCC patients, especially pCR patients.

[0155] To investigate the metabolic mechanisms affecting the efficacy of nCRT, differentially expressed metabolites (DEMs) were further analyzed before and after pCR, and before and after non-pCR. These metabolic pathways were enriched using KEGG. Figure 1 d and 1e). The results showed that nCRT interfered with amino acid metabolic pathways, such as arginine biosynthesis, in both the pCR and non-pCR groups. Figure 1 e). Furthermore, the TCA cycle and its related pathways, including glycolysis and pyruvate metabolism, were only affected by nCRT in the pCR group. Figure 1 e). In the pCR group, the relative abundance of the TCA cycle and its related intermediates (such as citric acid, α-ketoglutarate, malic acid, fumaric acid, and 2-hydroxyglutarate) increased after nCRT, while no such change was observed in the non-pCR group after nCRT. Figure 1 f), which indicates that the TCA cycle was disrupted in ESCC patients who achieved complete pathological remission via nCRT.

[0156] Example 2. Inhibition of OXPHOS can enhance the radiosensitivity of ESCC cells.

[0157] The TCA cycle is a central hub for ATP production in mitochondrial oxidative phosphorylation (OXPHOS). To investigate the effects of the TCA cycle or OXPHOS on radiosensitivity, radioresistant cells KYSE410R (410R) were generated by exposing KYSE410 cells to a dose gradient of up to 36 Gy. Figure 2 a). Compared to 410 cells, 410R cells showed a higher oxygen consumption rate (OCR). Figure 2 b). Both 410 and 410R cells showed increased oxygen consumption after irradiation, but the increase was more significant in 410R cells. Figure 2 b). Therefore, irradiation specifically induced ATP production in 410R cells ( Figure 2 c) This suggests that radioresistant cells may be more dependent on the TCA cycle and OXPHOS. Consistently, inhibitors blocking the TCA cycle (CPI-613, FH-IN-1, 3-NP) or OXPHOS (IACS-010759, rotenone, Gboxin, antimycin A) increased the radiosensitivity of human esophageal cancer cells. Figure 2 d-2e, Figure 9 a). It is noteworthy that TCA cycle inhibitors only interfere with the levels of one or two TCA cycle intermediates, while OXPHOS inhibitors block the entire TCA cycle, leading to the accumulation of multiple TCA cycle intermediates. Figure 2 f, Figure 9(b) This is consistent with changes in serum metabolites in nCRT pCR ESCC patients. Accumulation of TCA circulating intermediates was observed after irradiation in sensitive cell lines, while this phenomenon was not observed in resistant cell lines. Figure 9 c). Therefore, OXPHOS activity plays a crucial role in the radioresistance of ESCC cells.

[0158] Example 3. AREG and EREG modulate the radiosensitivity of ESCC via OXPHOS

[0159] To elucidate the mechanism by which radiotherapy regulates OXPHOS, researchers analyzed the transcriptomes of KYSE410 and KYSE410R cells before and after irradiation. Venn diagrams revealed significant differences in the expression of 55 genes (DEGs) across four comparisons. Figure 3 a). KEGG pathway analysis showed that these 55 DEGs were mainly concentrated in the colorectal cancer, ERBB, TGF-β, and MAPK signaling pathways, among which the ERBB pathway was the main signaling pathway in the radiation response (a). Figure 3 b). In the TCGA ESCA dataset, the expression of characteristic genes of the ERBB signaling pathway was positively correlated with the expression of characteristic genes of OXPHOS. Figure 3 c). AREG and EREG, two EGF-like growth factors that bind to ERBB to activate this pathway, are upregulated in ESCC cells due to irradiation. Figure 3 de), and in radiation-resistant 410R cells, the expression levels of these genes were significantly higher than in 410 cells (de), Figure 3 d). Furthermore, the ERBB pathway regulates OXPHOS in ESCC cells. First, knockdown of AREG or EREG significantly reduced oxygen consumption (OCR) and ATP production in ESCC cells. Figure 3 Secondly, AREG or EREG directly activate OXPHOS because they increase OCR and ATP production (fh). Figure 3 Third, blocking the ERBB pathway reduces OXPHOS activity because ERBB inhibitor treatment reduces OCR and ATP production. Figure 3 These data indicate that the ERBB pathway regulates OXPHOS in ESCC cells in response to irradiation. The expression of AKT / mTOR signaling pathway and mitochondrial complex proteins increased in radiation-resistant cell lines and after IR treatment. Figure 3 k).

[0160] Further investigation was conducted into the relationship between the ERBB signaling pathway and the radiosensitivity of esophageal squamous cell carcinoma (ESCC) cells. Firstly, by inhibiting the ERBB signaling pathway using varlitinib or dacomitinib, ESCC cells were made more sensitive to radiotherapy. Figure 3 Secondly, knocking down AREG or EREG gene expression enhanced the radiosensitivity of ESCC cells. Figure 3 Third, compared with pCR patients, the expression levels of AREG and EREG in tumor tissues of non-pCR patients undergoing nCRT were significantly increased, indicating that high levels of AREG or EREG are associated with resistance to nCRT treatment. Figure 3 Finally, baseline serum AREG levels in non-pCR patients were higher than those in pCR patients after nCRT. After nCRT, serum AREG and EREG levels in non-pCR patients were significantly higher than those in pCR patients, suggesting that AREG may serve as a liquid biopsy-based biomarker for assessing nCRT response in ESCC patients. Figure 3 In summary, these data indicate that AREG and EREG influence the radiosensitivity of ESCC cells by regulating the OXPHOS process through ERBB.

[0161] Example 4. AREG and EREG promote tumorigenesis in ESCC cells.

[0162] According to the TCGA database, AREG and EREG are upregulated in multiple cancer types, including ESCA ( Figure 10 a), and high expression of AREG and EREG is associated with a poorer prognosis ( Figure 10 b, 10c). Similarly, compared with adjacent normal tissue samples, the expression of AREG and EREG was significantly increased in ESCC tumor samples (b, 10c). Figure 4 a). High expression levels of AREG or EREG are associated with lower overall survival in ESCC patients. Figure 4 b). Knockdown of AREG or EREG using shRNAs significantly inhibited the proliferation of ESCC cells. Figure 4 c) Settlement formation ability ( Figure 4 d) Transferability ( Figure 4 These data indicate that AREG and EREG are oncogenes in ESCC.

[0163] Example 5. CEBPB regulates the expression of AREG and EREG in ESCC cells in response to irradiation.

[0164] The mRNA levels of AREG and EREG showed a high correlation in almost all cancer types. Figure 11a, 11b). In esophageal squamous cell carcinoma (ESCC) samples, the expression of both also showed a significant correlation ( Figure 5 a) This suggests that they may be co-regulated by the same transcription factor. Since IR simultaneously upregulates the expression of AREG and EREG, it is speculated that they share the same regulatory factor. After predicting the common transcription factor using three online software programs—CIST, GTRD, and ChEA3—CEBPB was found to be the only transcription factor capable of simultaneously regulating both. Figure 5 b). In the TCGA ESCA data, CEBPB expression was significantly correlated with AREG or EREG ( Figure 5 c). Knocking down CEBPB reduced the expression of AREG and EREG. Figure 5 d), while overexpression induces its expression in ESCC cells ( Figure 5 e). It is worth noting that CEBPB also showed a similar upward trend to AREG and EREG under radiation stimulation (e). Figure 5 f). More importantly, knockdown of CEBPB significantly inhibited radiation-induced AREG and EREG expression (f). Figure 5 g), indicating that in ESCC cells, the radiation response regulation of AREG and EREG mainly depends on CEBPB.

[0165] Example 6. CEBPB modulates the radiosensitivity of ESCC via OXPHOS

[0166] In the TCGA ESCA database, the expression level of CEBPB was significantly upregulated. Figure 6 a). Knockdown of CEBPB inhibits the proliferation, colony formation, wound healing, and migration abilities of ESCC cells. Figure 6 This effect (b-6e) is consistent with knockdown of AREG or EREG, indicating that CEBPB has an oncogenic effect in ESCC cells. Notably, knockdown of CEBPB also reduces oxygen consumption and ATP production in ESCC cells. Figure 6 f and 6g), while overexpression of CEBPB increases these indicators (f and 6g). Figure 6 h and 6i). Simultaneously, knocking down CEBPB also enhances the radiosensitivity of ESCC cells (h and 6i). Figure 6 Furthermore, compared with nCRT pCR patients, nCRT non-pCR patients had higher levels of CEBPB expression (j). Figure 6 These data indicate that CEBPB, as a transcriptional regulator of AREG and EREG, regulates the radiosensitivity of ESCC cells through oxidative phosphorylation.

[0167] Example 7. Inhibition of OXPHOS or AREG / EREG / ERBB can increase the radiosensitivity of ESCC tumors.

[0168] The ARB / EREG / ERBB axis regulates radiosensitivity in esophageal squamous cell carcinoma (ESCC) via oxidative phosphorylation (OXPHOS). Inhibiting the OXPHOS or ERBB signaling pathways may be an effective strategy to improve radiotherapy efficacy. Currently, inhibitors targeting OXPHOS complex I (IACS-010759) and ERBBs (dacomitinib) are being evaluated in clinical trials for their application in cancer treatment. IACS-0101759 or IR alone showed moderate inhibitory effects on subcutaneous xenograft tumors generated from KYSE30 cells; however, the combination of IACS-0101759 and IR significantly inhibited the growth of these tumors. Figure 7 TUNEL assays showed that the combination of IACS-0101759 and IR induced more cell death than either IR or IACS-0101759 alone. Figure 7 d). Similarly, dacomitinib significantly improved the radiosensitivity of subcutaneous xenograft tumors ( Figure 7 e.g., and the combination of dacrotinib and IR induced more cell death than IR or dacrotinib alone (Figure h). In summary, both OXPHOS inhibition and ERBB inhibition can enhance the radiosensitivity of subcutaneous ESCC tumors.

[0169] Example 8. Predicting the efficacy of neoadjuvant chemoradiotherapy in esophageal squamous cell carcinoma by serum and tissue AREG / EREG levels

[0170] Immunohistochemical scoring was used to detect the expression of AREG and EREGIHC in the patient's tumor tissue. ROC analysis showed that AREG and EREGIHC scores could predict pCR well, with AUC values ​​of 0.929 and 0.890, respectively, and a cutoff value of 10.5 for both. Figure 12 a, Table 2). Serum AREG and EREG levels in non-pCR patients were higher at baseline than in pCR patients. After radiotherapy, serum AREG and EREG levels in non-pCR patients increased significantly, resulting in significant differences in post-radiotherapy serum AREG and EREG levels between the pCR and non-pCR groups. Therefore, we used post-radiotherapy serum AREG and EREG levels to predict pCR. ROC analysis showed AUC values ​​of 0.846 and 0.938, respectively, and cutoff values ​​of 103.6 pg / ml and 150.1 pg / ml, respectively. Figure 12 b, Table 2).

[0171] Table 2. Tissue and serum AREG and EREG levels predict pCR in neoadjuvant chemoradiotherapy for esophageal squamous cell carcinoma.

[0172]

[0173] in conclusion

[0174] This application reveals a previously unknown metabolic mechanism in which mitochondrial oxidative phosphorylation (OXPHOS) is involved in the radiosensitivity of esophageal squamous cell carcinoma (ESCC) cells. CEBPB co-transcription factors regulate the AREG / EREG / ERBB axis, influencing radiosensitivity via OXPHOS. Inhibiting ERBB or OXPHOS may be an effective method to improve the radiosensitivity of ESCC cells. Furthermore, AREG and EREG, as biomarkers, can be used to assess the nCRT sensitivity of ESCC patients.

[0175] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A method for predicting the sensitivity of esophageal squamous cell carcinoma (ESCC) patients to neoadjuvant chemoradiotherapy, characterized in that, The method includes: A. Provide test samples from ESCC patients; B. Detect the levels of AREG and / or EREG proteins in the sample to be tested, and obtain the AREG level detection value C1 and the EREG level detection value C2; ​​and C. Compare C1 and C2 with the cutoff values ​​of AREG and / or EREG proteins to determine whether the ESCC patients are sensitive to neoadjuvant chemoradiotherapy; The serum cutoff value of the AREG protein was 103.6 pg / ml. The serum cutoff value of the EREG protein was 150.1 pg / ml.

2. The method as described in claim 1, characterized in that, The sample to be tested is selected from serum samples, cancer tissue samples, or a combination thereof.

3. The method as described in claim 1, characterized in that, If C1 > 103.6 pg / ml and / or C2 > 150.1 pg / ml, the patient is considered insensitive to neoadjuvant chemoradiotherapy; if C1 < 103.6 pg / ml and / or C2 < 150.1 pg / ml, the patient is considered sensitive to neoadjuvant chemoradiotherapy.

4. The method as described in claim 1, characterized in that, The cutoff values ​​for AREG and / or EREG proteins also include the cutoff values ​​for tissue immunohistochemistry scores, with the cutoff values ​​for both AREG and EREG proteins being 10.

5.

5. The method as described in claim 4, characterized in that, If the immunohistochemical score of AREG and / or EREG proteins in the sample is greater than 10.5, the patient is considered insensitive to neoadjuvant chemoradiotherapy; if the immunohistochemical score of AREG and / or EREG proteins in the sample is less than 10.5, the patient is considered sensitive to neoadjuvant chemoradiotherapy.

6. A method for improving the sensitivity of esophageal squamous cell carcinoma (ESCC) patients to neoadjuvant chemoradiotherapy (nCRT), characterized in that, The method includes: 1) Reduce the levels of CEBPB, AREG, and / or EREG proteins; 2) Inhibit mitochondrial oxidative phosphorylation (OXPHOS) and / or the TCA cycle; and / or 3) Inhibit the ERBB signaling pathway. In another preferred embodiment, the regulation of AREG and / or EREG protein levels is described.

7. The method as described in claim 6, characterized in that, The levels of the CEBPB, AREG, and / or EREG proteins were positively correlated with nCRT resistance.

8. The method as described in claim 6, characterized in that, The activity of OXPHOS is positively correlated with nCRT resistance.

9. The method as described in claim 6, characterized in that, Inhibiting the ERBB pathway reduces the activity of OXPHOS, thereby increasing the sensitivity of ESCC patients to nCRT.

10. An ESCC cell line highly sensitive to neoadjuvant chemoradiotherapy, characterized in that, The ESCC cell line is an ESCC cell line treated using the method described in claim 2.

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