Application of ER alpha biomarker in diagnosis, treatment and prognosis of ER + breast cancer bone metastasis

Abstract

The invention belongs to the technical field of biomedical detection, and particularly relates to application of an ER alpha biomarker in diagnosis, treatment and prognosis of ER + breast cancer bone metastasis. The gene sequence of the ERalpha is shown as NM000125.4 of an NCBI gene pool, and the ERalpha promotes the ER + breast cancer bone metastasis by promoting differentiation and maturation of osteoclast precursors, so that the risk of bone metastasis of a patient can be predicted, and the effect of preventing bone metastasis is achieved. According to the application of the ERalpha biomarker or target in preparation of the ER + breast cancer bone metastasis diagnosis, treatment and prognosis product, compared with an existing detection kit, detection can only be carried out after bone metastasis occurs; the ERalpha can be used for early predicting the occurrence of ER + breast cancer bone metastasis, diagnosing the ER + breast cancer bone metastasis, predicting the disease progress, evaluating the treatment effect, guiding the medicine use and prognosis evaluation and serving as a target for treating the bone metastasis in a more characteristic and more sensitive manner.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedical detection, and specifically relates to the expression of ERα biomarkers in ER + Application in the diagnosis, treatment and prognosis of breast cancer bone metastasis. Background Art

[0002] Breast cancer is one of the most common malignant tumors among women worldwide. The main cause of death in breast cancer patients is metastasis to bones, brain, lungs and other internal organs. Among these target organs, bones are the most common site of breast cancer metastasis. More than 70% of patients with advanced breast cancer will experience bone metastasis, leading to severe bone pain, fractures and pathogenic complications. Clinical studies have shown that most breast cancers overexpress estrogen receptors (ER), and ER-positive breast cancers are more likely to metastasize to bones. Patients with bone metastasis have a poor prognosis, with a median survival of only 16 months. Despite the overexpression of ER + Breast cancer patients who receive regular adjuvant endocrine therapy after surgery often have delayed recurrence of cancer cells in the bone, suggesting that bone metastatic cells have developed resistance to endocrine therapy. The long dormancy period also reduces the effectiveness of current preventive bone-targeted therapies due to their significant side effects. Therefore, it is necessary to identify potential targets that can be used to predict ER + Biomarkers for the risk of bone metastasis recurrence in breast cancer patients are very important. Although there is a strong association between ER positivity and bone metastasis, ER + How breast cancer cells acquire bone tropism is still unclear. Understanding the mechanism of this organ tropism is crucial for determining ER + Specific biomarkers and targets for prevention and treatment of breast cancer bone metastasis offer important opportunities.

[0003] Most ER + Breast cancer patients develop osteolytic lesions, which is mainly due to osteoclast-mediated bone resorption. Breast cancer cells interact with the bone microenvironment and release growth factors embedded in the bone matrix, which in turn stimulate cancer cell growth, forming a "vicious cycle." Recent preclinical data suggest that bisphosphonates and denosumab can reduce the release of tumor-promoting growth factors in bone and delay the growth of bone tumors by inhibiting osteoclast-mediated bone destruction. However, these drugs have been shown to alleviate symptoms but not prolong patient survival. Therefore, effective treatments are urgently needed to prevent and treat ER. + Breast cancer bone metastasis.

[0004] In order to survive in the bone microenvironment, breast cancer cells need to interact with bone microenvironment cells. There is increasing evidence that, in addition to cytokines, cancer cells are also able to interact with microenvironment cells through extracellular vesicles. These vesicles encapsulate tumor-specific contents and deliver them to environmental cells and circulation. They promote tumor growth or affect surrounding cells to produce metastatic niches by directly transforming tumor cells. Therefore, depending on the tumor type, the functional molecules loaded by vesicles can be detected in serum or urine and used to predict and diagnose the risk of tumor metastasis to specific organs, assess the patient's prognosis, and provide opportunities for treatment. Therefore, it is necessary to look for vesicle markers to predict ER + Breast cancer bone metastasis and its prognosis assessment are of vital importance. Summary of the invention

[0005] In view of the above problems, the present invention aims to provide an ERα biomarker in ER + Application in the diagnosis, treatment and prognosis of breast cancer bone metastasis.

[0006] The technical contents of the present invention are as follows:

[0007] The present invention provides an ERα as an ER + Application of biomarkers or targets for diagnosis, treatment and prognosis of breast cancer bone metastasis;

[0008] The gene sequence of ERα is shown in NM_000125.4 of NCBI gene library and in SEQ ID NO.1 of sequence listing;

[0009] ERα promotes the differentiation and maturation of osteoclast precursors, thereby promoting ER + Breast cancer bone metastasis can predict the patient's risk of bone metastasis, thereby preventing bone metastasis.

[0010] The present invention also provides a reagent for detecting the expression level of ERα in the preparation of ER + Application in a product for diagnosis and / or prognosis analysis of breast cancer bone metastasis, wherein the ERα is as shown above;

[0011] The method of detecting the expression level of ERα includes detecting in blood and tissues;

[0012] The method of detecting the expression level of ERα includes detecting the serum protein content in the blood;

[0013] The ER + Products for diagnosis and / or prognosis analysis of breast cancer bone metastasis include one or more of detection reagents, test kits, test strips, and gene chips;

[0014] The kit includes a western blot or ELISA kit;

[0015] The ER + The breast cancer bone metastasis diagnosis and / or prognosis analysis product has at least one of the following functions:

[0016] (1)ER + Prediction of the occurrence of breast cancer bone metastasis;

[0017] (2)ER + Diagnosis of breast cancer bone metastasis;

[0018] (3)ER + Prediction of progression of breast cancer bone metastasis;

[0019] (4)ER + Inhibition of breast cancer bone metastasis;

[0020] (5)ER + Screening and stratification of risk groups for breast cancer bone metastasis.

[0021] The present invention also provides an ERα inhibitor or target for preparing an ERα inhibitor + Application of drugs in the treatment of breast cancer bone metastasis.

[0022] The beneficial effects of the present invention are as follows:

[0023] The reagent for detecting the expression level of ERα of the present invention is used in the preparation of ER + Application of the biomarker ERα in the diagnosis and prognosis of breast cancer bone metastasis, wherein the biomarker ERα can be enriched in ER specific for bone metastasis + Large extracellular vesicles (LOs) secreted by breast cancer cell lines are expressed in the ER of specific bone metastases + ERα is significantly increased in the supernatant of breast cancer cell lines, and the ERα promotes the differentiation and maturation of osteoclast precursors, thereby promoting ERα + Breast cancer bone metastasis can predict the risk of bone metastasis in patients, thereby achieving the effect of preventing bone metastasis. The ERα biomarker or target of the present invention is used in the preparation of ER + Application in diagnosis, treatment and prognosis of breast cancer bone metastasis, the product includes a detection reagent or a kit. Compared with the existing detection kits that can only detect after bone metastasis occurs, the ERα of the present invention can more specifically and sensitively predict ERα at an early stage. + The occurrence and diagnosis of breast cancer bone metastasis + Breast cancer bone metastasis, a kit for predicting disease progression, evaluating treatment efficacy, guiding drug use and prognosis evaluation, and as a target for treating bone metastasis. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1The expression of ERα in breast tissue and different ER + Representative images of breast cancer tissues expressing;

[0025] Figure 2 Patients without bone metastasis and ER + Expression of ERα in bones, TRAP staining images and CT images of patients with breast cancer bone metastasis;

[0026] Figure 3 To analyze the ERα expression in the serum of healthy subjects and patients for ELISA experiments;

[0027] Figure 4 Patients without bone metastasis and ER + Expression of ERα and ALP staining images in the bones of patients with breast cancer bone metastasis;

[0028] Figure 5 Representative fluorescence images of ERα-enriched extracellular vesicles (LOs) secreted by breast cancer cells with specific bone metastasis;

[0029] Figure 6 Phase contrast micrographs and TRAP staining images of osteoclast precursor cells;

[0030] Figure 7 Representative mouse bone lesions and histological images at the pretreatment and metastasis stages;

[0031] Figure 8 Normalized bioluminescent imaging signals for bone metastasis and Kaplan-Meier survival curves without bone metastasis for mice in designated experimental groups;

[0032] Fig. 9 Representative TRAP staining images and fluorescence images of LOs-packaged inhibitor ISA-2011B inhibiting LOs-induced osteoclastogenesis;

[0033] Fig.10 Representative mouse bone lesions and histological images at the pretreatment stage and metastatic stage for the inhibitor ISA-2011B packaged with LOs;

[0034] Fig.11 Normalized bioluminescent imaging signals of bone metastasis and Kaplan-Meier survival curves of mice without bone metastasis in designated experimental groups. DETAILED DESCRIPTION

[0035] The present invention is further described in detail below through specific implementation cases and accompanying drawings. It should be understood that these embodiments are only used to illustrate the present invention and are not used to limit the scope of protection of the present invention. After reading the present invention, various equivalent forms of modifications to the present invention by those skilled in the art are all within the scope of the claims attached to this application.

[0036] Unless otherwise specified, all raw materials and reagents of the present invention are raw materials and reagents from the conventional market.

[0037] Example 1

[0038] One as ER + ERα: a biomarker for diagnosis, treatment and prognosis of breast cancer bone metastasis

[0039] like Figure 1 As shown in Figure a, ERα in situ in normal human breast tissue without bone metastasis + ER in situ in breast cancer tissues and bone metastases + ER in breast cancer tissue and bone metastasis + Representative images of immunohistochemistry in breast cancer tissue (left) and analysis of the correlation between ERα localization and bone metastasis (right), scale bar, 20μm. The breast cancer tissue of the patient was fixed with formalin and then prepared into paraffin sections for immunohistochemical staining. The results showed that compared with normal breast tissue and ERα without bone metastasis, + Compared with breast cancer tissues, ERα was localized on the cell membrane in breast cancer tissues with bone metastasis, and the expression of membrane-localized ERα was similar to that of ERα. + Breast cancer bone metastasis is positively correlated, which shows that ERα can be applied to ER + Diagnosis of breast cancer bone metastasis;

[0040] Figure b shows membrane-localized and non-membrane-localized ERα and ER + Kaplan-Meier analysis of bone metastasis-free survival in breast cancer patients (n=26; P=0.003, log-rank test) showed that membrane-localized ERα significantly promoted ER + Breast cancer bone metastasis, ERα can be applied to ER + Diagnosis and prognosis of breast cancer bone metastasis.

[0041] Figure 2 For breast cancer patients without bone metastasis and ER + Representative images of immunohistochemistry and TRAP staining of bone tissues from patients with breast cancer bone metastasis and CT images of patients. Scale bar, 20 μm. Results show: + In tissues of patients with breast cancer bone metastasis, ERα was localized on the osteoclast membrane and showed osteolytic lesions in the patients. In breast cancer patients without bone metastasis, ERα was only localized in the nucleus. This result suggests that membrane-localized ERα plays an important role in osteoclast differentiation.

[0042] The following is the detection of ERα levels in blood samples (serum) of breast cancer patients using an ERα enzyme-linked immunosorbent assay kit (DYC5715-2, R&D, San Diego, California, USA.):

[0043] 1) Blood samples from breast cancer patients were collected into serum separation tubes. After clot formation, the samples were centrifuged at 2000 g for 10 minutes, and the serum was collected. The samples were diluted 10 times and measured. The undiluted serum was stored below -20°C to avoid repeated freeze-thaw cycles.

[0044] 2) Test the blood sample according to the instructions of the ERα ELISA kit:

[0045] The serum samples and all the actual consumables in the experiment were left at room temperature for about 1 h;

[0046] The results are as follows Figure 3 The results show that the serum ERα level and ER + The correlation between ERα and bone metastasis of breast cancer is that compared with the serum of healthy people and breast cancer patients without bone metastasis, the level of ERα in the serum of breast cancer patients with bone metastasis is significantly increased, indicating that the level of ERα in serum increases before breast cancer bone metastasis. It can be seen that ERα in serum can be used for ER + Diagnosis of bone metastasis from breast cancer.

[0047] The ERα of the present invention promotes the differentiation and maturation of osteoclast precursors, thereby promoting ER + Breast cancer bone metastasis can predict the risk of bone metastasis in patients, thereby preventing bone metastasis. The results and analysis of the following figures show that:

[0048] Figure 4 For breast cancer patients without bone metastasis and ER + Representative images of immunohistochemistry and ALP staining of bone tissues from patients with breast cancer bone metastasis. Scale bar, 20 μm. The results showed that ERα had no effect on osteoblasts.

[0049] Figure 5 Representative fluorescence images of ERα-enriched extracellular vesicles (LOs) secreted by breast cancer cells with specific bone metastasis, compared with the control group n-ER + Compared with breast cancer cells, m-ER + In breast cancer cells and MCF-7 cells, ERα is present on LOs.

[0050] Figure 6 Osteoclast precursor cells were treated with LOs of the indicated cells, and then osteoclasts were stained with TRAP (top). It can be seen that compared with the LOs of the control cells, the m-ERα with high expression of membrane ERα+ LOs of breast cancer cells and MCF-7 cells promoted osteoclast differentiation; while n-ER + LOs of breast cancer cells inhibited osteoclast differentiation, which suggested that VAPA plays an important role in osteoclast differentiation. Figure 6 The bone slices were subjected to scanning electron microscopy (SEM) to detect the results of osteoclasts absorbing bone (below). The results showed that compared with the LOs of the control cells, the m-ERα highly expressed in the membrane + LOs in breast cancer cells and MCF-7 cells promoted osteoclasts to absorb bone; while n-ER + LOs of breast cancer cells inhibited osteoclasts from resorbing bone. This result proves that membrane ERα promotes the ability of osteoclasts to resorb bone.

[0051] Figure 7 Middle, left: μCT (trabecular bone) and histological (TRAP and ALP staining) images of representative mouse bone sites during the pretreatment phase (left), tumor metastasis phase, and injection of n-ER + BLI, μCT, and histological (H&E staining) images of mouse bone lesions after breast cancer cells (right);

[0052] Figure 8 The BLI signal (Bioluminescence Imaging) of the mice with bone metastasis and the Kaplan-Meier survival curve without bone metastasis in the designated experimental group (n=5 / group) were normalized. The LOs of tumor cells were injected into the mice by intraperitoneal injection for two weeks, and then n-ER was injected into the left ventricle. + Breast cancer cells were injected into mice and the metastasis of tumor cells in the mice was observed. The results showed that LOs of cells with high expression of membrane ERα promoted bone metastasis of ER-positive breast cancer cells, while LOs of cells with low expression of membrane ERα inhibited bone metastasis of ER-positive breast cancer cells. This result proves that membrane ERα promotes bone metastasis of ER-positive breast cancer cells.

[0053] Fig. 9 Representative images of TRAP staining and F-actin staining in preosteoclasts treated with vector or ISA-2011B from corresponding breast cancer cells. The inhibitor ISA-2011B was added to ERα-enriched LOs. LOs / ISA-2011B significantly inhibited the effect of ERα-enriched LOs on promoting preosteoclast fusion.

[0054] Fig.10 Mice were treated with LOs / vehicle or LOs / ISA-2011B for 14 days, followed by intracardiac injection of n-ER +Breast cancer cells. Left: μCT (trabecular bone) images and histology (TRAP and ALP staining) images of representative mouse bone sites during the pretreatment phase (left), tumor metastasis phase, and injection of n-ER + BLI, μCT, and histological (H&E staining) images of mouse bone lesions after breast cancer cells were injected into the bone marrow; LOs / ISA-2011B treatment significantly suppressed the educational effect of ERα-enriched LOs, showing smaller osteolytic lesions, less bone microarchitectural changes, and TRAP on the bone surface. + The number of osteoclasts decreases.

[0055] Fig.11 Normalized BLI signals (Bioluminescence Imaging) of bone metastasis and Kaplan-Meier survival curves of mice without bone metastasis in designated experimental groups (n=5 / group).

[0056] The above results demonstrate that ERα secreted by ER-positive breast cancer cells and loaded with LOs promotes osteolytic bone metastasis of ER-positive breast cancer. However, downregulating ERα significantly reduced the bone metastasis and osteolytic ability of ER-positive breast cancer cells, which was manifested as a lighter bone metastasis burden and smaller osteolytic lesions, indicating that targeting ERα is an important node for inhibiting bone metastasis of liver cancer.

[0057] Based on the above experimental results and the figures, the present invention shows that ERα as an ER + Role of diagnostic, therapeutic, and prognostic biomarkers or targets in breast cancer bone metastasis.

[0058] Compared with the existing detection kits that can only detect after bone metastasis occurs, the ERα described in the present invention can more specifically and sensitively predict ERα at an early stage. + The occurrence and diagnosis of breast cancer bone metastasis + Breast cancer bone metastasis, a kit for predicting disease progression, evaluating treatment efficacy, guiding drug use and prognosis evaluation, and as a target for treating bone metastasis.

Claims

1. One ERα as ER + Application of biomarkers or targets for diagnosis, treatment, and prognosis of breast cancer bone metastasis.

2. The use according to claim 1, characterized in that: The gene sequence of ERα is shown in NM_000125.4 of NCBI gene library and in SEQ ID NO.1 of sequence listing.

3. A reagent for detecting ERα expression level in the preparation of ER + Application in breast cancer bone metastasis diagnosis and / or prognosis analysis products, characterized in that, The ERα is as described in claim 1.

4. The use according to claim 3, characterized in that: The method of detecting the expression level of ERα includes detecting in blood and tissues.

5. The use according to claim 3, characterized in that: The ER + Breast cancer bone metastasis diagnosis and / or prognosis analysis products include one or more detection reagents, test kits, test strips, and gene chips.

6. The use according to claim 5, characterized in that: The kit includes a western blot or an ELISA kit.

7. The use according to claim 3, characterized in that: The ER + The breast cancer bone metastasis diagnosis and / or prognosis analysis product has at least one of the following functions: (1)ER + Prediction of the occurrence of breast cancer bone metastasis; (2)ER + Diagnosis of breast cancer bone metastasis; (3)ER + Prediction of progression of breast cancer bone metastasis; (4)ER + Inhibition of breast cancer bone metastasis; (5)ER + Screening and stratification of risk groups for breast cancer bone metastasis.

8. An ERα inhibitor or target in the preparation of an ERα inhibitor + Application of drugs in the treatment of breast cancer bone metastasis.

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