ERCC6L facilitates the onset of mammary neoplasia and promotes the high malignance of breast cancer by accelerating cell cycle

doi:10.21203/rs.3.rs-2954620/v1

Download PDF

Research Article

ERCC6L facilitates the onset of mammary neoplasia and promotes the high malignance of breast cancer by accelerating cell cycle

https://doi.org/10.21203/rs.3.rs-2954620/v1

This work is licensed under a CC BY 4.0 License

Journal Publication

published 04 Sep, 2023

Read the published version in Journal of Experimental & Clinical Cancer Research →

You are reading this latest preprint version

Breast cancer (BRCA) is the first cause of morbidity and the second cause of death in female malignant tumors. Although available drugs have been approved for the corresponding breast cancer subtypes (ER Positive, HER2⁺) currently, there are still no effective targeted drugs and treatment strategies for metastatic breast cancer or triple-negative breast cancer that lack targets. Therefore, it’s urgent to discover new potential targets. ERCC6L is an essential protein involved in chromosome separation during cell mitosis. However, it’s not unclear the effect of ERCC6L on tumorigenesis and progression of breast cancer. Here, we found that ERCC6L was highly expressed in breast cancer, especially in TNBC, which was closely related to poor outcomes of patients. ERCC6L conditional knockout mouse model was first carried out in this study and results that ERCC6L was required for the development of mammary gland and the tumorigenesis and progression of mammary gland cancers were confirmed. In vitro cell culture, ERCC6L acted as a tumor promoter in the malignant progression of breast cancer cells. Overexpression of ERCC6L could promote cell proliferation, migration and invasion, while knockdown of ERCC6L can cause the opposite results. Mechanistically, ERCC6L accelerated the cell cycle by regulating the G₂/M checkpoint signaling pathway. Additionally, we demonstrated that there is an interaction between ERCC6L and KIF4A, both of which are a pair of closely related factors in mitosis and involved in the malignant progression of breast cancer. Taken together, ERCC6L may be used as a promising target for the treatment of BRCA.

Breast cancer

ERCC6L

Cell mitosis

Conditional knockout mice

KIF4A

Breast cancer (BRCA) is a malignant tumor occurring in the glandular epithelium of the breast. According to the statistics of the latest cancer released by CA (A Cancer Journal for Clinicians), the incidence rate of breast cancer ranks first among female malignant tumors (297790/948000), and has been on the rise since the mid-2000s (an annual increase of about 0.5%). In addition, breast cancer is the second leading cause of cancer death in women (43,170/287,740) [1]. BRCA can be divided into Luminal A, Luminal B, HER2-enriched, basal-like and triple-negative (ER⁻, PR⁻, HER2⁻) subtypes based on the gene expression profile [2]. Although there are marketed drugs for some BRCA subtypes, metastatic BRCA is still considered incurable [3]. Triple negative breast cancer (TNBC), as a kind with highly aggressive and malignant degree with the worst prognosis, has no specific targeted drugs due to the lack of therapeutic targets. Therefore, it is still necessary to discover novel potential targets for breast cancer, especially those lacking therapeutic targets, such as TNBC.

Cell division is the process of dividing cells into two, which is of great significance for growth, tissue repair and regeneration, and asexual reproduction of single cells. It is a strictly regulated and highly ordered process that includes interphase and M phases, monitored by inherent cycle checkpoints within the cell to ensure the fidelity of cell replication [4]. It was reported that most breast cancers (especially in later stages of the disease) exhibited some degree of abnormal genomic structure, such as aneuploidy chromosomes, which may be related to genetic defects of mitotic spindle examination sites [5]. Therefore, targeting those defects to further induce tumor cell death may be a potentially desirable strategy. In addition, a variety of PARP inhibitors have been approved for the treatment of metastatic breast cancer [6–7]. Inhibiting PARP can cause impaired repair of DNA single strand breaks and damage the stability of replication forks, thereby inducing DNA damage. During mitosis, the accumulation of damaged DNA further leads to abnormal chromosome segregation, cytokinesis failure, polykaryocytosis, and ultimately cell death, further suggesting the potential to target the unique cell cycle progression of BRCA to interfere with tumorigenesis and progression.

ERCC Exception Repair 6 Like (ERCC6L), belonging to the SWItch/Sucrose Non-Fermentable (SWI/SNF2) protein family, is a DNA helicase closely related to cell mitosis [8]. It is an essential component of the spindle assembly checkpoint (one of the important mechanisms to ensure proper chromosome separation during mitosis). What’s more, ERCC6L, as a DNA transposase, can translocate to the endocentric and kinetochore regions of mitotic chromosomes to regulate spindle assembly checkpoint signals [9], and can also bind to ribosome DNA and ultra-fine DNA bridges (UFBs) to participate in sister chromatids separation during mitosis. Besides, ERCC6L carries an SNF2-like ATPase domain, and its ATPase activity is required for the decomposition of UFBs before cytokinesis [10–11]. Therefore, ERCC6L plays an important role in cell survival and proliferation.

Some studies reported that the deletion of ERCC6L could induce chromosomal abnormalities (chromatin bridges, micronuclei, dikaryon, etc.), embryonic lethality, DNA damage, p53 activation, and increased apoptosis [12–13]. Those, to some extent, revealed the feasibility of targeting ERCC6L. ERCC6L was also reported to be abnormally expressed in various tumors, such as renal cell carcinoma, liver cancer, laryngeal squamous cell carcinoma, and was involved in processes such as cell proliferation, migration, and invasion [14–16]. Huang et al. [17] reported the high expression of ERCC6L and its potential role in TNBC. However, there is still little information about the application of ERCC6L knockout mouse model in BRCA. Moreover, its role in BRCA tumorigenesis and progression remains unclear. Therefore, further exploration is warranted.

In the study, we first demonstrated the potential function of ERCC6L using ERCC6L conditional knockout mouse model. Compared with wild-type mice, ERCC6L deletion delayed mammogenesis. By hybridizing ERCC6L conditional knockout mice with MMTV-PyMT mice, it was found that ERCC6L deficiency can significantly inhibit the occurrence and development of mammary gland tumors. As for the respect of mechanism, ERCC6L could accelerate the cell cycle by regulating p53/p21/CDK1/Cyclin B and PLK/CDC25C/CDK1/Cyclin B signal pathways, thereby promoting the malignant progression of breast cancer cell lines. Additionally, we also revealed the potential relationship between KIF4A and ERCC6L, that is, there was a direct interaction between KIF4A and ERCC6L, and both are closely associated with mitosis and contribute to tumor growth and metastasis. Taken together, our results suggested that ERCC6L played a vital role in the occurrence and development of BRCA and might be a potential target for BRCA treatment.

1. Cell culture and tumor specimens

The BRCA cell lines, ZR-75-1, MCF-7, T47D, BT474, HCC1937, MDA-MB-231, MDA-MB-435, MDA-MB-453, MDA-MB-468, and SKBR3 were purchased from American Type Culture Collection (ATCC). The Dulbecco’s modified Eagle’s medium (DMEM) and RPMI 1640 (Gibco, CA, USA) with 10% fetal bovine serum (FBS) was applied to culture MCF-7, MDA-MB-231, MDA-MB-435, MDA-MB-453, MDA-MB-468, SKBR3 and BT474, HCC1937, T47D, ZR-75-1 cell, respectively. The serum was purchased from Procell Life Science&Technology (Wuhan, China). All cells were cultured in a humidified incubator with 5% CO₂ at 37℃. The human BRCA and adjacent normal tissue used in this article were gifted from Dr Bailin Zhang (National Cancer Center/National Clinical Research Center for Cancer/Cancer Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College), which have been approved by Institutional Review Board (IRB).

2. Construction of stable cell lines and Transfection

For the construction of MCF-7 and T47D cell lines stably overexpressing ERCC6L, control and ERCC6L lentiviruses were used to infect both cells for 72 h, followed by selection with 3 µg/mL puromycin. For stably silencing of ERCC6L, MDA-MB-231 cells were infected with control, shERCC6L#1 and shERCC6L#2 lentiviruses respectively. Subsequently, they were screened with 3 µg/mL puromycin. For transient overexpression of KIF4A, the human full-length KIF4A cDNA clone was transfected into 231 cell lines using Lipofectamine 3000 regent in accordance with manufacturer's instructions.

3. Transgenic mice model and Tumor monitoring

The construction of ERCC6L^flox/flox and MMTV-Cre/Line D mice were provided by Biocytogen Pharmaceuticals Co., Ltd. Mice were kept in the Laboratory Animal Center, Institute of Materia Medica, Chinese Academy of Medical Sciences under 12 h/d light, 22 ± 3 ° C temperature and 50 ± 5% relative humidity. All experimental conditions and experiments performed on animals were in accordance with the guidelines of Animal Ethics Committee of Institute of Materia Medica, Chinese Academy of Medical Sciences (Ethics number: 00008101).

The flox-Cre recombination system was applied to generate the mice with mammary epithelial-cell specific ERCC6L deletion. ERCC6L^flox/wt Cre^+/− mice were generated by crossing ERCC6L^flox/flox mice with MMTV-Cre mice. And then, ERCC6L^flox/wt Cre^+/− mice were hybridized with ERCC6L^flox/y Cre^+/− mice and ERCC6L^wt/y Cre^+/− mice to obtain ERCC6L^flox/flox Cre^+/− mice and ERCC6L^wt/wt Cre^+/− mice, respectively. At 6, 8 and 12 weeks, the fourth groin mammary glands of female mice were weighed and performed with whole mount assay.

To generate PyMT mice, we crossbred female ERCC6L mice of different genotypes with MMTV-PyMT mice. At 8 weeks, whole mount staining was performed on the fourth groin mammary gland of female mice to evaluate multifocal dysplasia. To assess palpable tumor onset, animals were palpated weekly. After the formation of palpable tumors, the body weight of mice was recorded and tumor volume was measured using vernier calipers within 42 days. At 16 weeks, the mice were dissected and their body weight, the number of tumors per mice, and the wet weight of each tumor were recorded. Tumor tissues, lungs, and spleens were soaked in 4% paraformaldehyde (PFC) for Immunohistochemistry (IHC) and Immunofluorescence (IF). The genotypes of all female mice were identified by specific PCR analysis of mouse tail genomic DNA. Primer information was listed in Supplementary Table 1.

4. Nude mice axillary xenograft tumor model

Female BALB/c-nu nude mice, purchased from Beijing Vital River Laboratory Animal Technology, were divided into three groups (n = 6) and separately injected with MDA-MB-231 shNC, MDA-MB-231 shERCC6L#1, and MDA-MB-231 shERCC6L#2 cells (1x10⁷/0.1 mL). After the tumor has grown (less than 1000 mm³), each mouse was dissected to measure the volume of tumors. The calculation formula is: V = 0.5xLxW² mm³ (L represents length and W represents width, Unit: mm).

5. Whole mount staining

The fourth inguinal mammary gland was removed, spread on a glass slide, and air-dried for 5–10 min. Fixation was performed with Carnoy's solution (60% ethanol, 30% chloroform, and 10% glacial acetic acid) for 2–4 h. The slides were then immersed in gradient concentrations of ethanol (75%-50%-30%) for 15 min, respectively. After immersion in ddH₂O for 5 min, the slides were immersed in a container with dye liquor (0.2% carmine and 0.5% aluminum potassium sulfate) for 1–2 days. They were then decolorized with 70% ethanol containing 2% hydrochloric acid for 2 h, immersed in gradient concentrations of ethanol (70%-95%-100%) for 15 min, defatted with xylene for 20 min, and finally stored in methyl salicylate.

6. The assays of cell viability, migration and invasion

CCK-8 was carried out to check cell proliferation. The cells were first seeded in 96-well plates and then incubated in incubator for 24, 48,72 and 96 h. Finally, the plates were placed in SpectraMax M5 and the absorbance value of each well was measured at 450 nm for quantification. In terms of migration and invasion assay, the cells were counted and inoculated in 8 µm polycarbonate membranes in Costar 24-Transwell plates. Then, being cultured for 3–4 h (after cell attachment), serum-free medium and 10% FBS medium were added to the upper and bottom chambers, respectively. After 19 h (for migration) and 22 h (for invasion), cells were fixed with 4% paraformaldehyde (PFA) and stained with 1% crystal violet. Subsequently, after cells on upper chamber were wiped off, the chambers were placed under a microscope for observation. Note that in the invasion assay, the upper chambers need to be coated with Matrigel™ diluted in serum-free medium (1:7) before seeding the cells.

7. Soft agar colony formation assay and 3D Matrigel cell culture

The low melting point agarose was prepared at 3.5% mother liquor which need to be autoclaved before use. The mother liquor was diluted 1:5 (0.7% agar) and 1:10 (0.35% agar), respectively, and successively spread in 6-well plates to form the bottom and upper layers. The upper layer contains prepared cells (about 3000/well). Before being placed into the incubator, plates were first put in a 4 ° C refrigerator for 30 min. After being plated in incubator for 2–3 weeks, cells were stained with MTT solution (5 mg/mL) and then counted. For the 3D Matrigel assay, the cell suspension and Matrigel were mixed in a 1:1 ratio and inoculated in 96-well plates. After two weeks, clones can be observed and photographed.

8. Edu assay

Cells were plated in 96-well plates. Edu reagent (1:1000 dilution) was added to plates and incubated for 2 h. The cells were then fixed with 4% PFA for 30 min, washed with 2 mg/mL glycine solution and PBS for 2–3 times, respectively, and then permeabilized with PBS with 0.5% Triton X. After the permeabilization, the prepared 1 x Apollo staining solution and hochest3342 staining solution were added successively, and the cells were incubated in the dark at room temperature for 30 min. Finally, the cells were observed under a microscope.

9. Flow cytometry for cell cycle assay

The cells were seeded in 60-mm medium dishes. After 48 h, cells were collected, washed with PBS, and fixed overnight with 70% ethanol at 4 ° C. Then, the cells were washed again with PBS and incubated successively in PBS with RNase (20 µg/mL) and PBS with 50 µg/mL PI and 0.5% Triton X for 30 min at room temperature in the dark. Finally, the cell suspension was filtered through a 300-mesh screen and its fluorescence was detected using Accuri C6. FlowJo v10 was used for data processing.

10. Immunohistochemistry (IHC) and Immunofluorescence (IF)

Human BRCA tissue samples and animal tissues were prefixed and made into paraffin sections. After roasted, dewaxed, and hydrated, the sections were infiltrated with preheated blocking permeabilizing solution (PBS with 30% H₂O₂ and Triton X) for 30 min to reduce endogenous peroxidase activity. Subsequently, the sections were antigen repaired and blocked using sodium citrate buffer (PH 6.0) and serum, respectively. ERCC6L, KIF4A, Ki67, F4/80, and CD31 (1:200 dilution) were added to the tissue sections for incubation at 4 ° C overnight. The next day, sections were washed and incubated with secondary antibodies for 1–2 h at 37 ° C. Diaminobenzidine (DAB), Mayer's Hematoxylin Stain Solution and neutral balata were subsequently applied to stain, counterstain (negative controls were treated with rabbit serum) and seal slides. Finally, the stained sections can be photographed under a Leica TCS SP8X confocal microscope. The staining density and positive rate of protein were analyzed by Image J (V1.8.0).

The cells were pre-seeded onto the glass bottom cell culture dishes, and 24 h later, the cells were fixed with 4% PFA for 20 min, permeabilized with 0.3% Triton X for 10 min, and blocked with 5% BSA for 30 min at room temperature. At the end of blocking, diluted KIF4A (1:100) and ERCC6L (1:100) antibodies were added to the bottom of the dish and cells were incubated overnight at 4 ℃. Subsequently, the cells were stained with the corresponding fluorescent secondary antibodies for 1 h at room temperature (Alexa 488 for ERCC6L and Alexa 574 for KIF4A). In the end, cells were further stained with DAPI for 30 min. A confocal microscope was used to acquire images.

11. Protein extraction and immunoblotting analysis by Western blotting (WB)

Total proteins in human BRCA tissue samples, animal tissues and cells were extracted by RIPA lysates with protease and phosphatase inhibitors (CwBio, Beijing) and subsequently quantified by BCA method. For immunoblot, the equal amounts of proteins were electrophoresed on 8%-10% SDS-PAGE and subsequently transferred to PVDF membranes. After blocking the membranes with 5% BSA or 5% defatted milk for 1 h at room temperature, the membranes were incubated with primary antibody overnight at 4 ° C. After being washed with TBST, membranes were incubated with commensurable secondary antibodies. Finally, the membrane was cleaned again with TBST and then exposed in Tanon 5200 (Shanghai, China) by dropping ECL regents on it. Data was handled by Image J V1.8.0. The used antibodies are shown in Supplementary Table 1.

12. Co-immunoprecipitation (Co-IP)

Cells seeded into 100 mm dishes were lysed using IP lysis buffer and centrifuged to obtain the supernatant. Part of the supernatant could be directly applied for Western blotting after denaturation. For Co-IP, the supernatant was mixed with protein A/G agarose beads and anti-DDK beads, and then rotated overnight at 4 ° C. Subsequently, the beads were washed, mixed with 2 x SDS-PAGE loading buffer, and heated at 95 ° C for 5 min for Western blotting analysis.

13. Statistical analysis

Mean ± standard deviation (SD) was used for the presentation of the results. P values were used to evaluate the significance of the differences, which were calculated by unpaired two-tailed Student’s t-test or one-/two-way ANOVA. There were statistically significant differences when P values < 0.05.

1. ERCC6L is highly expressed in BRCA patients and associated with poor outcome

To evaluate the expression of ERCC6L in BRCA and whether it is associated with the progression of BRCA, in silico assays were performed using UALCAN (https://ualcan.path.uab.edu/), Oncomine (www.oncomine.org), and GEPIA (http://gepia.cancer-pku.cn/) database. The results showed that ERCC6L was highly expressed in BRAC regardless of the BRCA subtypes compared with normal controls (Fig. 1A and Supplementary Fig. S1a). Among them, the ER-negative patients have higher expression level of ERCC6L than ER-positive patients (Fig. 1B). The mRNA levels of ERCC6L in patients with basal-like breast cancer or triple-negative breast cancer were higher than that in patients with other subtype breast cancer, respectively (Fig. 1C and Supplementary Fig. S1b-c). Additionally, as the grade of BRAC increases, ERCC6L presented a higher expression level which is closely related to its aggressiveness and poor prognosis of BRCA patients (Fig. 1D-E and Supplementary Fig. S1d-f). Therefore, we speculate that ERCC6L may be involved in the malignant progression of breast tumors. Subsequently, to further determine the level of ERCC6L in human BRCA tissue samples and its prognostic value in BRCA patients. Ten pairs of tumor tissues samples and adjacent normal brain tissues from BRCA patients was used to assessed the expression of ERCC6L by Western blotting and results showed that ERCC6L was more highly expressed in BRCA tissues than in normal controls (Fig. 1F). Moreover, tumor specimens from 145 breast cancer patients were also examined by Immunohistochemistry (IHC) and representative pictures were shown in Fig. 1G. Results from IHC showed that the expression level of ERCC6L was positively correlated with the grade of BRCA and the worse prognosis of BRCA patients (Fig. 1I). Besides, a higher proportion of patients with high ERCC6L expression were found in ER-negative BRCA patients than in ER-positive BRCA patients (Fig. 1H). All these results were consistent with the results of in silico assays as mentioned above. We also checked the expression of ERCC6L in 11 BRCA cell lines (6 non-triple-negative and 5 triple-negative cell lines) and found that most of BRCA cell lines had high expression levels of ERCC6L. it is worth mentioning that triple-negative BRCA cell lines had an overall higher expression level of ERCC6L (Fig. 1J). This result was consistent with ERCC6L mRNA data from Human Protein Atlas (HPA) database (https://www.proteinatlas.org/) (Fig. 1K). Taken together, ERCC6L was highly expressed in BRCA patients and closely associated with a malignant degree of BRCA and poor prognosis of BRCA patients.

2. ERCC6L promoted the development of breast cancer in vivo and in vitro

To explore the functional role of ERCC6L in BRCA, loss- and gain-function assays were carried out. The expression of ERCC6L in MDA-MB-231 was silenced using two distinct shRNAs lentiviral targeting constructs (named as 231 shERCC6L#1 and 231 shERCC6L#2). Control cells infected with scramble shRNA lentiviruses were named 231 shNC. BRCA cell lines MCF-7 and T47D stably overexpressing ERCC6L protein were constructed by infected pHBLV-ERCC6L lentivirus (named as MCF-7 ERCC6L and T47D ERCC6L respectively). Control cells were designated as MCF-7 NC and T47D NC. The effects of knockdown and overexpression of ERCC6L were determined by Western blotting (Supplementary Fig. S2a). Then, proliferation assay, colony-forming method, 3D-matrigel culture model and Transwell assay were conducted to evaluate the functional role of ERCC6L on BRCA in vitro. Indeed, ERCC6L overexpression significantly promoted the proliferation, colony formation and cell growth in 3D Matrigel of both MCF-7 and T47D cells whereas ERCC6L knockdown had an inhibitory effect in MDA-MB-231 cells (Fig. 2A-C). The Edu assay revealed that ERCC6L could enhance DNA synthesis. Conversely, ERCC6L knockdown in MDA-MB-231 cells resulted in the opposing effect (Fig. 2D-E). Besides, migration and invasion were suppressed in MDA-MB-231 cells with ERCC6L knockdown, whereas both of them were enhanced in MCF-7 and T47D cells with ERCC6L overexpression (Fig. 2F and Supplementary Fig. S2b). To further confirm the effects of ERCC6L in vivo, human BRCA xenografts in nude mice were established using 231 shERCC6L cells. Compared with being inoculated with control cells, the tumor growth in nude mice inoculated with ERCC6L- knockdown 231 cells was significantly inhibited (Fig. 2G-H). The ERCC6L knockout efficiency of inoculated 231 cells was shown in Supplementary Fig. S2c. Altogether, ERCC6L is essential for the tumor progression and can promoted the development of breast cancer.

3. ERCC6L accelerates G ₂ /M phase transition of BRCA cells by regulating p53/p21/CDK1/Cyclin B and Aurora A/PLK/CDC25C/CDK1/Cyclin B pathways

ERCC6L is known as a protein involved in mitotic process. To explore its regulatory role in cell cycle, Flow cytometry was carried out. It was shown in Fig. 3A and B that ERCC6L overexpression reduced the proportion of MCF-7 and T47D cells in G₂/M phase, which indicated an accelerated rate of cell mitosis. In contrast, ERCC6L knockdown induced significant G₂/M phase arrest, which may further lead to the death of 231 cells. In line with this, the RNA-Seq data in 231 shERCC6L cells also showed an enrichment in the apoptosis signal pathway (Supplementary Fig. S3a). Based on the above results, ERCC6L may act as an important regulator in cell cycle progression of BRCA cells. To identify the potential mechanisms, we checked proteins at the G₂/M checkpoint using Western blotting. It was shown in Fig. 3C and D that overexpression of ERCC6L increased the expression level of CDK1 and Cyclin B and promoted the phosphorylation of CDK1 at Tyr 15 whereas ERCC6L knockdown led to adverse results. The Cyclin B-CDC2 (CDK1) complex, which is extremely essential for G₂ phase transition, is regulated by two parallel upstream cascades, PLK/CDC25C and p53/p21 respectively. Overexpression of ERCC6L downregulated the expression levels of p53 and p21, upregulated the levels of CDC25C and Aurora A, and promoted the phosphorylation of PLK at Thr 210 and CDC25C at Ser 216. Overall, ERCC6L regulated cell cycle though p53/p21/CDK1/Cyclin B and Aurora A/PLK/CDC25C/CDK1/Cyclin B pathways (Fig. 3E and F). All in all, these results suggested that ERCC6L accelerates G₂/M phase transition of BRCA cells by regulating p53/p21/CDK1/Cyclin B and Aurora A/PLK/CDC25C/CDK1/Cyclin B pathways.

4. ERCC6L is vital for the proper mouse ductal network development

To assess the function of ERCC6L in mammary gland development, a specific conditional knockout model of ERCC6L in the mammary gland through Cre/loxp-mediated recombination was generated (Fig. 4A). Floxed ERCC6L mice were intercrossed with the MMTV-Cre mice and then ERCC6L gene was specifically deleted due to the Cre recombinase expressed in the hybrid mice (Supplementary Fig. S4a). Specific primers for various ERCC6L alleles (i.e. wild-type, floxed and deleted) and for Cre were used in genotyping of female offspring. They were designated as ERCC6L^fl/fl Cre^+/− (ERCC6L^−/−), ERCC6L^fl/wt Cre^+/− (ERCC6L^+/−) and ERCC6L^wt/wt Cre^+/− (ERCC6L^+/+) (Fig. 4B). The deletion efficiency of ERCC6L in the mammary glands was confirmed by quantitative RT-qPCR, Western blotting of whole breast protein extracts and IHC of mammary gland tissues, respectively (Fig. 4C-D and Supplementary Fig. S4b).

Then, we dissected the fourth inguinal mammary glands from three genotype mice aged 6, 8, and 12 weeks, which were subsequently weighed and stained with alum carmine. Mouse mammary gland terminal end buds (TEB), having a high ability of division and proliferation, is composed of cap cells, myoepithelial cells and luminal epithelial cells and are closely related to mammogenesis. When the mammary gland enters a period of rapid development, the duct can extend towards the mammary fat pad under the guidance of the TEB and then form secondary and tertiary branches [18]. Therefore, TEB number and the branching complexity can indicate the developmental status of the mammary gland to a certain extent. As shown in Fig. 4E-H, the TEB number and the branching number of 6- and 8-week-old ERCC6L^+/+ mice were both higher than ERCC6L^+/− and ERCC6L^−/− mice aged 6 and 8 weeks. Furthermore, the amount of TEBs and branches in 6- and 8-week-old ERCC6L^+/− mice were also significant more than ERCC6L^−/− mice, respectively. But there was no difference in the mammary gland organ index among three genotypes of mice (Fig. 4I), which ruled out the impact of mouse self-growth on mammary duct development.

Additionally, the length of straight line from initiation point to TEB represents the ductal outgrowth to some extent. It was shown in Fig. 4J-K that the TEBs of most ERCC6L^+/+ mice aged 6 and 8 weeks penetrated the lymph node (LN). However, at 6 weeks, the TEBs of most ERCC6L^+/− mice were located near the LN, while those of most ERCC6L^−/− mice remained at the initiation site of the ductal and even approached LN at 8 weeks. Moreover, regardless of whether at 6 or 8 weeks, mammary fat pads of ERCC6L^+/+ mice had longer ductal outgrowth than ERCC6L^+/− and ERCC6L^−/− mice (Fig. 4L-O). Nonetheless, there were no significant differences in mammary gland organ index, TEB number, TEB bifurcation number and duct distance among the different genotypes aged 12 weeks (Supplementary Fig. S4c). It is speculated that the effect of their self-growth on mammoplasia exceeds the regulatory effect of ERCC6L knockout on mammogenesis in 12-week-old mice. Collectively, these results indicated that the deletion of ERCC6L could cause dysplasia of mammary gland.

Apart from organ index of mammary gland, other organ indexes were also detected in three genotypes of mice aged 6 and 8 weeks, including the heart, liver, spleen, lung, and kidney. However, there were no significant discrepancies in different organ indexes between these genotype mice (Supplementary Fig. S4d), except for spleen organ index in 8-week-old mice (Supplementary Fig. S4e-f). Considering that the spleen is a vital immune organ, we speculated whether ERCC6L has a certain correlation with immunity. To this end, bioinformatics analyses and immunofluorescence (IF) staining of the spleen in different genotypes mice at the age of 8 weeks were carried out. It was observed that ERCC6L was closely related to B cells, CD8⁺ T cells, especially macrophages. The deletion of ERCC6L could induce an increase of the amount of CD8⁺ T cells and macrophages (Supplementary Fig. S4g-h), which was also confirmed by IF (Supplementary Fig. S4i). Therefore, the cause of splenomegaly in 8-week-old ERCC6L^−/− mice may be due to the accumulation of T cells and macrophages in the spleen. Together, ERCC6L may affect the immunity of mice.

5. Effect of ERCC6L deletion on MMTV/PyMT tumor onset and growth

To assess the role of ERCC6L in breast tumorigenesis in vivo, a spontaneous breast cancer mouse model was constructed by crossing three genotype mice with MMTV-PyMT mice, named ERCC6L^+/+ PyMT, ERCC6L^+/− PyMT and ERCC6L^−/− PyMT respectively (Fig. 5A). MMTV-PyMT mouse was a widely-applied model for BRCA study due to its ability to generate spontaneous breast cancer tumors which were closely similar to the progression and morphology of human breast cancer [19]. PyMT could be identified by specific primers in this study (Supplementary Fig. S5a). And then we used palpation to investigate the mammary tumor onset in female mice and observed that both ERCC6L^−/− PyMT and ERCC6L^+/− PyMT mice slowed down mammary tumor onset, with the median tumor latency (T₅₀) of 78.5 days (n = 19) and 68.5 days (n = 10), respectively, significantly higher than that of ERCC6L^+/+ PyMT mice (T₅₀ = 44 days; n = 10) (Fig. 5B). Meanwhile, Whole mount staining of the mammary glands of 8-week-old non-tumor-bearing mice showed that ERCC6L deletion could inhibit early mammary hyperplastic lesion to some extent (Supplementary Fig. S5b).

The tumor growth rate of different genotypes of tumor-bearing mice was monitored to determine the effect of ERCC6L on tumor development. The results showed that the tumor growth speed of ERCC6L^+/+ PyMT mice was significantly faster than that of mice with ERCC6L deletion, and there was also a statistically significant difference in tumor growth rate between ERCC6L^+/− PyMT and ERCC6L^−/− PyMT mice (P = 0.0016) without significant change in body weight (Fig. 5C-D).

At 16 weeks, the mice were dissected and the tumor multiplicity (the number of tumors per mice), tumor burden, and individual tumor weight (the largest tumor weight) per mice were counted. Compared with ERCC6L^+/+ PyMT group, the number of tumors were significant reduced in ERCC6L deletion groups, especially in ERCC6L^−/− PyMT group (Fig. 5E). As shown in Fig. 5F and Supplementary Fig. S5c, there were less than three tumors in most ERCC6L^−/− PyMT mice, while ERCC6L^+/+ PyMT mice mostly carried more than six tumors. Besides, ERCC6L deletion mice had lower tumor burden and individual tumor weight than ERCC6L^+/+ PyMT mice with significant statistical discrepancies (Fig. 5G-I). However, there were no significant diversities in organ index of cardiac, liver, spleen, lung, and kidney (Fig. 5J). All these suggested that ERCC6L deletion has an effect on the occurrence and development of mammary gland tumors in vivo.

Subsequently, IHC was carried out in mammary gland tumor tissue samples in diverse genotype mice. Immunolabeling markers of Ki67, F4/80, and CD31 were used to evaluate cell proliferation, immune infiltration, and tumor angiogenesis in three groups of mice, respectively. IHC staining clearly showed that the percentages of Ki67-positive, F4/80-positive and CD31-positive cells were significantly reduced in the ERCC6L deletion group compared with ERCC6L^+/+ PyMT mice (Fig. 5K). We also detected the expression level of kinesin family member 4 (KIF4A) in tumor tissues, which also play a role in chromosome disjunction during mitosis, and found that KIF4A was highly expressed in tumor tissues with high ERCC6L expression level (Supplementary Fig. S5d). Pearson correlation analysis demonstrated that there was a strong positive correlation between ERCC6L and KIF4A (Supplementary Fig. S5e). These findings suggested that ERCC6L could facilitate the malignant progression in mammary gland tumor growth.

Since the spontaneous tumors of MMTV/PyMT mice tend to metastasize to the lung at a later stage [20], and our earlier data also showed that ERCC6L could promote the migration and invasion of BRCA cells. Therefore, to investigate whether ERCC6L deletion played a causal role in metastasis suppression in vivo, the lungs of each mouse were examined during dissection. Noted that not every mammary gland tumor-bearing mice occurred lung metastasis, but among different genotype mice with lung metastasis, there were significant differences in the number and size of metastasis (Fig. 5L-N). In summary, ERCC6L deletion inhibited onset and growth of MMTV/PyMT tumor.

6. KIF4A is closely related to ERCC6L in BRCA

As we found earlier, a strong correlation was observed between the expression of KIF4A and ERCC6L in mammary gland tumor tissues. Thus, we focused on KIF4A in BRCA. KIF4A is a member of the kinesin superfamily proteins (KIFs), whose members are involved in the transport of membranous organelles, protein complexes, mRNA and other substances [21]. Furthermore, KIF4A is also an ATP-dependent microtubule-based motor protein that participates in chromosome arms condensation and the maintenance of chromosome integrity during mitosis. In addition, it is reported that KIF4A is involved in central spindle formation and centrosome assembly prior to cytokinesis [22–23]. In summary, KIF4A is crucial for maintaining proper mitosis and its abnormal expression has been reported in various tumors [24–27]. To verify the potential association of KIF4A with ERCC6L in BRCA, in silicon assay was carried out and found that the expression of KIF4A was strongly related to ERCC6L (Pearson value = 0.880) (Fig. 6A). Moreover, KIF4A was observed to have a similar expression trend with ERCC6L in different types of BRCA (Supplementary Fig. S6a), and be highly expressed in high-grade tumors (Fig. 6B). This high expression of KIF4A was associated with poor outcome of BRCA patients (Fig. 6C). To explore the possible effect of KIF4A on the BRCA cells, three siRNAs were purchased and two siRNAs with high silencing efficiency were selected for experiments (Supplementary Fig. S6b). It was turned out that interfering the expression of KIF4A by siRNAs could inhibit the growth and migration of MDA-MB-231 cells (Supplementary Fig. S6c-d). Those indicated that KIF4A was also involved in progress of BRCA.

Nonetheless, what role ERCC6L and KIF4A play in tumor progression remains unclear. To check if there is an interaction between KIF4A and ERCC6L, immunofluorescence assay and Co-IP were carried out. Firstly, the confocal immunofluorescence assay confirmed that there is a co-localization of KIF4A and ERCC6L (Fig. 6D-E and Supplementary Fig. S6e). The reciprocal immunoprecipitation (IP) strongly determined a direct binding between KIF4A and ERCC6L (Fig. 6F). The Western blotting assay was performed in protein extracts from various cells that received transfection with KIF4A and ERCC6L plasmid, respectively, and showed that compared with the control group, KIF4A overexpression significantly upregulated the protein level of ERCC6L, while there was no significant change in the protein level of KIF4A after the overexpression of ERCC6L (Fig. 6G). Similarly, as shown in Supplementary Fig. S6f, KIF4A levels were also detected in MCF-7 and T47D cells with stable overexpression of ERCC6L and MDA-MB-231 cells with stable knockdown of ERCC6L, and the results showed that overexpression or knockdown of ERCC6L had little effect on KIF4A levels. However, after transfection of KIF4A plasmid in T47D, MCF7 and 231 cells, ERCC6L protein and mRNA levels were up-regulated (Supplementary Fig. S6g-h), further elucidating the potential relationship between ERCC6L and KIF4A. Therefore, we hypothesized that there was an interaction between KIF4A and ERCC6L.

To further validate the regulatory role of KIF4A on ERCC6L and whether KIF4A exerts its effect on cell functions and downstream pathways through ERCC6L, we transfected KIF4A plasmid into 231 shERCC6L and 231 shNC cells respectively. The expression level of KIF4A and ERCC6L in 231 cells was shown in Fig. 6H. The assay of proliferation, EDU and Transwell all showed that KIF4A overexpression could further promote the growth, proliferation, migration and invasion of 231 shNC cells and effectively reduced the inhibitory effect of ERCC6L knockout (Fig. 6I-M). Western blotting analysis also revealed that KIF4A overexpression could upregulate the expression of CDC25C and the phosphorylation of PLK, CDK1, and CDC25C, as well as downregulate the level of p53 and p21 in both 231 shNC and 231 shERCC6L cells (Fig. 6N-O). Overall, KIF4A could facilitate the malignant progression of BRCA cells by regulating the expression of ERCC6L. Collectively, ERCC6L interacted with KIF4A, which facilitates the malignant progression of BRCA.

BRCA has received much attention due to its high incidence in women, which is highly heterogeneous and can be divided into numerous subtypes. Drug reactivity and patient prognosis also vary according to BRCA subtypes. However, there is still no cure for advanced metastatic breast cancer and TNBC. Thus, discovering new potential targets is of great significance for the development of targeted drugs and prognosis improvement of patients. In the study, ERCC6L was found to be highly expressed in BRCA patients, and its expression level was positively correlated with the malignant grade of the tumor and the poor prognosis of patients, which to some extent suggested that ERCC6L played an essential role in the malignant progression of BRCA. Moreover, a higher expression level of ERCC6L was discovered in TNBC patients’ tissue and cell lines compared to other subtypes, indicating the potential value of ERCC6L in TNBC. Cell experiments in vitro were also performed and found that ERCC6L could facilitate the malignant progression of BRCA cells by promoting cell proliferation, migration and invasion. Knocking down ERCC6L could significantly inhibit tumor growth in nude mice. In brief, using both cell culture in vitro and nude mouse models in vivo, we demonstrated the important role of ERCC6L in breast tumorigenesis. Taken together, ERCC6L is a novel, potential target needed to be explored urgently.

Although a few studies have indicated that the knockdown of ERCC6L could induce cell cycle arrest, chromosome instability, and cell death in breast cancer cell lines. However, to our best knowledge, there is currently no in vivo study that directly examined the role of ERCC6L in breast tumorigenesis using ERCC6L conditional knockout mice. In our study, we explored the effects of ERCC6L on the development of mammary gland using an ERCC6L conditional knockout mouse model for the first time. Our results firstly showed that ERCC6L deletion significantly reduced the number of TEBs and ductal branches and significantly inhibited the extension of mammary branch ducts into the fat pad in both 6-week-old and 8-week-old mice, which suggested mammary dysplasia. However, mammary gland development in 12-week-old mice was not affected by ERCC6L deletion, and it is speculated that the effect of mouse self-growth and hormone levels exceeded the regulatory effect of ERCC6L on development. Interestingly, splenomegaly was found in 8-week-old ERCC6L^−/− mice, as indicated by increased infiltration of T cells, especially macrophages, in splenic tissues. One possible reason was that ERCC6L deletion mediated the generation of cells with a complex karyotype (aneuploid), which have been reported to induce self-elimination by the immune system [28]. However, the potential relationship between ERCC6L and immunity has not been reported, so further confirmation and exploration are still needed.

Notably, ERCC6L, a key mitotic checkpoint-associated protein, is theorized to induce increased chromosome missegregation and chromosomal instability (CIN) when absent, which can contribute to tumorigenesis [29]. However, ERCC6L^−/− or ERCC6L^+/− mice did not develop mammary gland tumors, which suggested that ERCC6L knockout does not induce tumorigenesis alone and it might not be a breast tumor susceptibility gene like BRCA1/2. But, when crossed with MMTV-PyMT mice, ERCC6L knockout significantly suppressed mammary tumorigenesis. The possible reason was that because PyMT itself could induce breast tumorigenesis, loss of ERCC6L caused PymT-mediated mammary gland tumor cells to occur G₂/M phase checkpoint disorder. Thereby, they could not repair the damaged DNA and directly enter mitosis, resulting in "mitotic catastrophe", which caused aneuploidy, micronuclei, etc. and eventually lead to tumor cell death. This was consistent with the results verified by Huang et al. [17] on breast cancer cell lines in vitro.

Given that a high proportion of MMTV-PyMT mice have observed lung metastasis [20]. Therefore, we also investigated the effect of ERCC6L on lung metastasis of mammary gland tumors. ERCC6L knockdown was first found to significantly suppress the number and area of lung metastases in tumor-bearing mice. However, further deep exploration is still needed on how ERCC6L plays a role in lung metastasis.

Another interesting finding in our study was that there was a direct interaction between ERCC6L and KIF4A. However, it remained unclear what consequences could be mediated by the interaction between ERCC6L and KIF4A. Moreover, ERCC6L and KIF4A may synergistically promote breast cancer cell proliferation, migration and invasion. But, how KIF4A regulates ERCC6L levels remains unknown. It is worth mentioning that Mazumdar et al. [30] found that knockdown of KIF4 resulted in changes in the mRNA levels of above 500 genes and could increase the expression of normally silent repetitive elements in heterochromatin domains. This indicates that KIF4 could be involved in transcriptional regulation, and this mechanism of transcriptional control was related to the interactions between proteins such as Poly (ADP-Ribose) Polymerase 1 (PARP-1), DNA methyltransferase 3B (DNMT3B) and the histone deacetylase HDAC1 [31]. Therefore, KIF4A may indirectly regulate the level of ERCC6L by regulating some related proteins. In summary, various evidences suggested that KIF4A was strongly associated with ERCC6L, however, the relationship between them still deserves further investigation.

Mechanistically, ERCC6L has been reported to act as a binding partner and substrate of polo-like kinases (PLKs), including PLK1, involved in centromeric chromatin remodeling [32–33]. And PLK1 is a key kinase for DNA damage checkpoint [34]. Hence, we investigated the role of ERCC6L in cell cycle regulation and found that ERCC6L knockdown could suppress the activation of PLK/CDC25C/CDK1/Cyclin B signaling pathway, thereby mediating cell cycle arrest. Furthermore, another important pathway for DNA damage checkpoint, p53/p21/CDK1/Cyclin B, was significantly regulated in ERCC6L knockdown cells.

In summary, we first demonstrated the role of ERCC6L in breast tumorigenesis and development in vivo using the ERCC6L conditional knockout mouse model, that is, ERCC6L is required for mammary gland tumorigenesis induced by MMTV-PyMT. Besides, ERCC6L, as a tumor promoter, could promote the proliferation, migration and invasion of breast cancer cells, and accelerate cell cycle progression by activating PLK/CDC25C/CDK1/Cyclin B and p53/p21/CDK1/Cyclin B signaling pathways. It is worth mentioning that ERCC6L was also demonstrated to have a close relationship with KIF4A, including interactions and regulatory relationships, which might be correlated with mitotic process. Taken together, targeting ERCC6L could be a promising strategy for the treatment of BRCA.

Acknowledgment and Funding Statement:

This work was supported by Beijing Natural Science Foundation (7212157, China), CAMS Innovation Fund for Medical Sciences (2021-I2M-1-029 and 2022-12M-JB-011, China) and National Natural Science Foundation of China (81703536, China).

Acknowledgment and Funding Statement:

Author contributions:

Hong Yang and Xiangjin Zhen were responsible for most of the experiments. Wan Li and Liwen Ren proofread the data. Yizhi Zhang, Sen Zhang, Binbin Ge and Yue Hao help to assist with animal sample collection. Guanhua Du modified manuscript. Jinhua Wang was responsible for conceive and design the work. All the authors warrant that the article has not received prior publication and is not under consideration for publication elsewhere.

Availability of data and material:

All data in this study are included in this published article, including its supplementary information files.

Ethics approval and consent to participate:

All experimental conditions and experiments performed on animals were in accordance with the guidelines of Animal Ethics Committee of Institute of Materia Medica, Chinese Academy of Medical Sciences, CAMS&PUMC (Ethics number: 00008101).

Consent for publication:

All authors agree with the publication of this manuscript in Journal of Experimental & Clinical Cancer Research.

Conflicts of Interest:

The authors declare that they have no conflicts of interest to report regarding the present study.

Siegel RL, Miller KD, Wagle NS et al. Cancer statistics, 2023 [J]. CA: a cancer journal for clinicians, 2023, 73(1): 17–48. https://doi.org/10.3322/caac.21763.
The Cancer Genome Atlas Network. Comprehensive molecular portraits of human breast tumours [J]. Nature. 2012;490(7418):61–70. https://doi.org/10.1038/nature11412.
Iwata H. Future treatment strategies for metastatic breast cancer: curable or incurable? [J]. Breast Cancer. 2012;19:200–5. https://doi.org/10.1007/s12282-011-0267-4.
Scholey JM, Brust-Mascher I, Mogilner A. Cell division [J]. Nature. 2003;422(6933):746–52. https://doi.org/10.1038/nature01599.
Yoon DS, Wersto RP, Zhou W, et al. Variable levels of chromosomal instability and mitotic spindle checkpoint defects in breast cancer [J]. Am J Pathol. 2002;161(2):391–7. https://doi.org/10.1016/S0002-9440(10)64194-6.
Tutt ANJ, Garber JE, Kaufman B, et al. Adjuvant olaparib for patients with BRCA1-or BRCA2-mutated breast cancer [J]. N Engl J Med. 2021;384(25):2394–405. https://doi.org/10.1056/NEJMoa2105215.
Litton JK, Rugo HS, Ettl J, et al. Talazoparib in patients with advanced breast cancer and a germline BRCA mutation [J]. N Engl J Med. 2018;379(8):753–63. https://doi.org/10.1056/NEJMoa1802905.
Ryan DP, Owen-Hughes T. Snf2-family proteins: chromatin remodellers for any occasion [J]. Curr Opin Chem Biol. 2011;15(5):649–56. https://doi.org/10.1016/j.cbpa.2011.07.022.
Baumann C, Körner R, Hofmann K, et al. PICH, a centromere-associated SNF2 family ATPase, is regulated by Plk1 and required for the spindle checkpoint [J]. Cell. 2007;128(1):101–14. https://doi.org/10.1016/j.cell.2006.11.041.
Biebricher A, Hirano S, Enzlin JH, et al. Mol Cell. 2013;51(5):691–701. https://doi.org/10.1016/j.molcel.2013.07.016. PICH: a DNA translocase specially adapted for processing anaphase bridge DNA [J].
Pitchai GP, Kaulich M, Bizard AH, et al. A novel TPR–BEN domain interaction mediates PICH–BEND3 association [J]. Nucleic Acids Res. 2017;45(19):11413–24. https://doi.org/10.1093/nar/gkx792.
Albers E, Sbroggiò M, Pladevall-Morera D, et al. Loss of PICH results in chromosomal instability, p53 activation, and embryonic lethality [J]. Cell Rep. 2018;24(12):3274–84. https://doi.org/10.1016/j.celrep.2018.08.071.
Nielsen CF, Huttner D, Bizard AH, et al. PICH promotes sister chromatid disjunction and co-operates with topoisomerase II in mitosis [J]. Nat Commun. 2015;6(1):8962. https://doi.org/10.1038/ncomms9962.
Zhang G, Yu Z, Fu S, et al. ERCC6L that is up-regulated in high grade of renal cell carcinoma enhances cell viability in vitro and promotes tumor growth in vivo potentially through modulating MAPK signalling pathway [J]. Cancer Gene Ther. 2019;26(9–10):323–33. https://doi.org/10.1038/s41417-018-0064-8.
Chen H, Wang H, Yu X, et al. ERCC6L promotes the progression of hepatocellular carcinoma through activating PI3K/AKT and NF-κB signaling pathway [J]. BMC Cancer. 2020;20:1–10. https://doi.org/10.1186/s12885-020-07367-2.
Cui M, Chang Y, Wang J, et al. ERCC6L facilitates the progression of laryngeal squamous cell carcinoma by the binding of FOXM1 and KIF4A [J]. Cell Death Discovery. 2023;9(1):41. https://doi.org/10.1038/s41420-023-01314-3.
Huang Y, Li W, Yan W, et al. Loss of PICH promotes chromosome instability and cell death in triple-negative breast cancer[J]. Cell Death Dis. 2019;10(6):428. https://doi.org/10.1038/s41419-019-1662-6.
Macias H, Hinck L. Mammary gland development [J]. Wiley Interdisciplinary Reviews: Developmental Biology. 2012;1(4):533–57. https://doi.org/10.1002/wdev.35.
Lin EY, Jones JG, Li P, et al. Progression to malignancy in the polyoma middle T oncoprotein mouse breast cancer model provides a reliable model for human diseases [J]. Am J Pathol. 2003;163(5):2113–26. https://doi.org/10.1016/S0002-9440(10)63568-7.
Guy CT, Cardiff RD, Muller WJ. Induction of mammary tumors by expression of polyomavirus middle T oncogene: a transgenic mouse model for metastatic disease [J]. Mol Cell Biol. 1992;12(3):954–61. https://doi.org/10.1128/mcb.12.3.954-961.1992.
Hirokawa N, Noda Y, Tanaka Y, et al. Kinesin superfamily motor proteins and intracellular transport [J]. Nat Rev Mol Cell Biol. 2009;10(10):682–96. https://doi.org/10.1038/nrm2774.
Kurasawa Y, Earnshaw WC, Mochizuki Y, et al. Essential roles of KIF4 and its binding partner PRC1 in organized central spindle midzone formation [J]. EMBO J. 2004;23(16):3237–48. https://doi.org/10.1038/sj.emboj.7600347.
Hu CK, Coughlin M, Field CM, et al. KIF4 regulates midzone length during cytokinesis [J]. Curr Biol. 2011;21(10):815–24. https://doi.org/10.1016/j.cub.2011.04.019.
Taniwaki M, Takano A, Ishikawa N, et al. Activation of KIF4A as a prognostic biomarker and therapeutic target for lung cancer [J]. Clin Cancer Res. 2007;13(22):6624–31. https://doi.org/10.1158/1078-0432.CCR-07-1328.
Wang H, Lu C, Li Q, et al. The role of Kif4A in doxorubicin-induced apoptosis in breast cancer cells [J]. Mol Cells. 2014;37(11):812. https://doi.org/10.14348/molcells.2014.0210.
Minakawa Y, Kasamatsu A, Koike H, et al. Kinesin family member 4A: a potential predictor for progression of human oral cancer [J]. PLoS ONE. 2013;8(12):e85951. https://doi.org/10.1371/journal.pone.0085951.
Gao J, Sai N, Wang C, et al. Overexpression of chromokinesin KIF4 inhibits proliferation of human gastric carcinoma cells both in vitro and in vivo [J]. Tumor Biology. 2011;32:53–61. https://doi.org/10.1007/s13277-010-0090-0.
Santaguida S, Richardson A, Iyer DR, et al. Chromosome mis-segregation generates cell-cycle-arrested cells with complex karyotypes that are eliminated by the immune system [J]. Dev Cell. 2017;41(6):638–51. https://doi.org/10.1016/j.devcel.2017.05.022. e5.
Duesberg P, Rausch C, Rasnick D et al. Genetic instability of cancer cells is proportional to their degree of aneuploidy [J]. Proceedings of the National Academy of Sciences, 1998, 95(23): 13692–13697. https://doi.org/10.1073/pnas.95.23.1369.
Mazumdar M, Sung MH, Misteli T. Chromatin maintenance by a molecular motor protein[J]. Nucleus. 2011;2(6):591–600. https://doi.org/10.4161/nucl.2.6.18044.
Geiman TM, Sankpal UT, Robertson AK, et al. Isolation and characterization of a novel DNA methyltransferase complex linking DNMT3B with components of the mitotic chromosome condensation machinery[J]. Nucleic Acids Res. 2004;32(9):2716–29. https://doi.org/10.1093/nar/gkh589.
Leng M, Besusso D, Jung SY, et al. Cell Cycle. 2008;7(10):1480–9. https://doi.org/10.4161/cc.7.10.5951. Targeting Plk1 to chromosome arms and regulating chromosome compaction by the PICH ATPase [J].
Kurasawa Y, Yu-Lee L. Mol Biol Cell. 2010;21(7):1188–99. https://doi.org/10.1091/mbc.e09-11-0950. PICH and cotargeted Plk1 coordinately maintain prometaphase chromosome arm architecture [J].
Holtrich U, Wolf G, Bräuninger A et al. Induction and down-regulation of PLK, a human serine/threonine kinase expressed in proliferating cells and tumors [J]. Proceedings of the National Academy of Sciences, 1994, 91(5): 1736–1740. https://doi.org/10.1073/pnas.91.5.1736.

Download PDF

Journal Publication

published 04 Sep, 2023

Read the published version in Journal of Experimental & Clinical Cancer Research →

Editorial decision: Major revision
04 Jul, 2023
Reviewers agreed at journal
06 Jun, 2023
Reviewers invited by journal
29 May, 2023
Editor assigned by journal
29 May, 2023
First submitted to journal
25 May, 2023

You are reading this latest preprint version

ERCC6L facilitates the onset of mammary neoplasia and promotes the high malignance of breast cancer by accelerating cell cycle

Status:

Journal Publication

Version 1

Abstract

Figures

Introduction

Materials and Method

Results

1. ERCC6L is highly expressed in BRCA patients and associated with poor outcome

4. ERCC6L is vital for the proper mouse ductal network development

5. Effect of ERCC6L deletion on MMTV/PyMT tumor onset and growth

6. KIF4A is closely related to ERCC6L in BRCA

Discussion

Conclusion

Declarations

References

Supplementary Files

Status:

Journal Publication

Version 1