Reciprocal regulation of forkhead box C1 and L1 cell adhesion molecule contributes to triple-negative breast cancer progression

doi:10.21203/rs.3.rs-2730531/v1

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Research Article

Reciprocal regulation of forkhead box C1 and L1 cell adhesion molecule contributes to triple-negative breast cancer progression

https://doi.org/10.21203/rs.3.rs-2730531/v1

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published 06 Jan, 2024

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Purpose

Forkhead box C1 (FOXC1) may act as a therapeutic target for triple-negative breast cancer (TNBC) but without a comprehensive understanding of its regulations, especially at the upstream. L1 cell adhesion molecule (L1CAM) is a transmembrane glycoprotein that may involve in brain metastasis. Indicated by a positive correlation between FOXC1 and L1CAM transcripts, this study aims to further examine their relation in the process of TNBC.

Methods

FOXC1 and L1CAM transcripts were downloaded from two public datasets, and their proteins were examined in four TNBC cell lines. FOXC1 and L1CAM were separately knocked down in BT549 cells; L1CAM was overexpressed in BT549-shFOXC1, MDA-MB-231, and HCC1937 cells. CCK-8, transwell and wound healing assays were conducted in these cells, so was immunohistochemical staining in tumors.

Results

L1CAM and FOXC1 transcripts were positively correlated in public datasets. BT549-shFOXC1 cells showed a decreased L1CAM expression both at the transcriptional and protein levels. Intriguingly, BT549-siL1CAM cells displayed decreased FOXC1 proteins, but exerted little effect on FOXC1 transcripts. Conversely, overexpression of L1CAM resulted in upregulation of FOXC1 protein without substantial change in FOXC1 mRNA, that consistently observed in BT549-shFOXC1, MDA-MB-231-L1CAM and HCC1937-L1CAM cells. Additionally, decreased or increased capacities of cell proliferation, migration, and invasion were seen along with down- or up-regulation of FOXC1 or L1CAM. Finally, a positive correlation between L1CAM and FOXC1 proteins was observed in human TNBC tumors.

Conclusion

FOXC1 and L1CAM display coregulation at the protein level but not mRNA level to positively affect cell proliferation, migration and invasion in TNBC.

triple-negative breast cancer

forkhead box C1

L1 cell adhesion molecule

reciprocal regulation

cell proliferation

metastasis

In 2020, breast cancer overtook lung cancer as the most commonly diagnosed malignancy in the world [1]. As a group of highly heterogeneous tumors with distinct biological behaviors, breast cancer consists of several subtypes, among which triple-negative breast cancer (TNBC) accounts for about 15% [2]. TNBC, characterized by estrogen receptor (ER)-, progesterone receptor (PR)- and human epidermal growth factor receptor 2 (HER2)-negativity, has a predilection for visceral metastases, such as lung and brain [3]. This lack of ER and HER2 expression renders endocrine therapy and anti-HER2-targeted therapy ineffective for TNBC, and breast cancer patients with brain metastasis have an average survival time of less than two years [4]. Until now, combined chemotherapy is still the primary treatment for patients with TNBC. Therefore, additional therapeutic targets are needed for TNBC.

FOXC1 (forkhead box C1) belongs to the FOX family of transcription factors [5]. Physiologically, FOXC1 plays an important role in different stages throughout embryonic development [6]. Pathologically, its role in tumor development and progression across a series of cancers has been demonstrated. Compared with other molecular subtypes of breast cancer, FOXC1 is a consistent feature of basal-like breast cancer, an intrinsic subtype with 60–90% comprising triple-negative tumors [7]. The detrimental role of FOXC1 may be indicated by its involvement in activating NF-κB signaling [8], inducing epithelial-to-mesenchymal transition [9], increasing breast cancer stem cell properties [10], and downregulating ER expression [11]. FOXC1 expression is positively associated with breast cancer brain and lung metastasis [12]. Previously, we found that FOXC1 enhanced TNBC cell invasion and motility in vitro and metastasis to the lung in vivo, probably through activating the transcription of chemokine receptor-4 at the transcriptional level [13]. Despite these studies, a more comprehensive understanding of the up- and down-stream regulators of FOXC1 in TNBC still needs further investigation [5].

L1 cell adhesion molecule (L1CAM), a member of the immunoglobulin (Ig) superfamily, is a transmembrane glycoprotein [14]. Physiologically, the most distinctive molecular actions of L1CAM relate to neuronal migration, axon growth and synapse formation in the developing and adult brain. During the last two decades, L1CAM is constantly detected in tumors, and may involve in tumor progression [14]. Regarding breast cancer, L1CAM can be expressed in all molecular subtypes, but has a preference for TNBC, and is associated with aggressive behavior [15, 16]. Valiente et al. demonstrated an involvement of L1CAM in vascular co-option in brain metastasis [17]. However, the trigger in cancer cells for expressing L1CAM remains as an open question. One recent work from Ganesh et al. showed E-cadherin–REST transcriptional derepression of L1CAM in colorectal cancer could be caused by disruption of intercellular epithelial contacts, but that is not always observed in all cells within tumors [18]. Thus, more mechanisms of regulating L1CAM expression remain to be unraveled.

Through utilizing public RNA sequencing profiles from two cohorts of patients with breast cancer, we observed a positive correlation between FOXC1 and L1CAM (Pearson correlation coefficient = 0.370, 0.244). In view of their shared properties in tumor progression, the possibility exists of an association of L1CAM with FOXC1 in TNBC. We observed that (1) knockdown of FOXC1 led to decreased L1CAM, probably through transcriptional inactivation; (2) either down- or up-regulating L1CAM resulted in corresponding decrease or increase of FOXC1 in a manner that does not primarily rely on transcriptional levels; (3) either FOXC1 or L1CAM dysregulation positively affected TNBC cell proliferation, migration and invasion in vitro. Collectively, these results suggest that there may be reciprocal co-regulation of FOXC1 and L1CAM that at least partially contributes to the aggressiveness of TNBC cells.

Public database

Two cohorts of 1108 and 379 patients with breast cancer (TCGA, Firehose legacy; The Metastatic Breast Cancer Project, Provisional, December 2021) were investigated from the cBioportal database (https://www.cbioportal.org/, assessed on November 18th, 2022) [19, 20]. Messenger RNA values were determined using RNA-Seq by Expectation-Maximization (RSEM) on a log-2 scale. Pearson’s correlation analyses were separately performed in these two cohorts of 960 and 157 patients, who had complete information both for L1CAM and FOXC1. Furthermore, in the TCGA cohort, L1CAM transcription was compared between high (n = 375) or low (n = 585) FOXC1 transcription groups, which were determined using the mean as the cutoff.

Cell Culture And Specimens

Four human TNBC cell lines SUM149, HCC1937, MDA-MB-231 and BT549, were obtained from the ATCC (American Type Culture Collection, Manassas, VA, USA). Cells were cultured in RPMI1640 (Gibco, ThermoFisher, Waltham, USA) supplemented with 10% fetal bovine serum (FBS, Biological Industry, Kibbutz BeitHaEmek, Israel).

Transient Transfection

Twenty-four hours prior to transfection, approximately 2×10⁵ cells (BT549, MDA-MB-231, or HCC1937) were seeded into 6-well plates and allowed to reach 80–90% confluence by the second day. To knock down L1CAM, BT549 cells were transiently transfected with L1CAM siRNA (BT549-siL1CAM) or control scramble siRNA (BT549-siNC) using Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s instructions. The siRNA targeting L1CAM and the control scramble siRNA were purchased from HIPPOBIO (Shenzhen, China), with the following sequences: si-L1CAM: forward: 5′-GAACGGCAACCUCUACUUUTT-3′ and reverse: 5′-AAAGUAGAGGUUGCCGUUCTT-3′; siNC: 5′-UUCUCCGAACGUGUCACGUTT-3′ and reverse: 5’-ACGUGACACGUUCGGAGAATT-3’. To deplete FOXC1, BT549 cells were transfected with the shRNA of FOXC1 (BT549-shFOXC1) or scrambled sequence as a control (BT549-shNC), that were cloned into the pLV-hU6-Neongreen construct (SyngenTech, Beijing, China). Cells with stably decreased FOXC1 were then selected with 1 µg/mL puromycin. Furthermore, to overexpress L1CAM, BT549-shFOXC1, MDA-MB-231 and HCC1937 cells were transfected with a full length human L1CAM mRNA (NM_000425), that was inserted into the GV358Vector (GeneChem, Shanghai, China) and were selected with 400 µg/mL, 1mg/mL and 1mg/mL Geneticin (BT549-shFOXC1-L1CAM, MDA-MB-231-L1CAM and HCC1937-L1CAM), respectively.

Wound Healing Assay

BT549-shFOXC1, BT549-siL1CAM, BT549-shFOXC1-L1CAM, MDA-MB-231-L1CAM and HCC1937-L1CAM and their respective control cells were seeded at a density of 5 × 10⁵ cells per well in 6-well plates. The monolayer of cells was scratched with a 1 mL plastic pipette tip to create a uniform wound. After 24 hours of incubation, the wound width was then examined under a phase-contrast microscope (NIKON; Konan, Tokyo, Japan). Photographs of at least 3 random fields were taken, and in each filed, the closure of the gap distance was calculated at 0 and 24 hours. Cell migration percentages (%) were calculated using the following formula: [(0-hour-gap distance − 24-hour-gap distance) / 0-hour-gap distance] × 100. All distances were measured by ImageJ software.

Migration And Invasion Assays

Transwell chambers (Corning, NY, USA) were used to detect cell migration and invasion. The bottom of the transwell chamber was made of a polycarbonate membrane with 8 µm pore diameter. For invasion assays, an additional Matrigel (50 µl BD biosciences, Franklin Lakes, NewJersey, US) was used to coat the surface of the polycarbonate membrane. For each cell line, 5×10⁴ cells were suspended in 200 µl serum-free RPMI1640 medium, and seeded into the upper chamber, and 600 µl 10% FBS-supplemented RPMI1640 medium was added to the lower chamber. After 24 hours, the upper chamber and cells on the upper surface of the membrane were removed. Cells that had migrated to the lower surface of the membrane were stained with 0.1% crystal violet, and the number of cells was counted under a Leica microscope under 100× magnification (DM3000, Wetzlar, Germany). Four fields from each sample were randomly selected for counting cells. Results were summarized by the average of three replicates, and each experiment was repeated three times.

Cell Proliferation Assay

To measure cell viability, cells were separately seeded into 96-well plates at a concentration of 2000 cells per well; then after four to five hours, cells were first incubated with 20 µl CCK-8 solution (C0038, Beyotime Biotechnology, China) for 2.5 hours in a humidified chamber at 37℃, and then absorbance was detected (OD value: optical density) at a wavelength of 450 nm using a microplate reader (Multiskan MK3, ThermoFisher, CA, US). Results were summarized by four technical replicates, and each experiment was repeated for triple times.

Real-time Polymerase Chain Reaction

Total RNAs were extracted from cells using TRIzol (cat#15596026) and reverse transcription of purified RNAs was performed using oligo (dT) priming and Superscript III reverse transcriptase according to the manufacturer’s instructions (TaKaRa, Tokyo, Japan). Real-time PCR was performed using a SYBR Premix kit (TaKaRa, Tokyo, Japan). β-Actin was used as a loading control. The reactions were undertaken with a 7300 Real-Time PCR System (Applied Biosystems, Waltham, MA, USA). The primer pairs used for target genes were purchased from SangonBiotech (Shanghai, China) as follows:

FOXC1 forward, 5′-TCACAGAGGATCGGCTTGAAC-3′,

FOXC1 reverse, 5′-TCCTGCTTTGGGGTTCGATT-3′,

L1CAM forward, 5′-CCCCGAGGAATTGATGGAGC-3′,

L1CAM reverse, 5′-GGTTCTGGTAGGTGACACGG-3′,

β-actin forward, 5′-CATGTACGTTGCTATCCAGGC-3′,

β-actin reverse, 5′-CTCCTTAATGTCACGCACGAT-3′.

Western Blot Analysis

Cells were first lysed with a cell lysis buffer containing phenylmethylsulfonyl fluoride (Beyotime, Shanghai, China). Proteins (30 µg) of each cell lysate were then separated by SDS-PAGE and transferred onto a polyvinylidene difluoride (PVDF, Millipore, Bedford, MA, USA) membrane followed by blocking with bovine serum albumin, and incubating at 4℃ overnight with primary antibodies anti-FOXC1 (1:500, ab223850; Abcam, Cambridge, UK), anti-L1CAM (1:1000, ab270455; Abcam, Cambridge, UK), anti-GAPDH (1:2000, sc-47724; Santa Cruz Biotechnology, Dallas, TX, USA), and anti-β-tubulin (1:3000, sc-5274; Santa Cruz Biotechnology, Dallas, TX, USA) in blocking buffer. Following washes with TBST (Tris-buffered saline with 0.2% Tween20), the blots were incubated with horseradish peroxidase-labelled anti-rabbit (1:10000, ab205718; Abcam, Cambridge, UK) or anti-mouse (1:5000, ab6728; Abcam, Cambridge, UK) at room temperature for two hours, washed with TBST, followed by signal detection using chemiluminescence. Protein bands were visualized using an ECL western blotting substrate (cat#32109, ThermoFisher, USA).

Patients And Tumor Specimens

Paraffin-embedded archival pathological specimens from 44 patients diagnosed with TNBC were available, along with complete clinicopathological features. The patients had undergone biopsy without preoperative therapy at the Cancer Hospital of Shantou University Medical College between January 2013 and December 2019. Of this cohort, most of patients (n = 34) were diagnosed with early stages (I/II), while the rest were at the advanced stages (III/IV) except for one with unknown stage. Clinical tumor stage (TNM stage) was grouped in accordance with the American Joint Committee on Cancer, 6th Edition Cancer Staging Manual (2002). Participants gave their informed consent after being notified of the details and potential consequences. This study of tumor samples was approved by the medical ethics committee of the Cancer Hospital of Shantou University Medical College (approval number: 2019024).

Immunohistochemistry

Immunohistochemistry (IHC) for detecting FOXC1 and L1CAM in TNBC was carried out. Briefly, sections (4-µm thick) were fixed in 10% buffered formalin and embedded in paraffin. After deparaffinization and rehydration, endogenous peroxidase activity was blocked with 0.3% hydrogen peroxide for 30 minutes. Then tissue sections were autoclaved at 121°C in citrate buffer (pH 6.0) for 10 minutes, and incubated with rabbit anti-FOXC1 monoclonal antibody (1:50, ab223850; Abcam, Cambridge, UK) or L1CAM antibody (1:100, ab270455; Abcam, Cambridge, UK). IHC staining was carried out with an EnVision antibody complex (anti-mouse/rabbit) method using an ElivisionTM plus Polymer HP (Mouse/Rabbit) IHC Kit (MXB Biotechnologies, Fujian, China) and 3,3’-diaminobenzidine as the chromogen substrate. IHC staining for FOXC1 and L1CAM were scored, by a combination of intensity (0, no staining; 1, weak staining; 2, moderate staining; 3, strong staining) and proportion (0, < 5% of tumor cells stained; 1, 5–25% positive cells; 2, 26–50% positive cells; 3, 51–75% positive cells; 4, more than 76% positive cells). If the product of multiplication between staining intensity and the proportion of positive cells was > 4, expression was defined as positive. Two pathologists independently assessed the cellular location and intensity of immunostaining in each section.

Statistical Analyses

Statistical analyses were performed using GraphPad Prism 8.0 software (San Diego, CA). Data are expressed as mean ± standard deviation (SD). Comparisons of transcriptional levels, cell viability and cell mobility between constructed cells and their controls were conducted using t-tests. For all tests, a value of P < 0.05 was considered significant.

L1CAM is down-regulated at the transcription and protein level after FOXC1 knockdown

To initially learn the relation between L1CAM and FOXC1, a correlation analysis was conducted using publicly available mRNA profiles from two cohorts of patients diagnosed with breast cancer. As seen in Fig. 1a, in patients with a high FOXC1 level, L1CAM expression was higher than that in those with a low FOXC1 level (P < 0.001). In addition, L1CAM was significantly and positively correlated with FOXC1 (r = 0.37, P < 0.001, Fig. 1b; r = 0.244, P = 0.002, Fig. 1c). Then, both L1CAM and FOXC1 proteins were examined in four TNBC cell lines (Fig. 1c). Since BT549 had observable expression of these two genes, we primarily used this cell line for the following experiments. BT549-shFOXC1 cells showed a considerable decrease of FOXC1 mRNA and protein expression (Fig. 1d-1e), reduction of L1CAM mRNA and protein levels was also observed (Fig. 1d-1e), indicating that FOXC1 may regulate L1CAM expression at the transcriptional level, at least in part. Compared to its parental cells, capacity for cell proliferation, invasion and migration were impaired in BT549-shFOXC1 cells, which is consistent with previous findings (Online Resource Fig. S1).

Down regulation of L1CAM suppresses capacity for proliferation, invasion and migration in TNBC cells along with reducing FOXC1 protein levels

To examine the role of L1CAM in TNBC cells, as well as its impact on FOXC1, BT549-siL1CAM cells, with reduced L1CAM mRNA and protein, were constructed (Fig. 2a-2b). Intriguingly, we observed knockdown of L1CAM simultaneously reduced FOXC1 at the protein level (Fig. 2a). At the level of FOXC1 mRNA, only a slight decrease was seen (0.11-fold decrease, Fig. 2b) in comparison to controls. This indicates L1CAM may reciprocally regulate FOXC1 expression, in a manner independent of transcription.

CCK-8 assays showed that the viability of BT549-siL1CAM cells decreased after 48 (OD values: 0.523 vs. 0.644, P < 0.001, Fig. 2c), 72 (0.676 vs. 0.851, P < 0.001), and 96 hours (0.811 vs. 1.205, P < 0.001), compared to the parental cells. Transwell assays revealed an impaired capacity for metastasis in BT549-siL1CAM cells (number of migrated cells: 54 ± 7 vs. 128 ± 13, P < 0.001; number of invaded cells: 14 ± 5 vs. 62 ± 3, P < 0.001, Fig. 2d). Similarly, the wound healing assays confirmed a reduced migration of BT549-siL1CAM cells (migration area: 38.5% vs. 88.0%, P < 0.001, Fig. 2e). These results indicated that BT549-siL1CAM cells decreased both cell viability and metastasis, which may be related to a reduction of FOXC1 proteins.

Overexpression of L1CAM promotes cell invasion and migration through FOXC1

To further examine the role of L1CAM in TNBC cells, as well as its impact on FOXC1, BT549-shFOXC1 cells overexpressing L1CAM (BT549-shFOXC1-L1CAM) were engineered (Fig. 3a-3b). Notably, in these cells, FOXC1 was significantly increased at the protein level (Fig. 3a), whereas only a negligible change at the mRNA level. Compared with controls (BT549-shFOXC1-NC), CCK-8 assay indicated a significant increase in the proliferation of BT549-shFOXC1-L1CAM cells (e.g., BT549-shFOXC1-L1CAM-OA1: 24 hours: 0.319 vs. 0.414, P < 0.001; 48 hours: 0.501 vs. 0.707, P < 0.001; 72 hours: 0.848 vs. 1.225, P < 0.001; Fig. 3d-3e). Transwell assays indicated increased migration and invasion by BT549-shFOXC1-L1CAM cells (e.g., BT549-shFOXC1-L1CAM-OA1: number of migrated cells: 50 ± 15 vs. 96 ± 18, P < 0.001; number of invaded cells: 41 ± 29 vs. 123 ± 32, P < 0.001, Fig. 3f-3g). Wound healing assays confirmed an elevated capacity of migration in BT549-shFOXC1-L1CAM cells (Fig. 3h-3i), compared to controls. These findings suggest that the restoration of proliferation, invasion and migration in BT549-shFOXC1-L1CAM could be through up-regulation of FOXC1.

Similar findings were seen in two other TNBC cell lines, MDA-MB-231-L1CAM and HCC1937-L1CAM, which overexpress L1CAM. As shown in Online Resource Fig. S2a-S2b and Fig. S3a-S3b, MDA-MB-231-L1CAM and HCC1937-L1CAM cells displayed increased FOXC1 protein levels but only a moderate increase of FOXC1 mRNA. CCK-8 assays and wound healing demonstrated that L1CAM overexpression in MDA-MB-231 and HCC1937 cells promoted cell proliferation and migration (Online Resource Fig. S2d-S2e, Fig. S3d-S3e), whereas transwell assays failed to show an increased capacity of invasion in MDA-MB-231-L1CAM (Online Resource Fig. S2d).

L1CAM and FOXC1 at the protein levels were correlated in human breast cancer

To examine the relation between FOXC1 and L1CAM in TNBC, their expressions at the protein levels were investigated by IHC in tumors from 44 patients diagnosed with TNBC. Examples for typical FOXC1 and L1CAM staining are shown in Fig. 4a-4c. FOXC1 was significantly correlated to L1CAM expression (r = 0.451, P = 0.004, Fig. 4d). Clinic-pathological information of these samples is shown in Fig. 4e. These data indicate that L1CAM expression is positively correlated to FOXC1 in TNBC tissues.

In this study, we first observed a decline of L1CAM both at the protein and transcriptional levels after FOXC1 knockdown. Then of note, down- or up-regulating L1CAM in return led to a decrease or an increase, respectively, of FOXC1 mainly at the protein level, concomitant with impaired or enhanced capacities of proliferation, migration and invasion of TNBC cells. Taken together, there may be a reciprocal regulation between FOXC1 and L1CAM, that promotes proliferation, migration and invasion in TNBC cells.

FOXC1 and L1CAM have been found in a variety of cancers; their detrimental roles in cancer development have been demonstrated in vitro experiments, and their associations with poor prognosis of patients with cancers have been constantly observed [14, 21]. Regarding breast cancer, expression of FOXC1 and L1CAM both have a preference for TNBC as opposed to other subtypes. It is known that FOXC1 boosts distant metastasis in TNBC to both lung and brain [12, 13, 22], and L1CAM may be the key molecular in vascular co-option in brain metastasis [17, 23]. However, a recent IHC study failed to observe L1CAM expression in an entire set of thirty resected breast cancer brain metastases by IHC [24]. One possible explanation the authors gave is that, after developing macro-metastases in the brain, L1CAM is no longer necessary, and then downregulated [24]. Herein, we offered a possibility that L1CAM downregulation could be due to FOXC1 silencing. Although FOXC1 was not examined in that study, most brain metastases are ER-positive or HER2-positive tumors, that are less likely to express FOXC1 [24].

FOXC1 and L1CAM may share some common mechanisms in the progression of breast cancer. Regarding FOXC1, how it is regulated is still not well understood, despite extensive functional studies in tumor progression [5]. In this study, we observed a substantial change in FOXC1 protein but not transcripts, followed by a corresponding change in L1CAM expression. Thus, we assume that NF-κB and MAPK signaling pathways may possibly act as the mediators between these two genes. NF-κB is one of the most distinctive pathways in basal-like tumors, as opposed to ER-positive breast cancer [25]. In vitro, L1CAM has been shown to act as a ligand in MDA-MB-231 cells for integrins thereby promoting NF-κB activation [26]. Of note, NF-κB can bind to the FOXC1 promoter to initiate transcription [27]. Thus, NF-κB may bring L1CAM and FOXC1 together. Intriguingly, we did not observe a striking decrease or increase of FOXC1 transcripts in contrast to the large change in FOXC1 protein level. That indicates transcriptional regulation may not the predominant means by which L1CAM regulates FOXC1 expression. Recently, one study pointed out that FOXC1 was regulated by p38 MAPK, specially Ser241 and Ser272 of FOXC1 might be key p38 phosphorylation sites that are critically required for the stability of FOXC1 protein, but do not influence mRNA levels [28]. Of note, inhibition of L1CAM expression by L1CAM-specific siRNA suppressed the activation of MAPKs such as ERK and p38 [29, 30]. Thus, maybe p38 or its analogues may contribute to the underlying mechanism by which L1CAM affects FOXC1 expression at the protein levels, but not transcriptional level. However, these are not yet confirmed in the breast cancer cells, and we are still investigating the possibility.

Other than a positive influence of L1CAM on FOXC1, we still observed a reduction of FOXC1 concomitant with decreased L1CAM probably through transcriptional downregulation. The underlying mechanism is unclear. Earlier work suggests that β-catenin is a direct transcriptional target of FOXC1 [31–33], and L1CAM is a target gene of β-catenin signaling [34]. This suggests β-catenin may be involved in the link between them. However, whether FOXC1 directly or indirectly regulates L1CAM remains to be further investigated in breast cancer cells.

Upstream regulation of L1CAM and FOXC1 expression is well summarized in these reviews [5, 14]. However, in terms of their potential as a therapeutic target, a more comprehensive understanding of their regulation in TNBC still requires further investigations. In this study, we demonstrate the possibility that there may be a reciprocal regulation between FOXC1 and L1CAM, that involved in proliferation, migration, and invasion in TNBC cells. Since their functions in TNBC have been separately and extensively investigated, herein we focused on their relation in TNBC cells. As far as we know, this is the first study that suggests L1CAM may be a regulator of FOXC1, and vice versa, and the former may not rely on transcriptional regulation. One weakness of this study is that, the specific regulatory mechanisms underlying FOXC1 and L1CAM are not clear, which we will try to clarify in subsequent studies.

In conclusion, our results suggest a positive relation between FOXC1 and L1CAM for TNBC. FOXC1 and L1CAM might be mutually regulated and jointly promote cell proliferation and metastasis in TNBC. Further studies will be needed to evaluate the underlying mechanisms between them.

ER, estrogen receptor; FOXC1: forkhead box C1; HER2: human epidermal growth factor receptor 2; IHC: immunohistochemistry; L1CAM: L1 cell adhesion molecule; OD, optical density; PR: progesterone receptor; RSEM: RNA-Seq by Expectation Maximization; SD: standard deviation; TNBC, triple-negative breast cancer.

Acknowledgments

We thank Professor Stanley Li Lin for the language editing.

Funding

This work was supported by grants from the Natural Science Foundation of Guangdong Province of China (Grant No. 2020A1515010094); the Special Fund for Science and Technology of Guangdong Province in 2020 (Grant No. 200110115891683); the Special Fund for Science and Technology of Guangdong Province in 2021 (Grant No. 210729156901814), and the National Natural Science Foundation of China (Grant No. 82272670).

Competing Interests

The authors have no relevant financial or non-financial interests to disclose.

Author Contributions

Fan Zhang and Yukun Cui contributed to the study conception and design. Yue Xu and Jiediao Lin conducted the cells experiments. Fan Zhang and Yue Xu drafted the first manuscript. Yue Xu and Hongchao Pan collected clinic-pathological information of patients and conducted immunohistochemistry. All authors contributed to the data interpretation, critically reviewed each draft of the manuscript, and approved the final version for submission.

Data Availability

The public datasets analysed during the current study are available in cBioportal, https://www.cbioportal.org/.

The data generated during the current study is available from the corresponding author on reasonable request.

Ethics approval

This study was performed in line with the principles of the Declaration of Helsinki. Approval was granted by the medical ethics committee of the Cancer Hospital of Shantou University Medical College (Date 2019-05-29/No. 2019024).

Consent to participate

Informed consent was obtained from all individual participants included in the study.

Ferlay J, Colombet M, Soerjomataram I, Parkin DM, Pineros M, Znaor A, and Bray F (2021) Cancer statistics for the year 2020: An overview. Int J Cancer. https://doi.org/10.1002/ijc.33588.
Waks AG and Winer EP (2019) Breast Cancer Treatment: A Review. JAMA 321: 288-300.https://doi.org/10.1001/jama.2018.19323.
Yin L, Duan JJ, Bian XW, and Yu SC (2020) Triple-negative breast cancer molecular subtyping and treatment progress. Breast Cancer Res 22: 61. https://doi.org/10.1186/s13058-020-01296-5.
Jin J, Gao Y, Zhang J, Wang L, Wang B, Cao J, Shao Z, and Wang Z (2018) Incidence, pattern and prognosis of brain metastases in patients with metastatic triple negative breast cancer. BMC Cancer 18: 446.https://doi.org/10.1186/s12885-018-4371-0.
Han B, Bhowmick N, Qu Y, Chung S, Giuliano AE, and Cui X (2017) FOXC1: an emerging marker and therapeutic target for cancer. Oncogene 36: 3957-3963.https://doi.org/10.1038/onc.2017.48.
Sasaki H and Hogan BL (1993) Differential expression of multiple fork head related genes during gastrulation and axial pattern formation in the mouse embryo. Development 118: 47-59.https://doi.org/10.1242/dev.118.1.47.
Ray PS, Wang J, Qu Y, Sim MS, Shamonki J, Bagaria SP, Ye X, Liu B, Elashoff D, Hoon DS, Walter MA, Martens JW, Richardson AL, Giuliano AE, and Cui X (2010) FOXC1 is a potential prognostic biomarker with functional significance in basal-like breast cancer. Cancer Res 70: 3870-3876.https://doi.org/10.1158/0008-5472.CAN-09-4120.
Wang J, Ray PS, Sim MS, Zhou XZ, Lu KP, Lee AV, Lin X, Bagaria SP, Giuliano AE, and Cui X (2012) FOXC1 regulates the functions of human basal-like breast cancer cells by activating NF-kappaB signaling. Oncogene 31: 4798-4802.https://doi.org/10.1038/onc.2011.635.
Taube JH, Herschkowitz JI, Komurov K, Zhou AY, Gupta S, Yang J, Hartwell K, Onder TT, Gupta PB, Evans KW, Hollier BG, Ram PT, Lander ES, Rosen JM, Weinberg RA, and Mani SA (2010) Core epithelial-to-mesenchymal transition interactome gene-expression signature is associated with claudin-low and metaplastic breast cancer subtypes. Proc Natl Acad Sci U S A 107: 15449-15454.https://doi.org/10.1073/pnas.1004900107.
Han B, Qu Y, Jin Y, Yu Y, Deng N, Wawrowsky K, Zhang X, Li N, Bose S, Wang Q, Sakkiah S, Abrol R, Jensen TW, Berman BP, Tanaka H, Johnson J, Gao B, Hao J, Liu Z, Buttyan R, Ray PS, Hung MC, Giuliano AE, and Cui X (2015) FOXC1 Activates Smoothened-Independent Hedgehog Signaling in Basal-like Breast Cancer. Cell Rep 13: 1046-1058.https://doi.org/10.1016/j.celrep.2015.09.063.
Yu-Rice Y, Jin Y, Han B, Qu Y, Johnson J, Watanabe T, Cheng L, Deng N, Tanaka H, Gao B, Liu Z, Sun Z, Bose S, Giuliano AE, and Cui X (2016) FOXC1 is involved in ERalpha silencing by counteracting GATA3 binding and is implicated in endocrine resistance. Oncogene 35: 5400-5411.https://doi.org/10.1038/onc.2016.78.
Jensen TW, Ray T, Wang J, Li X, Naritoku WY, Han B, Bellafiore F, Bagaria SP, Qu A, Cui X, Taylor CR, and Ray PS (2015) Diagnosis of Basal-Like Breast Cancer Using a FOXC1-Based Assay. J Natl Cancer Inst 107.https://doi.org/10.1093/jnci/djv148.
Pan H, Peng Z, Lin J, Ren X, Zhang G, and Cui Y (2018) Forkhead box C1 boosts triple-negative breast cancer metastasis through activating the transcription of chemokine receptor-4. Cancer Sci 109: 3794-3804.https://doi.org/10.1111/cas.13823.
Altevogt P, Doberstein K, and Fogel M (2016) L1CAM in human cancer. Int J Cancer 138: 1565-1576.https://doi.org/10.1002/ijc.29658.
Doberstein K, Milde-Langosch K, Bretz NP, Schirmer U, Harari A, Witzel I, Ben-Arie A, Hubalek M, Muller-Holzner E, Reinold S, Zeimet AG, Altevogt P, and Fogel M (2014) L1CAM is expressed in triple-negative breast cancers and is inversely correlated with androgen receptor. BMC Cancer 14: 958.https://doi.org/10.1186/1471-2407-14-958.
Moisini I, Zhang H, D'Aguiar M, Hicks DG, and Turner BM (2021) L1CAM Expression in Recurrent Estrogen Positive/HER2 Negative Breast Cancer: A Novel Biomarker Worth Considering. Appl Immunohistochem Mol Morphol 29: 287-292.https://doi.org/10.1097/PAI.0000000000000909.
Valiente M, Obenauf AC, Jin X, Chen Q, Zhang XH, Lee DJ, Chaft JE, Kris MG, Huse JT, Brogi E, and Massague J (2014) Serpins promote cancer cell survival and vascular co-option in brain metastasis. Cell 156: 1002-1016.https://doi.org/10.1016/j.cell.2014.01.040.
Ganesh K, Basnet H, Kaygusuz Y, Laughney AM, He L, Sharma R, O'Rourke KP, Reuter VP, Huang YH, Turkekul M, Er EE, Masilionis I, Manova-Todorova K, Weiser MR, Saltz LB, Garcia-Aguilar J, Koche R, Lowe SW, Pe'er D, Shia J, and Massague J (2020) L1CAM defines the regenerative origin of metastasis-initiating cells in colorectal cancer. Nat Cancer 1: 28-45.https://doi.org/10.1038/s43018-019-0006-x.
Gao J, Aksoy BA, Dogrusoz U, Dresdner G, Gross B, Sumer SO, Sun Y, Jacobsen A, Sinha R, Larsson E, Cerami E, Sander C, and Schultz N (2013) Integrative analysis of complex cancer genomics and clinical profiles using the cBioPortal. Sci Signal 6: pl1.https://doi.org/10.1126/scisignal.2004088.
Cerami E, Gao J, Dogrusoz U, Gross BE, Sumer SO, Aksoy BA, Jacobsen A, Byrne CJ, Heuer ML, Larsson E, Antipin Y, Reva B, Goldberg AP, Sander C, and Schultz N (2012) The cBio cancer genomics portal: an open platform for exploring multidimensional cancer genomics data. Cancer Discov 2: 401-404.https://doi.org/10.1158/2159-8290.CD-12-0095.
Elian FA, Yan E, and Walter MA (2018) FOXC1, the new player in the cancer sandbox. Oncotarget 9: 8165-8178.https://doi.org/10.18632/oncotarget.22742.
Han B, Zhou B, Qu Y, Gao B, Xu Y, Chung S, Tanaka H, Yang W, Giuliano AE, and Cui X (2018) FOXC1-induced non-canonical WNT5A-MMP7 signaling regulates invasiveness in triple-negative breast cancer. Oncogene 37: 1399-1408.https://doi.org/10.1038/s41388-017-0021-2.
Garcia-Gomez P and Valiente M (2020) Vascular co-option in brain metastasis. Angiogenesis 23: 3-8.https://doi.org/10.1007/s10456-019-09693-x.
Karpathiou G, Camy F, Dumollard JM, and Peoc'h M (2021) Immunohistochemical analysis of L1 cell adhesion molecule and high endothelial venules in breast cancer brain metastasis. Pathol Res Pract 223: 153484.https://doi.org/10.1016/j.prp.2021.153484.
Yamaguchi N, Ito T, Azuma S, Ito E, Honma R, Yanagisawa Y, Nishikawa A, Kawamura M, Imai J, Watanabe S, Semba K, and Inoue J (2009) Constitutive activation of nuclear factor-kappaB is preferentially involved in the proliferation of basal-like subtype breast cancer cell lines. Cancer Sci 100: 1668-1674.https://doi.org/10.1111/j.1349-7006.2009.01228.x.
Kiefel H, Bondong S, Pfeifer M, Schirmer U, Erbe-Hoffmann N, Schafer H, Sebens S, and Altevogt P (2012) EMT-associated up-regulation of L1CAM provides insights into L1CAM-mediated integrin signalling and NF-kappaB activation. Carcinogenesis 33: 1919-1929.https://doi.org/10.1093/carcin/bgs220.
Chung S, Jin Y, Han B, Qu Y, Gao B, Giuliano AE, and Cui X (2017) Identification of EGF-NF-kappaB-FOXC1 signaling axis in basal-like breast cancer. Cell Commun Signal 15: 22.https://doi.org/10.1186/s12964-017-0180-3.
Zhang Y, Liao Y, Chen C, Sun W, Sun X, Liu Y, Xu E, Lai M, and Zhang H (2020) p38-regulated FOXC1 stability is required for colorectal cancer metastasis. J Pathol 250: 217-230.https://doi.org/10.1002/path.5362.
Jo DH, Lee K, Kim JH, Jun HO, Kim Y, Cho YL, Yu YS, Min JK, and Kim JH (2017) L1 increases adhesion-mediated proliferation and chemoresistance of retinoblastoma. Oncotarget 8: 15441-15452.https://doi.org/10.18632/oncotarget.14487.
Yi YS, Baek KS, and Cho JY (2014) L1 cell adhesion molecule induces melanoma cell motility by activation of mitogen-activated protein kinase pathways. Pharmazie 69: 461-467.
Cao Q, Wang X, Shi Y, Zhang M, Yang J, Dong M, Mi Y, Zhang Z, Liu K, Jiang L, Wang N, and Wang P (2019) FOXC1 silencing inhibits the epithelial‑to‑mesenchymal transition of glioma cells: Involvement of beta‑catenin signaling. Mol Med Rep 19: 251-261.https://doi.org/10.3892/mmr.2018.9650.
Cao S, Wang Z, Gao X, He W, Cai Y, Chen H, and Xu R (2018) FOXC1 induces cancer stem cell-like properties through upregulation of beta-catenin in NSCLC. J Exp Clin Cancer Res 37: 220.https://doi.org/10.1186/s13046-018-0894-0.
Sun Y, Lin C, Ding Q, and Dai Y (2022) Overexpression of FOXC1 Promotes Tumor Metastasis by Activating the Wnt/beta-Catenin Signaling Pathway in Gastric Cancer. Dig Dis Sci 67: 3742-3752.https://doi.org/10.1007/s10620-021-07226-5.
Cheriyamundath S and Ben-Ze'ev A (2020) Wnt/beta-Catenin Target Genes in Colon Cancer Metastasis: The Special Case of L1CAM. Cancers (Basel) 12.https://doi.org/10.3390/cancers12113444.

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Reciprocal regulation of forkhead box C1 and L1 cell adhesion molecule contributes to triple-negative breast cancer progression

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