Summary

Graphical abstract

Open in a separate window

Introduction

Breast cancer is the second most frequently diagnosed malignancy, just behind lung cancer, and the leading cause of cancer death in women with more than 2 million new cases annually (Siegel et al., 2021). Although standard therapy, consisting of surgical removal of the tumor followed by adjuvant radiation and chemotherapy, has decreased breast cancer mortality, around 12% of patients are still at high risk of relapse (DeSantis et al., 2016). Breast cancer metastasis is one of the major causes of eventual mortality; thus, novel therapeutic targets to halt or delay metastasis are of critical need.

Generally, it is recognized that cancer cells responsible for developing distant organ metastases have changed their phenotype to adjust and facilitate colonization (Lambert et al. 2017). G protein-coupled receptors (GPCRs) are overexpressed and aberrantly activated during different metastatic phases including dissemination from the primary site and colonization at other sites (Dorsam and Gutkind 2007; O'Hayre et al., 2013). GPCRs are activated by a diverse array of ligands including inorganic ions, proteins, small molecules, and GPCR sensory stimuli such as light, taste, and odorants (Neves et al. 2002). This activation transduces a wide range of extracellular signals into intracellular messages resulting in cell proliferation, invasion, migration, survival, angiogenesis, and metastasis (Dorsam and Gutkind 2007; Hutchings et al., 2017; Wu et al., 2019).

The human olfactory receptor (OR) gene family is a crucial member of GPCRs typically expressed in the sensory neurons and plays an essential role in various physiological processes outside the olfactory epithelium, including cancer (Lee et al. 2019). For instance, OR51E2 (also called prostate-specific G-protein coupled receptor) is overexpressed in prostate cancer tissues with a crucial role in accelerating tumor cell invasiveness through loss of PTEN (Neuhaus et al., 2009). OR2AT4 regulates cell proliferation, apoptosis, and differentiation of human myelogenous leukemia (Manteniotis et al., 2016). OR2B6 and OR2W3 are abundant in invasive breast tumors, whereas OR2T6 is overexpressed in breast cancer tissues, contributing to metastasis (Li et al., 2019; Masjedi et al., 2019a, 2019b; Weber et al., 2018a). Thus, ORs might play a vital role in cancer progression and metastasis. Despite these observations, the function of the OR family in breast cancer metastasis and its underlying molecular mechanisms are still largely unknown.

In this study, we evaluated the expression profile of ORs in primary and different breast cancer metastatic sites including brain, lung, and bone. Among the 20 differentially expressed ORs, OR5B21 was upregulated in all organ metastases, with the highest being the brain. We show that OR5B21 plays a vital role in breast cancer cell invasion/migration and metastasis to different sites, especially the brain, by activating epithelial to mesenchymal transition through the STAT3/NF-κB/CEBPβ signaling axis.

Results

Olfactory receptors are associated with breast cancer metastasis

We established a breast cancer metastasis model by intracardiac injection of MDA-MB-231 (231P) cells in immunocompromised mice. We then produced a series of derivative cell lines from developed metastatic sites including the brain-seeking clone (231BR), the bone-seeking clone (231BO), and the lung-seeking clone (231LM) (Figure 1A). We screened these different cell lines for 20 common OR transcripts by quantitative real-time polymerase chain reaction (qRT-PCR) and found them to be differentially expressed in 231BR, 231BO, and/or 231LM, compared with the parental line (Figure 1B). OR5B21 transcript, in particular, was significantly increased in all three organ metastases, being 15-fold higher in 231BR (p < 0.0001), 10-fold higher in 231LM (P < 0.0001), and 3-fold higher in 231BO (p = 0.0082), compared with primary 231P (P < 0.0001; Figure 1C). These results were confirmed in another model where OR5B21 was enriched in two different clones of the brain-seeking mouse metastatic line, ErbB2-BrM1 and ErbB2-BrM2, compared with the primary ErbB2-P line (P < 0.01; Figure 1D). In addition, we analyzed differential expression of OR5B21 in healthy and tumor tissue using the Database: TCGA-BRCA and found OR5B21 to be upregulated in breast cancer compared with normal breast tissue (P < 0.001, Figure S1A). We therefore chose to focus on OR5B21 for further analyses in breast cancer metastasis.

Open in a separate window

Figure 1

ORs are differentially expressed in breast cancer metastasis

(A) Breast cancer metastasis model was developed by intracardiac injection of human breast cancer cell line MDA-MB-231 (231P). Brain-seeking clone (231BR), lung-seeking clone (231LM), and bone-seeking clone (231BO) were harvested and cultured.

(B) Transcript abundance of 20 olfactory receptor genes in primary and different metastatic clones.

(C) Relative transcript abundance of OR5B21 in various MDA-MB-231 metastatic sites with respect to the primary tumor.

(D) OR5B21 transcript abundance in two different brain-seeking clones of ErbB2 versus primary cells.

Data presented as mean ± SEM (n = 3); p < 0.01, p < 0.001 by one-way ANOVA.

OR5B21 induces breast cancer invasion/migration

To evaluate the role of OR5B21 in breast cancer invasion/migration, we knocked down OR5B21 in different metastatic lines (231BR, 231LM, and 231BO; Figure S1B) and performed a scratch wound-healing assay. OR5B21 knockdown significantly reduced the invasion ability of all three metastatic cell lines (p < 0.01; Figures 2A and 2B). Similar results were obtained using Transwell migration assay (Figures 2C and 2D). To further validate our results, we increased OR5B21 transcription level in 231P as well as MCF7, a luminal non-metastatic human breast cancer cell line (Figure S1C), and analyzed cell invasion and migration. The increasing of OR5B21 transcript abundance led to a significant increase in cell migration and invasion in both cell lines (p < 0.05; Figures 2E and 2F). Interestingly, OR5B21 overexpression in 231P or knockdown in 231BR, 231LM, and 231BO did not affect cell proliferation as analyzed by colony formation assay and the secreted Vluc reporter assay (Figures S2A and S2B). These data demonstrate that OR5B21 participates in the invasion and migration of breast cancer cells.

Open in a separate window

Figure 2

OR5B21 regulates breast cancer invasion/migration

(A and B) 231BR, 231LM, and 231BO expressing either shCTRL or shOR5B21 were plated as a monolayer, scratched, and analyzed by bright-field microscopy at different time points. Representative images (A) and relative quantification of migratory cells (B) are shown (n = 4).

(C and D) 231P cells overexpressing OR5B21 (or control cells) or 231BR, 231LM, 231BO cells transduced with shOR5B21 or scrambled shRNA (shCTRL) were plated on Matrigel-coated Transwell chambers. Forty-eight hours later, cells that migrated to the bottom well were stained with crystal violet and counted (n = 4).

(E and F) 231P and MCF7 control cells or overexpressing OR5B21 were plated, scratched, and analyzed at different time points (n = 3).

Data presented as mean ± SEM; p < 0.05, p < 0.01 by unpaired Student's t test. Scale bar, 150 μm (A and E), 100 μm (C).

OR5B21 promotes breast cancer metastasis

Our in vitro data revealed that OR5B21 was highly associated with pro-metastatic traits. To confirm the role of OR5B21 in breast cancer metastasis, we knocked down OR5B21 in the brain-seeking highly metastatic breast cancer line 231BR expressing firefly luciferase (Fluc) and injected them into the left ventricle of athymic mice. Tumor metastasis to different sites was monitored by bioluminescence imaging weekly. Although control mice formed multiple extensive metastases, OR5B21 knockdown led to a significant decrease in overall metastasis (Figures 3A and 3B). On day 21 post injection, the overall Fluc mean signal was 3.2 × 10⁹ for the control group versus 1.14 × 10⁹ for the OR5B21 knockdown group (p < 0.01, n = 10/group; Figure 3B). In addition, OR5B21 knockdown decreased the incidence rate of the brain, lung, and bone metastases (Figure 3C). For instance, 5/10, 6/10, and 7/10 mice formed brain metastasis in the control group compared with 3/10, 4/10, and 5/10 mice in the OR5B21 knockdown group, at days 7, 14, and 21 post tumor cells injection, respectively (Figure 3C). Importantly, OR5B21 knockdown resulted in a significant increase in the overall mice survival where the median survival for the shOR5B21 group was 29 days versus 23.5 days for the shCTRL group (n = 10/group; p = 0.0024; Figure 3D). Collectively, these data suggest that OR5B21 plays a vital role in breast cancer metastasis to different tissues, especially the brain.

Open in a separate window

Figure 3

OR5B21 knockdown inhibits breast cancer metastasis

231BR cells expressing Fluc and either shCTRL or shOR5B21 were injected intracardially into athymic mice, and metastasis was monitored over time by bioluminescence imaging.

(A) Mouse images at days 7, 14, and 21 post cell injections.

(B) Overall whole-body bioluminescence signals at different time points; data presented as mean ± SEM; p < 0.01 (n = 10 mice/group), unpaired Student’s t test.

(C) Brain, lung, and bone metastasis incidence rate for each group over time.

(D) Kaplan-Meir survival curves for each group, p = 0.0024 Log rank (Mantel-cox) test (n = 10 mice/group).

OR5B21 induces breast cancer metastasis by activating STAT3/NF-κB/CEBPβ signaling

To identify the potential underlying mechanism responsible for the observed effect on breast cancer metastasis, we evaluated the outcome of OR5B21 genetic manipulation on markers associated with invasion/migration and epithelial to mesenchymal transition (EMT), typically involved in metastasis. As expected, MMP1, MMP2, MMP9, Snail, α-SMA, and/or N-cadherin were highly expressed in metastatic breast cancer lines compared with primary 231P (Figure S3). Knockdown of OR5B21 led to a significant decrease in MMP1, MMP2, and MMP9 (markers associated with invasion/migration) as well as SNAIL, α-SMA, and N-cadherin (associated with EMT) in 231BR, 231LM, and 231BO metastatic lines (Figure 4A). Since NF-κB activation is an important characteristic of EMT and cancer metastasis (Azam et al. 2020; Huber et al., 2004; Shostak and Chariot 2011; Smart et al. 2020), we evaluated the effect of OR5B21 on this pathway as well as two transcription factors known to co-operate with NF-κB as synergistic initiators and master regulators of EMT, the signal transducer and activator of transcription 3 (STAT3) and the CCAAT/enhancer binding protein b (C/EBPβ) (Bagnato and Rosanò 2019; Carro et al., 2010; Grivennikov and Karin 2010; Shackleford et al., 2011; Zahnow 2002, 2009). C/EBPβ promotes both STAT3 expression and NF-κB signaling (Canino et al., 2015; Cappello et al., 2009; Shackleford et al., 2011). Of interest, phosphorylation of STAT3 and upregulation of C/EBPβ in different metastatic breast cancer lines (231BR, 231LM, 231BO) coincided with phosphorylation of NF-κB p65 (Figure 4B). Knockdown of OR5B21 reversed STAT3 and p65 phosphorylation and decreased C/EBPβ expression in all three metastatic lines (Figure 4B). On the other hand, OR5B21 overexpression in 231P and MCF7 showed the opposite effect (Figures 5A–5C). Altogether, these data suggest that OR5B21 promotes breast cancer metastasis to different tissues by activating EMT through the STAT3/NF-κB/C/EBPβ signaling axis, revealing OR5B21 as a potential target for breast cancer therapy.

Open in a separate window

Figure 4

OR5B21 regulates invasion/migration through STAT3/NF-κB/CEBPβ signaling pathway

(A) Relative mRNA levels of MMP1, MMP2, MMP9, SNAIL, α-SMA, N-CAD (normalized to β-Actin) in 231BR, 231LM, and 231BO expressing shCTRL or shOR5B21 (n = 7).

(B) 231BR, 231LM, and 231BO cells expressing shCTRL or shOR5B21 were analyzed by western blotting for p-STAT3, STAT3, p-P65, P65, C/EBP-β, and β-Actin loading control. Representative blots from three independent experiments are shown, while bar graphs represent relative quantification of each target, normalized to β-Actin.

Data presented as mean ± SEM (n = 3); p < 0.05, p < 0.01, p < 0.001 by unpaired Student's t test.

Open in a separate window

Figure 5

OR5B21 overexpression increases invasion/migration of BC cells

(A) Relative mRNA levels of MMP1, MMP2, MMP9, SNAIL, α-SMA, N-CAD (normalized to β-Actin) in 231P control cells (CTRL) or overexpressing OR5B21.

(B and C) 231P and MCF7 control cells or overexpressing OR5B21 were analyzed by western blotting for p-STAT3, STAT3, p-P65, P65, C/EBP-β, and β-Actin loading control. Representative blots from three independent experiments are shown (B), while bar graphs represent relative quantification of each target, normalized to β-Actin (C).

Data presented as mean ± SEM (n = 3); p < 0.05, p < 0.01, p < 0.001 by unpaired Student's t test.

Discussion

The human OR repertoire contains 851 loci and mediates olfactory recognition by activating a G-protein-mediated transduction cascade in olfactory sensory neurons (Firestein 2001; Kato and Touhara 2009). In recent years, it has become clear that ORs play an essential role in physiological processes outside the nasal epithelium, some of which are ectopically overexpressed in different cancers (Flegel et al., 2013; Gelis et al., 2016; Kalra et al., 2020; Neuhaus et al., 2009; Rodriguez et al., 2014). Thus, ORs have emerged as markers for cancer diagnostics and potential therapeutic targets (Lee et al., 2019). For instance, 34 ORs were observed in breast cancer with the CHEK2 1100delC mutation (Muranen et al., 2011), whereas OR2T6 showed a key role in breast cancer progression (Li et al., 2019). OR transcript abundance revealed that OR2W3 is associated with invasion and basal-like subtype, whereas OR2B6 correlates with proliferation and luminal A subtype (Masjedi et al., 2019a, 2019b). Activation of different ORs can regulate various signaling pathways leading to either enhanced migration and proliferation or apoptosis. Expression of OR51E1 and closely related OR51E2 is upregulated in prostate cancer (Lee et al., 2019; Maβberg et al., 2016; Neuhaus et al., 2009; Sanz et al., 2014; Xia et al., 2001; Xu et al., 2000). Overexpression or stimulation of OR51E2 promotes prostate cancer invasiveness and progression (Neuhaus et al., 2009; Rodriguez et al., 2016; Sanz et al., 2014). OR51E2 was identified as a potential tumor antigen for CD8+ cytotoxic T cells with implications in cancer immunotherapy (Matsueda et al., 2012). OR51E1 activation negatively regulates prostate cancer cell growth (Maβberg et al., 2016), whereas OR51E2 inhibits melanoma cell proliferation/migration and induces apoptosis (Gelis et al., 2017). Similarly, OR2J3 is overexpressed in lung cancer, and its activation results in apoptosis through ERK1/ERK2/PI3K signaling (Kalbe et al., 2017). Activation of OR1A2 inhibits cell proliferation of liver cancer cells via p38 MAPK signaling (Maβberg et al., 2015). OR51B4 activation in colorectal cancer and OR2AT4 in acute and chronic myelogenous leukemia inhibits cell migration and induces apoptosis (Manteniotis et al., 2016; Weber et al., 2017). In addition to these, several other olfactory receptors serve as potential biomarkers or therapeutic targets in different cancers, such as OR2C3 for melanoma and OR10H1 for bladder cancer (Lee et al., 2019; Weber et al., 2018b). Here, we provide evidence that OR5B21 is ectopically expressed in different breast cancer metastatic sites, with the most upregulation observed in the brain. Interfering with OR5B21 expression leads to inhibition of breast cancer invasion, migration, and metastasis through the STAT3/NF-κB/C/EBPβ signaling axis, serving as a potential therapeutic target for breast cancer.

ORs are a member of GPCR family known to directly or indirectly stimulate STAT transcription factor activity (Ram et al. 2000; Wu et al. 2003; Yu et al., 2014). STAT3, one of the seven members of the STAT family, is constitutively activated in all breast cancer subtypes and is most often associated with triple-negative breast cancer (Furth 2014; Song et al., 2008; Yu et al., 2014). Aberrant STAT3 signaling promotes breast tumor metastasis by interacting with the MMP9 promoter through Fra-1 and c-Jun (Song et al., 2008). On the other hand, STAT3 activation is associated with Snail expression, an interlined event (Saitoh et al., 2016; Xie et al., 2020). Furthermore, several studies showed that STAT3 and C/EBPβ transcription factors act synergistically and co-operate with NF-κB to induce mesenchymal transformation, stimulating cancer metastasis (Cappello et al., 2009; Carro et al., 2010; Schweiger et al., 2020; Shackleford et al., 2011; Xia et al., 1997). In our breast cancer model, aberrant STAT3 signaling correlated with overexpression of MMP9, Snail, and activation of NF-κB in breast cancer metastasis to different tissues (brain, bone, and lung). Of importance, OR5B21 knockdown inhibited phosphorylation of STAT3 and reversed the expression of MMP2, MMP9, and Snail, which coincided with a decrease in both C/EBPβ levels and activation of NF-κB, whereas overexpression of OR5B21 had the opposite effect, endorsing the role of C/EBPβ/STAT3 in OR5B21-induced metastasis. Certainly, additional mechanisms could also be involved. The ligands to which OR5B21 responds to are unknown, and how exactly these ligands affect breast cancer metastasis through OR5B21 remains unclear.

In summary, we identified that OR5B21 plays a vital role in breast cancer metastasis by inducing EMT through the STAT3/NF-κB/C/EBPβ signaling pathway. Downregulation of OR5B21 significantly inhibited breast cancer metastasis to different tissues, especially the brain. Thus, we propose OR5B21 as a potential target for adjuvant breast cancer therapy.

STARMethods

Acknowledgments

Notes

Published: December 17, 2021

Footnotes

References