Involvement of Prokineticin 2-Expressing Neutrophil Infiltration In

Author Manuscript Published OnlineFirst on April 11, 2018; DOI: 10.1158/1535-7163.MCT-17-0845 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Involvement of prokineticin 2-expressing neutrophil infiltration in

5-fluorouracil-induced aggravation of breast cancer metastasis to lung

Soichiro Sasaki1, Tomohisa Baba1, Hayato Muranaka2, Yamato Tanabe1, Chiaki Takahashi2, Seiichi Matsugo3, Naofumi Mukaida1

1Division of Molecular Bioregulation and 2Division of Oncology and Molecular Biology, Cancer Research Institute, Kanazawa University, Kanazawa 920-1192, Japan, 3School of Natural System, College of Science and Engineering, Kanazawa University, Kanazawa

920-1192, Japan.

Running Title: Promotion of lung metastasis by 5-FU treatment

Key words: breast cancer, lung metastasis, neutrophil, 5-FU, prokineticin 2

Corresponding author: Naofumi Mukaida, MD, Ph.D. Division of Molecular Bioregulation, Cancer Research Institute, Kanazawa University, Kakuma-machi, Kanazawa, Ishikawa 920-1192, Japan TEL: +81-76-264-6735; Fax: +81-76-234-4520; Email: [email protected]

Disclosure of conflicts: The authors declare no potential conflicts of interest.

Word count: 4980 excluding References Total Figures and tables: 6 Figures, 15 Supplementary Figures and 1 Supplementary Table

Abstract: 249 words

Downloaded from mct.aacrjournals.org on September 28, 2021. © 2018 American Association for Cancer Research. Author Manuscript Published OnlineFirst on April 11, 2018; DOI: 10.1158/1535-7163.MCT-17-0845 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Abstract

Adjuvant chemotherapy is used for human breast cancer patients, even after curative surgery of primary tumor, in order to prevent tumor recurrence primarily as a form of metastasis. However, anti-cancer drugs can accelerate metastasis in several mouse metastasis models. Hence, we examined the effects of post-surgical administration with

5-fluorouracil (5-FU), doxorubicin, and cyclophosphamide, on lung metastasis process, which developed after the resection of the primary tumor arising from the orthotopic injection of a mouse triple-negative breast cancer cell line, 4T1. Only 5-FU markedly increased the numbers and sizes of lung metastasis foci, with enhanced tumor cell proliferation and angiogenesis as evidenced by increases in Ki67-positive cell numbers and CD31-positive areas, respectively. 5-FU-mediated augmented lung metastasis was associated with increases in intrapulmonary neutrophil numbers and expression of neutrophilic chemokines, Cxcl1 and Cxcl2 in tumor cells, with few effects on intrapulmonary T cell or macrophage numbers. 5-FU enhanced Cxcl1 and Cxcl2 expression in 4T1 cells in a NF-B-dependent manner. Moreover, the administration of a neutrophil-depleting antibody or a Cxcr2 antagonist, SB225002, significantly attenuated

5-FU-mediated enhanced lung metastasis with depressed neutrophil infiltration.

Furthermore, infiltrating neutrophils and 4T1 cells abundantly expressed prokineticin-2

(Prok2) and its receptor, Prokr1, respectively. Finally, the administration of 5-FU after the resection of the primary tumor, failed to augment lung metastasis in the mice receiving

Prokr1-deleted 4T1 cells. Collectively, 5-FU can enhance lung metastasis by inducing tumor cells to produce Cxcl1 and Cxcl2, which induced the migration of neutrophils expressing Prok2 with a capacity to enhance 4T1 cell proliferation.

Introduction Breast cancer is still the most commonly diagnosed cancer in women but can be frequently diagnosed at its early stage with improvement in diagnostic measures such as mammogram and ultrasonography (1,2). As a consequence, breast cancer can often be curatively resected with surgery and nearly 90 % patients can survive for longer than 5 years after the surgery (3). However, even after curative surgery, breast cancer sometimes relapses primarily as a form of metastasis to distant organs including lung, liver, and bone, probably originating from micrometastasis foci in these organs, which are undetected at surgery (4). Thus, in order to reduce the risk of recurrence as a form of metastasis, breast cancer patients generally receive various chemotherapeutic regimens consisting mainly of cyclophosphamide (CTX), doxorubicin (DOX), or 5-fluorouracil (5-FU), as an adjuvant therapy after the surgery (5). It is widely accepted that metastasis can proceed by the interaction of tumor cells and local tissue microenvironments (6). Accumulating evidence indicates that chemotherapeutic drugs can have impacts on resident normal cells such as immune cells besides its direct anti-proliferative effects on cancer cells (7), raising the possibility that the administration of chemotherapeutic drugs may modulate tumor metastasis. Consistently, several independent groups reported that chemotherapy exacerbated rather than attenuated distant metastasis in several mouse breast cancer metastasis models and human patients (8-11). However, these models utilized either immune-deficient mice injected with a human breast cancer cell line, or mice intravenously injected with a cancer cell line, and as a consequence, cannot recapitulate accurately the process of post-surgical adjuvant chemotherapy for breast cancer in immune competent human patients. Hence, it is necessary to examine the effects of chemotherapeutic drugs on spontaneous lung metastasis model in immunocompetent mice, the model which can reproduce more precisely the situations observed in human breast cancer patients undergoing post-surgical adjuvant therapy. A mouse triple-negative breast cancer (TNBC) cell line, 4T1, can metastasize to lung after the complete resection of primary tumor arising from its orthotopic injection into

mammary fat pad (MFP) at the time when cancer cells were hardly detected in lungs (12). This situation resembles lung metastasis, which develops in breast cancer patients with latency after curative surgery of the primary tumors. Hence, we examined the effects of representative chemotherapeutics used for post-surgery adjuvant therapy, CTX, DOX, and 5-FU, on this lung metastasis formation. We proved that 5-FU but neither CTX nor DOX accelerated lung metastasis development by inducing massive migration of neutrophils expressing a potent growth factor, prokineticin-2, in this model. Collectively, we unraveled hitherto unknown and paradoxical effects of 5-FU on breast cancer metastasis to lungs.

Materials and methods Cell lines A mouse mammary carcinoma cell line, BALB/c-derived 4T1 (CRL-2539) (13), and human breast cancer cell line, BT-20 (HTB-19) (14), were purchased in 2007 from American Type Culture Collection. TS/A, another mouse mammary carcinoma cell established from BALB/c mammary adenocarcinoma (15), was obtained in 2010 from Dr. Nanni. All these cell lines were cultured at 37°C under 5 % CO2 in a RPMI 1640 medium supplemented with 10 % fetal bovine serum (FBS) and used within 15 passages of receiving from the source. Mycoplasma and authentication tests of all cell lines were not performed before use.

Lung metastasis models Parental or prokineticin receptor (Prokr) 1-deleted 4T1 cells, or TS/A cells were suspended at a cell density of 1.5 x 106 cells/ml in HBSS and 100 l suspensions were injected into the secondary MFP. Tumor growth was daily monitored and the resultant primary tumor was surgically removed 12 days after the tumor injection, when it reached a diameter of 8.0 to 10.0 mm. Two days after the surgery, the mice received intraperitoneal injection of 5-FU (50 mg/kg) or CTX (150 mg/kg), or intravenous administration of DOX (5 mg/kg). Mice were sacrificed at the indicated time intervals for macroscopical inspection, histological analysis, and total RNA extraction. To deplete neutrophils, mice received intraperitoneally

500 g of a rat anti-Ly6G monoclonal antibody (clone 1A8) in 500 l or PBS once every 4 days after 5-FU injection. In some experiments, mice received intraperitoneally 200 g of a rat anti-Gr-1 monoclonal antibody (clone RB6-8C5) in 200 l PBS or vehicle once every 3 days after 5-FU injection. In another series of experiments, SB225002 (16) was administered at a dose of 0.5 mg/kg body weight once every day after 5-FU treatment.

Statistical analysis The means + SD were calculated for all parameters determined. Statistical significance was evaluated using one-way ANOVA, followed by Tukey-Kramer posthoc test or

Mann-Whitney’s U test. p values less than 0.05 were considered statistically significant.

Supplementary information The Supplementary Methods provided detailed information on mice, reagents and antibodies, generation of Prokr1-deleted 4T1 cells, immunohistochemical analyses of lung tissues, flow cytometric analysis of single cell suspensions obtained from lung or bone marrow, in vitro cell proliferation assay and cell culture of 4T1, TS/A, or BT-20 cells, quantitative (q) RT-PCR analysis, chromatin immunoprecipitation (ChIP) assay, determination of intracellular reactive oxygen species (ROS) generation, isolation of mouse CD11b+Ly6G+ myeloid cells and tumor cells from lung tissue, cell cycle analysis, co-culture of tumor cells and CD11b+Ly6G+ myeloid cells using non-migration transwell system, transwell migration assay, Preparation of H2O2 and H2O2 treatment, In vitro treatment of mouse CD11b+Ly6G+ neutrophils, Senescence-associated β-galactosidase (SA β-gal) staining and clinical database analysis.

Results 5-FU-treatment enhances lung metastasis of mouse breast cancer cell lines We administered chemotherapeutic drugs used for post-surgical adjuvant therapy, DOX, CTX, and 5-FU, to the mice after the surgical removal of the primary tumors arising from the injection of a mouse breast cancer cell line, 4T1, into MFP, in order to investigate their effects on lung metastasis development (Fig. 1A). We observed that a single injection of 5-FU had adverse effects on survival, compared with that of CTX or DOX (Fig. 1B). Moreover, at 28 days after tumor cell injection, only a small number of tumor foci were detected in lungs of untreated mice. By contrast, 5-FU but neither CTX nor DOX, progressively increased the numbers of metastatic tumor foci in lungs (Fig. 1C, D and E). Moreover, 5-FU treatment increased Ki67-positive proliferating tumor cell numbers (Fig.

1F) and CD31-positive vascular areas inside metastatic foci (Fig. 1G) with few effects on ssDNA-positive apoptotic cell numbers (Fig. 1H). Likewise, 5-FU increased lung metastasis focus numbers even when another mouse breast cancer cell line, TS/A, was used (Supplementary Fig. S1). Thus, 5-FU treatment can accelerate lung metastasis when administered after the resection of the primary breast cancer.

5-FU-mediated enhanced lung metastasis is associated with massive neutrophil infiltration We next evaluated the effects of 5-FU on the cells infiltrating into lungs. Immunohistochemical analysis identified Ly6G+ myeloid cells but neither F4/80+ macrophages nor CD3+ T lymphocytes as a predominant type of cells present in lungs in mice treated with 5-FU (Fig. 2A). Consistently, among the cell types that we examined, a flow cytometric analysis revealed the increases only in CD11b+Ly6G+ myeloid cells in lungs from mice treated with 5-FU, at 26 and 28 days after the tumor injection (Fig, 2B and Supplementary Fig. S2A). These CD11b+Ly6G+ myeloid cells exhibited typical neutrophilic morphological features on Giemsa staining (Supplementary Fig. S3A) and did not express granulocyte-myeloid-derived suppressor cell (G-MDSC) markers, CD115 and CD244 (Supplementary Fig. S3B). These observations identified CD11b+Ly6G+ cells as

neutrophils but not G-MDSC. By contrast, CD11b+Ly6G+ neutrophils were not increased in bone marrow in 5-FU-treated tumor-bearing mice or in lung of 5-FU-treated mice without tumor injection (Fig. 2B and Supplementary Fig. S2B). In order to define the contribution of neutrophils to lung metastasis, we next treated mice with anti-Gr-1 antibody. The antibody treatment reduced the numbers of intrapulmonary Ly6G+ neutrophils (Supplementary Fig. S2C) and metastatic foci, particularly those with a diameter larger than 2.0 mm (Fig. 3A and B), together with reductions in Ki67-positive proliferating tumor cell numbers and CD31-positive neovascular areas, but not ssDNA-positive apoptotic cell numbers in metastatic foci (Fig. 3C, D, and E). Similarly, the treatment of a neutrophil-specific antibody, anti-Ly6G, reduced lung metastasis formation (Fig. 3F and G). Massive neutrophil infiltration further prompted us to examine the expression of chemokines with a potent neutrophil chemotactic activity, Cxcl1 and Cxcl2, in lungs. Indeed, Cxcl1 and Cxcl2 mRNA expression was enhanced marginally in lungs of mice after the resection of the primary tumor, but subsequent 5-FU administration augmented their mRNA expression in whole lung tissues (Fig. 4A) and tumor cell fraction purified from lung 28 days after tumor injection (Supplementary Fig. S4A). Immunohistochemical analysis further detected Cxcl1 and Cxcl2 proteins in intrapulmonary tumor cells in 5-FU-treated mice (Fig. 4B). The treatment with a Cxcr2 antagonist, SB225002, reduced the numbers of intrapulmonary Ly6G+ neutrophils (Supplementary Fig. S4B) and the numbers and sizes of metastatic foci (Fig. 4C and D), together with reductions in Ki67-expressing proliferating tumor cell numbers and CD31-positive neovascular areas, but not ssDNA-positive apoptotic cell numbers in metastatic foci (Fig. 4E, F and G), similarly as anti-Gr-1 antibody did. These observations implicated the association of 5-FU-mediated enhanced lung metastasis with 5-FU-induced neutrophilic chemokine expression in tumor cells and subsequent Ly6G+ myeloid cell infiltration.

5-FU induces CXCL1 and CXCL2 expression in breast cancer cells through NF-B signaling via ROS elevation We next examined the effects of 5-FU, CTX, or DOX on Cxcl1 and Cxcl2 expression in

mouse breast cancer cell lines, 4T1 and TS/A. 5-FU enhanced mRNA expression of Cxcl1 and Cxcl2 in 4T1 and TS/A cells in a dose-dependent manner (Fig. 5A and Supplementary

Fig. S5A), while CTX or DOX failed to do (Supplementary Fig. S5B and C). In line with the previous report to indicate the crucial role of NF-B activation in inducible Cxcl1 and Cxcl2 mRNA expression (17), IKK inhibitor, CID-2858522 (18), and NF-B inhibitor, bortezomib, inhibited 5-FU-induced enhancement in Cxcl1 and Cxcl2 mRNA expression (Fig. 5B and Supplementary Fig. S5D). Similarly, 5-FU enhanced the mRNA expression of CXCL1 and CXCL2, and their related molecule, CXCL8, in a human breast cancer cell line, BT-20, and their mRNA expression was reduced by bortezomib (Supplementary Fig. S6A and B). Moreover, ChIP assay revealed that 5-FU increased the binding of a component of

NF-B, p65, to Cxcl1 and Cxcl2 promoters in 4T1 cells (Fig. 5C). The capacity of ROS to activate NF-B incited us to examine the effects of 5-FU on ROS generation in 4T1 cells. Consistently, 5-FU increased intracellular ROS concentration in 4T1 cells (Fig. 5D). An antioxidant, N-acetyl cysteine (NAC), reduced 5-FU-induced p65 binding to Cxcl1 and Cxcl2 promoters (Fig. 5C) and attenuated 5-FU-induced Cxcl1 and Cxcl2 mRNA expression in 4T1 cells (Fig. 5E) together with reduced ROS levels (Fig. 5D). These observations would indicate that 5-FU increased intracellular ROS levels, thereby activating NF-B and subsequent Cxcl1 and Cxcl2 expression.

5-FU-mediated metastasis depends on the Prok2-expressing neutrophils Increases in intrapulmonary tumor cells can be induced by their enhanced invasion into lungs and/or their augmented proliferation, and infiltrating neutrophils may be able to provide cues to induce tumor cells to invade or proliferate. However, neutrophil-derived supernatants failed to induce 4T1 cells to invade (Supplementary Fig. S7A). Thus, we next investigated the expression of various growth factors in lungs. Among the protumorigenic growth factors that we examined (19), only Prok2 expression was enhanced in lungs of mice who received 5-FU after the resection of the primary tumor (Fig. 6A). Of interest is that its enhanced expression was observed only at 26 and 28 days after the tumor injection (Supplementary Fig. S7B) when neutrophil infiltration into lungs became evident (Fig. 1E).

Prok2 mRNA was expressed selectively in CD11b+Ly6G+ neutrophils of lung tissues derived from 5-FU-treated mice (Fig. 6B). Moreover, Prok2 protein was detected in

Ly6G-positive cells obtained from 5-FU-treated but not untreated animals (Fig. 6C and Supplementary Fig. S7C). Furthermore, anti-Gr-1 antibody or SB225002 significantly reduced 5-FU-mediated enhancement in Prok2 mRNA expression in lungs (Fig. 6D). These observation prompted us to explore the involvement of Prok2 in 5-FU-induced and neutrophil-mediated enhancement in lung metastasis. 4T1 cells expressed Prokr1 but not Prokr2 among the receptors for Prok2 (Fig. 6E). Prok2 enhanced the in vitro proliferation and cell cycle progression of parental cells but Prokr1 deletion abrogated Prok2-induced enhancement in in vitro cell proliferation (Fig. 6F and Supplementary Fig. S8A). Moreover, a transwell assay demonstrated that in vitro 4T1 cell proliferation was enhanced in the presence of the CD11b+Ly6G+ neutrophils obtained from the lungs from 5-FU-treated tumor bearing mice, but not those from 5-FU-untreated tumor bearing mice (Fig. 6G left panel). The enhancement was abrogated when Prokr1-deleted 4T1 cells were used instead of the parental cells (Fig. 6G right panel). Furthermore, the administration of 5-FU after the resection of the primary tumor, failed to augment lung metastasis when mice were injected with Prokr1-deleted 4T1 cells (Fig. 6H and Supplementary Fig. S8B) with few effects on Ki67-positive cell numbers, CD31-positive areas, and ssDNA-positive cell numbers (Supplementary Fig. S8C, D and E). Thus, 5-FU induced the infiltration of Prok2-expressing neutrophils through the action of Cxcl1 and Cxcl2, thereby promoting the intrapulmonary growth of breast cancer cells expressing Prokr1 (Supplementary Fig. S9).

Discussion Breast cancer can be frequently diagnosed at its early stage, leading to curative resection with surgery (20). However, breast cancer can often recur with a long latency after the surgery primarily as a form of metastasis, which is presumed to originate from a minute number of dormant cancer cells in the organ (21). Thus, post-surgical adjuvant chemotherapy is commonly employed to reduce the risk of recurrence, but biological heterogeneities in breast cancer can cause difficulties in evaluating the efficacy of the adjuvant therapy as a whole. Moreover, some chemotherapeutics can rather aggravate metastasis in murine experimental breast cancer models as well as human patients (8-11). Hence, we evaluated the effects of chemotherapeutics on lung metastasis model arising from orthotopic injection of breast cancer cells. We proved that 5-FU but neither DOX nor

CTX accelerated lung metastasis together with enhanced neutrophil infiltration. Moreover, neutrophil infiltration was mediated by 5-FU-induced NF-B-dependent production of neutrophilic chemokines, Cxcl1 and Cxcl2, in cancer cells and was associated with enhanced expression of a growth factor, Prok2, with a capacity to augment tumor cell growth. Thus, under some circumstances, 5-FU can accelerate lung metastasis in mice when administered after the resection of primary breast cancer. Furthermore, a similar mechanism may work in some cases of post-surgical adjuvant therapy for breast cancer since 5-FU enhanced the expression of neutrophilic chemokines in a human breast cancer cell line, BT-20, in a NF-B-dependent manner. 5-FU, DOX, and CTX exhibit cytotoxic activities against proliferating cells in distinct manners. 5-FU, an analogue of uracil (22), promptly enters the cells and is intracellularly converted to its active metabolites, several fluoronucleotides, which are incorporated into DNA and RNA to inhibit the synthesis (22). DOX and CTX interfere with DNA synthesis by intercalating with (23) and alkylating DNA (24), respectively. In addition to the inhibition of DNA synthesis, 5-FU-derived fluoronucleotides can inhibit the nucleotide synthetic enzyme, thymidine synthetase (TS). The inhibition can prevent the conversion of dUDP to dTTP, resulting in the imbalance of dNTP amounts and the accumulation of dUTP, both of which cause DNA damage and eventually p53 activation

(22). Through activation of p53, 5-FU can augment the activities of Ataxia telangiectasia mutant (Atm) kinase to repair DNA damage (25), induce senescence to counteract cell damages (26), and enhance ROS generation (27). Evidence is accumulating to indicate that 5-FU can activate a transcription factor,

NF-B (28). We also observed that 5-FU induced mouse and human breast cancer cell lines to express neutrophilic chemokines, whose enhancer regions possess canonical NF-B binding sites (29,30). Moreover, 5-FU enhanced the binding of NF-B p65 to these NF-B binding sites, indicating that 5-FU-induced chemokine expression was mediated by NF-B activation. NF-B activation can be observed in cellular senescence provoked by p53 activation (31), but 5-FU failed to increase senescence-associated -galactosidase staining, a characteristic feature of cellular senescence, in 4T1 and TS/A cells (Supplementary Fig.

S10). Activated p53 can also activate Atm kinase, which can sequentially activate NF-B essential modifier (NEMO), a component of the IKK complex with a capacity to induce nuclear translocation and subsequent activation of NF-B p65/p50 complex (32,33). The involvement of this mechanism, however, was negated by our observation that a specific Atm inhibitor, KU-60019 (34), failed to abrogate 5-FU-induced expression of Cxcl1 and Cxcl2 in mouse breast cancer cell lines, 4T1 and TS/A cells (Supplementary Fig. S11). By contrast, 5-FU increased intracellular ROS amounts and NAC-mediated reduction in ROS significantly reduced 5-FU-induced NF-B binding to its cis-elements in mouse Cxcl1 and

Cxcl2 genes, and their subsequent mRNA expression. Consistently, H2O2 enhanced Cxcl1 and Cxcl2 mRNA expression in mouse breast cancer cell lines (Supplementary Fig. S12).

Thus, 5-FU can activate NF-B via ROS elevation and eventually augment the expression of NF-B target genes such as Cxcl1 and Cxcl2. Accumulating evidence indicates that as metastasis proceeds, lungs are infiltrated by inflammatory cells, which can contribute to metastasis progression by providing growth factors and suppressing immune responses (35). Among inflammatory cells, the roles of macrophages in lung metastasis have been extensively examined. Intrapulmonary macrophages can be classified into interstitial and alveolar macrophages, which reside in interstitial and alveolar spaces, respectively (36). Metastasis-associated macrophages

(MAMs) are defined as those which migrate from the circulation into interstitial space of metastatic lungs (37-39) and can promote lung metastasis. Another intrapulmonary macrophage subpopulation, alveolar macrophages, can also foster lung metastasis through their immunosuppressive actions (40). However, we could not detect a significant increase in macrophage numbers in the present metastatic lungs, making it a remote possibility of the contribution of macrophages to this accelerated lung metastasis process. Other types of infiltrating inflammatory cells include neutrophils (41) and myeloid-derived suppressor cells (MDSCs) (42). MDSCs are immature myeloid cells in contrast to terminally differentiated myeloid cells such as neutrophils. Mouse MDSCs express simultaneously CD11b and Gr-1 (also known as Ly6G/Ly6C), and function as immunosuppressive cells. In mice, surface markers are shared by neutrophils and MDSCs, but MDSCs can be discriminated from neutrophils by using the combination of several markers (43). It is presumed that MDSCs are recruited from bone marrow to a target organ to provide a niche to support cancer cell growth before the arrival of cancer cells (44). By contrast, in the present lung metastasis model, we showed that CD11b+Ly6G+ myeloid cells were increased in lungs only at the late phase of metastasis progression but lacked characteristic G-MDSC features. Thus, it is more likely that the increased CD11b+Ly6G+ cells represented neutrophils but not G-MDSCs. Several lines of evidence indicate that infiltrating neutrophils can promote metastasis by producing metalloproteinase (45) and growth factors such as Prok2 (46). Indeed, our unbiased analysis revealed a selectively enhanced expression of Prok2 by neutrophils infiltrating into metastatic lungs. Prok1 and Prok2 are highly conserved small peptides (47) and mammalian homologues of Bv8, which was originally identified from amphibian skin (48). Both of them were identified as a potent contractor of gastrointestinal smooth muscle (47), and subsequent studies revealed that Prok1 and Prok2 have diverse effects on feeding, drinking, neuron migration and survival, angiogenesis, hematopoiesis, and inflammation by binding their specific G protein-coupled receptors, Prokr1 and Prokr2 (49). Accumulating evidence further indicates the crucial involvement of Prok1 and Prok2 in neovascularization observed in various types of cancers (50,51). Prok2 or

Prok2-expressing neutrophils in vitro augmented, in a dose-dependent manner, the proliferation of 4T1 cells, which expressed Prokr1. Moreover, the proliferative response was abrogated by Prokr1 gene deletion in 4T1 cells. Furthermore, 4T1 cells deficient in Prokr1 gene did not exhibit 5-FU-mediated enhanced lung metastasis formation. Thus, the Prok2-Prokr1 axis can directly regulate tumor cell proliferation, thereby contributing to tumor progression. This assumption may be supported by the analysis using a public database, PrognoScan, which demonstrates that high PROKR1 and PROK2 gene expression is associated with shorter overall survival and relapse-free survival, respectively (Supplementary Fig. S13A and B). Of interest is that enhancement of Prok2 expression was detected in neither neutrophils isolated from the lungs of untreated mice (Supplementary Fig. S2B) nor neutrophils stimulated in vitro with Cxcl1 or Cxcl2 (Supplementary Fig. S14). Thus, 5-FU-induced production of these chemokines may not be sufficient to fully activate neutrophils to express Prok2. We observed that granulocyte colony-stimulating factor

(G-CSF), a representative NF-B target molecule (52), was abundantly detected in lungs after 5-FU administration (Supplementary Fig. S15), and that G-CSF can in vitro induce Prok2 expression in CD11b+Ly6G+ neutrophils (Supplementary Fig. S14), consistent with the previous reports (50,53). Thus, 5-FU-induced enhanced lung metastasis can be mediated by the cooperative actions of neutrophilic chemokines and G-CSF, both of which are

NF-B target, and therefore, NF-B may be a candidate target molecule to improve the efficacy of post-surgical neoadjuvant chemotherapy for breast cancer.

Acknowledgements We would like to express our sincere appreciation to Professors Noriko Gotoh (Kanazawa

University) and Tsuneyasu Kaisho (Wakayama Medical University) for providing us with a material support and advising us on CRIPSR-Cas 9 methodology, respectively. This work was supported partly by the Grant-in-Aid for Scientific Research (C) from the Japan Society for the Promotion of Science (JSPS) KAKENHI Grant Number 17K07159.

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Legends to Figures

Figure 1. 5-FU-induced increase in lung metastasis of a mouse breast cancer cell line, 4T1. (A) Schematic representation of experimental procedure of orthotopic lung metastasis model. Mice were injected with CTX (light gray circles), DOX (dark gray circles), or 5-FU (closed circles) along with figure A. (B) Kaplan-Meier survival curves for mice which were injected with CTX, DOX or 5-FU as chemotherapeutic drugs (n=10). p < 0.05; n.s., not significant. (C, D) The lungs were removed 28 days after 4T1 cell injection to inspect the macroscopic appearance of the lung (C) and determine the tumor focus numbers (D). Each symbol represents the metastatic tumor focus numbers of an individual mouse (n = 5). The bars represent the average of each group. **, p < 0.01; n.s., not significant. (E) Animals were sacrificed at each time point until day 28 after 4T1 cell injection with or without 5-FU treatment, to determine tumor focus numbers of lung. The data represent the mean + SD (n = 3). *, p < 0.05; **, p < 0.01. (F-H) Twenty-eight days after 4T1 cell injection, lung was removed from mice to determine the numbers of Ki67-positive cells (F), CD31-positive areas (G), and ssDNA-positive cells (H). All values represent the mean + SD (n = 4). *, p < 0.05; **, p < 0.01; n.s., not significant.

Figure 2. Effects of 5-FU treatment on cells infiltrating into lungs. (A) Lung tissue were obtained from mice 28 days after 4T1 cell injection and were subjected to H&E staining or immunohistochemical analysis using anti-Ly6G, anti-F4/80, or anti-CD3 antibodies. Representative results from 5 independent experiments are shown here. Insets indicate enlarged area of lungs. Original magnification, x 100; bars, 100 m. (B) CD11b+Ly6G+ myeloid cell numbers of lung or bone marrow were counted by flow cytometry. Animals were sacrificed at each time point until 28 days after 4T1 cell injection, to determine the CD11b+Ly6G+ myeloid cell numbers. Data represent the mean + SD (n = 4). *, p < 0.05; **, p < 0.01.

Figure 3. Effects of neutrophil depletion on 5-FU-induced increase in lung metastasis. (A to E) Mice were injected intraperitoneally with anti-Gr-1 antibody (200 g/ head) or PBS once every 3 days, starting 14 days after 4T1 cell injection. The lungs were removed 28 days after 4T1 cell injection to determine the tumor numbers (A) and sizes (B). Each symbol represents the tumor numbers per mouse, and bars represent the average for each group (n = 6). *, p < 0.05; **, p < 0.01; n.s., not significant. (C-E) Ki67-positive cell numbers (C), CD31-positive areas (D), and ssDNA-positive cell numbers (E) were determined with immunohistochemical analysis. All values represent the mean + SD (n = 5). *, p < 0.05; n.s., not significant. (F to G) Mice were injected intraperitoneally with anti-Ly6G antibody (500

g/ head) or PBS once every 4 days. The lungs were removed 28 days after 4T1 cell injection to determine the tumor numbers (F) and sizes (G). Each symbol represents the tumor numbers per mouse, and bars represent the average for each group (n = 10). *, p < 0.05; **, p < 0.01;

Figure 4. Involvement of the Cxcl1 and Cxcl2 in 5-FU-mediated lung metastasis enhancement. (A) Cxcl1 and Cxcl2 mRNA expression in the lungs of 5-FU untreated (open squares) or treated mice (closed squares). Expression levels were normalized to Hprt mRNA levels. Data represent the mean + SD (n = 4). *, p < 0.05; **, p < 0.01. (B) Lungs were removed from

5-FU-treated mice 28 days after the tumor injection. The obtained tissues were fixed and immunostained with Cxcl1 (upper panel) or Cxcl2 (lower panel) antibody. Representative results from three independent experiments are shown here. Original magnification, x 100; bar, 100 m. (C-G) Mice were injected intraperitoneally with SB225002 (0.5 mg/kg) or vehicle, once every day, starting 14 days after 4T1 cell injection. The lungs were removed 28 days after 4T1 cell injection to determine the tumor numbers (C) and sizes (D). Each symbol represents the tumor numbers per mouse, and bars represent the average for each group (n = 10). (E-G) Ki67-positive cell numbers (E), CD31-positive areas (F), and

ssDNA-positive cell numbers (G) were determined with immunohistochemical analysis. All values represent the mean + SD (n = 5). *, p < 0.05; **, p < 0.01; n.s., not significant.

Figure 5. Molecular mechanisms underlying 5-FU-induced Cxcl1 and Cxcl2 expression. (A) 4T1 cells were treated with the indicated concentrations of 5-FU for 24 hrs. The levels of mRNA were quantified by qRT-PCR. All values represent the mean + SD (n = 3). *, p < 0.05; **, p < 0.01. (B) 4T1 cells were pre-treated for 1 hr with the indicated concentrations of CID-2858522 and were further incubated with 5-FU (5 g/ml) for 24 hrs. The levels of mRNA were quantified by qRT-PCR. All values represent the mean + SD (n = 3). **, p <

0.01. (C) NF-B p65 binding to the promoter region of the mouse Cxcl1 and Cxcl2 was assessed by ChIP assay in untreated or 5-FU-treated 4T1 cells with or without pre-treatment with NAC. Input or eluted chromatin was subjected to qRT-PCR analysis using promoter-specific primers. Data are represented as the % input of the immunoprecipitated chromatin for each gene from three separate chromatin preparations. Data represent the mean + SD (n = 3). (D) Intracellular ROS levels were determined. 4T1 cells were incubated in the absence or the presence of 5-FU (5g/ml) for 24 hrs together with 1 hr pretreatment with NAC. Representative results from 3 independent experiments are shown here. (E) 4T1 cells were incubated in the absence or the presence of 5-FU (5

g/ml) for 24 hrs after 1 hr pre-treatment with indicated concentrations of NAC. The levels of mRNA were quantified by qRT-PCR. All values represent the mean + SD (n = 3). *, p <

0.05.

Figure 6. Involvement of Prok2-expressing CD11b+Ly6G+ neutrophils in 5-FU-induced increase in lung metastasis. (A) Intrapulmonary growth factor expression was determined in mice administered with or without 5-FU after the resection of the primary tumor. Twenty-eight days after 4T1 cell injection, total RNAs were extracted from lung and subjected to qRT-PCR to determine the mRNA expression levels of the indicated growth factors. All

values represent mean + SD (n = 4). *, p < 0.05; **, p < 0.01. (B) Prok2 mRNA expression in intrapulmonary neutrophils. Twenty-eight days after 4T1 cell injection, CD11b+Ly6G+ neutrophils and non-neutrophils populations were purified from whole lung cells by a flow cytometry. All values represent mean + SD (n = 3). *, p < 0.05. (C) Double-color immunofluorescence analysis of lungs were conducted with the anti-Ly6G and anti-Prok2 antibodies. Representative results are shown from 3 independent experiments. Original magnification, x 200; bar, 50 m. (D) Prok2 mRNA expression was determined in mice injected intraperitoneally with anti-Ly6G antibody (500 g/head) or SB225002 (0.5 mg/kg) starting 14 days after 4T1 cell injection. Twenty-eight days after 4T1 cell injection, total RNAs were extracted from lung. All values represent mean + SD (n = 3). **, p < 0.01. (E) Prokr1 and Prokr2 mRNA expression in 4T1 cells. The cells were untreated (open squares) or treated (closed squares) with 5-FU (5 g/ml) for 24 hrs. Data represent the mean + SD (n = 3). *, p < 0.05. (F) In vitro proliferation rates of control vector-treated 4T1 (left) or Prokr1-deleted 4T1 cells (right). The cells were cultured with the indicated concentrations of Prok2 for 48 hrs, to determine cell proliferation rates using the cell counting kit-8. The ratios of cell numbers were determined by comparing the OD value of untreated cells. All values represent the mean + SD (n = 5). *, p < 0.05; **, p < 0.01; n.s., not significant. (G) The CD11b+Ly6G+ neutrophils were purified from untreated or 5-FU-treated mice and were placed in the upper well of 0.4 µM pore transwells while parental or Prokr1-deleted 4T1 cells were placed in the lower wells of 96-well. After 48 hrs, the upper well were removed to determine cell proliferation rates of control vector 4T1 (left panel) or

Prokr1-deleted 4T1 cells (right panel) using the cell counting kit-8. The ratios of cell numbers were determined by comparing the OD value of untreated cells. All values represent the mean + SD (n = 5). *, p < 0.05; n.s., not significant. (H) Mice were injected intraperitoneally with 5-FU 14 days after control vector-treated 4T1 (Control) or

Prokr1-deleted 4T1 cells (Prokr1) injection to determine the tumor numbers. Each symbol represents the tumor numbers per mouse, and bars represent the average for each group (n = 5). *, p < 0.05; n.s., not significant.

Involvement of prokineticin 2-expressing neutrophil infiltration in 5-fluorouracil-induced aggravation of breast cancer metastasis to lung

Soichiro Sasaki, Tomohisa Baba, Hayato Muranaka, et al.

Mol Cancer Ther Published OnlineFirst April 11, 2018.

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