Menahydroquinone-4 may play a key role in regulating CCL5 expression induced by epidermal growth factor receptor inhibitors

Epidermal growth factor receptor (EGFR) inhibitors frequently cause severe skin rash as a side effect, which is a critical burden for patients who continuously receive drug treatments. Several recent clinical trials have shown that vitamin K is effective against these side effects; however, the underlying mechanisms remain unclear. EGFR inhibitors induce C–C motif chemokine ligand 5 (CCL5) in dermopathy. We hypothesized that menahydroquinone-4 (MKH), the active form of menaquinone-4 (MK-4, vitamin K2(20)), supplied by biosynthesis or external delivery, is essential for the suppressive effect on CCL5. The aim of this study was to explore the underlying mechanisms governing the relieving effects of MKH against skin rashes caused by EGFR inhibitors. The responses generated by EGFR inhibitors and the effect of MKH derivatives (two ester derivatives and MK-4) on them were evaluated using human skin cell lines (HaCaT and HSC-1). EGFR inhibitors downregulated UbiA prenyltransferase domain-containing protein-1 (UBIAD1, MKH synthetase) expression and MKH biosynthesis. Knockdown of UBIAD1 or γ-glutamyl carboxylase and treatment with warfarin upregulated CCL5 expression. MKH derivatives suppressed the CCL5 expression induced by EGFR inhibitors. Our data strongly suggest that MKH is involved in suppressing CCL5 expression and alleviating the skin damage caused by EGFR inhibitors.

In recent years, epidermal growth factor receptor (EGFR)-targeted agents have primarily been used for cancer treatment.EGFR inhibitors consist of tyrosine kinase inhibitors (TKIs) and monoclonal antibodies, which are employed for the treatment of colorectal cancer and lung cancer, respectively.However, although EGFR inhibitors generate strong anti-cancer effects, they often induce skin rashes, which substantially affect quality of life and impact medication adherence [1][2][3][4] .The occurrence of skin rashes is highly correlated with the efficacy of EGFR inhibitors 1,5,6 .Nevertheless, severe skin rashes can cause both physical and psychological distress, prompting dose reduction or discontinuation of anti-cancer therapy 5,6 .Therefore, meticulous management of such skin rashes becomes imperative.
To date, no prophylactic methods for EGFR inhibitor-induced skin rashes have been established.The general approach to skin rash is symptomatic treatment using topical steroids and antibiotic agents 7 ; however, more efficacious treatment strategies are required as certain patients continue suffering from severe skin rashes 8 .Furthermore, increased focus on drug resistance and the side effects associated with long-term drug use is warranted.
Both PK and Vitamin K 2 (menaquinone-4, MK-4) obtained from the diet are converted into vitamin K 3 (menadione) in the gastrointestinal tract and then reduced to menadiol (Fig. 1).Subsequently, menadiol is prenylated to menahydroquinone-4 (MKH) via UbiA prenyltransferase domain-containing protein 1 (UBIAD1) [12][13][14] .MKH is the fully reduced and active form of MK-4 and functions as a cofactor for γ-glutamyl carboxylase (GGCX), an enzyme that converts γ-glutamate (Glu) residues to γ-carboxyglutamate (Gla) residues during the post-translational modification of vitamin K-dependent proteins (VKDPs) 15,16 .MKH promotes the conversion of Glu to Gla in VKDPs via GGCX, after which MKH is stoichiometrically converted to menaquinone-4 2,3-epoxide (MKO).MKO is subsequently regenerated to MK-4 via the action of vitamin K epoxide reductase complex subunit 1 (VKORC1) or vitamin K epoxide reductase complex subunit 1-like 1 (VKORC1L1).This series of redox reactions is termed the vitamin K cycle.Vitamin K is involved in various biological functions, including blood clotting, bone formation, and anti-inflammatory effects, through the conversion of Glu to Gla in VKDPs 17 .Therefore, we hypothesized that adequate MKH delivery to the skin may suppress EGFR inhibitor-induced skin rashes.
Herein, using a normal human keratinocyte cell line (HaCaT cells) and human skin squamous cell carcinoma cell line (HSC-1), we aimed to explore the mechanisms underlying the relieving effects of MKH against skin rashes caused by EGFR inhibitors.In the present study, a novel strategy for managing skin toxicity caused by EGFR inhibitors has been delineated.

EGFR inhibitors suppressed MKH biosynthesis
As EGFR inhibitors downregulate UBIAD1 expression in HaCaT cells, we hypothesized that MKH biosynthesis would also be hindered.To assess the effect of EGFR inhibitors on MKH biosynthesis, we measured MKH concentration in the S9 fraction of HaCaT cells cultured with gefitinib (1 µM), erlotinib (1 µM), or cetuximab (100 µg/mL).Enzymatic reactions were initiated by adding menadione (100 µM), GGPP (100 µM), and DTT (1 mM) into S9 fraction-containing tubes.The MKH produced was easily oxidized to MK-4 during extraction.Consequently, MK-4 concentration was measured using LC-MS/MS and regarded as the MKH concentration.In each group, MK-4 concentrations increased in a time-dependent manner and plateaued after 1 h.S9 fractions cultured with EGFR inhibitors showed significantly lower MK-4 levels than the control (Fig. 3).Gefitinib and erlotinib equally suppressed MKH production, to a greater extent than cetuximab.To reaffirm the obtained result, we showed a concentration-dependent increase in uncarboxylated matrix Gla protein (ucMGP), an indicator of decreased conversion of Glu to Gla in VKDPs, in gefitinib-treated HaCaT cells (Supplementary Fig. 2a).Of note, no significant effect of gefitinib on VKORC1, VKORC1L1 and GGCX mRNA expression were observed (Supplementary Fig. 2b-d).As MKH promotes the conversion of Glu to Gla in VKDPs if GGCX is functional, an increased ucMGP content implies the suppression of the biosynthesis of MKH.Altogether, these results indicate that EGFR inhibitors suppress the biosynthesis of MKH by downregulating UBIAD1 expression and affecting the conversion of Glu to Gla in VKDPs, which could lead to upregulated CCL5 mRNA expression.

Suppression of MKH levels and Glu to Gla conversion in VKDPs lead to the upregulation of CCL5 expression
The anticoagulant warfarin inhibits the activities of VKORC1 and VKORC1L1 24,25 .These enzymes catalyze the reduction of MKO and MK-4 to MKH, which are required for the Glu to Gla conversion in VKDPs.Consequently, the inhibition of VKORC1 and VKORC1L1 restricts the availability of MKH, leading to the suppression of Glu to Gla conversion in VKDPs.To confirm whether reduced levels of MKH affect CCL5 expression, we evaluated the effect of warfarin on CCL5 mRNA expression in HaCaT cells.The inhibition of the activity of VKORC1 was confirmed by the increased intracellular MKO concentration via the administration of 3 µM MK-4 following treatment with 200 µM warfarin (Fig. 4a).Furthermore, treatment with warfarin (50, 100, and 200 µM) upregulated CCL5 mRNA expression in a dose-dependent manner (Fig. 4b).Next, to investigate whether conversion of Glu to Gla in VKDPs affects CCL5 expression, we evaluated the effects of GGCX knockdown on CCL5 mRNA expression.The decrease in GGCX activity was confirmed by the decreased intracellular MKO concentration via the administration of 10 µM MK-4 following siGGCX treatment (Fig. 4c).Treatment with siGGCX suppressed GGCX mRNA expression and upregulated CCL5 mRNA expression (Fig. 4d,e).These results indicate that MKH functions as a cofactor of GGCX and promotes the conversion of Glu to Gla in VKDPs, thereby regulating CCL5 expression.Graphic summary depicting changes in intracellular MKO levels following warfarin or siGGCX treatment and the modulation of CCL5 expression by Gla proteins are presented in Fig. 4f.

Discussion
EGFR plays a pivotal role in epidermal development and physiology 26 .Blocking EGFR signaling in skin cells downregulates several pathways, including the mitogen-activated protein kinase, phosphatidylinositol-3-kinase, and stress-activated protein kinase pathways.EGFR inhibition causes cell growth arrest and apoptosis, which interfere with the structural development of the skin.It also induces CCL2, CCL3, CCL5, CCL18, CXC motif chemokine ligand (CXCL) 9, CXCL10, XCL1, fractalkine (CX3CL1), and CXC chemokine receptor type 4 production [21][22][23] .These chemokines promote the recruitment and activation of leukocytes, such as neutrophils, www.nature.com/scientificreports/monocytes, and lymphocytes, at the skin rash sites.Consequently, the barrier function of the skin is impaired, exacerbating inflammation 21 .
Recently, vitamin K has emerged as a novel therapeutic candidate for EGFR inhibitor-induced skin rashes.Menadione activates ErbB, a member of the TK receptor family that also includes EGFR, through the phosphatase-inhibitory effect of menadione 27 .Perez-Soler and Ling disclosed a patent for a method to activate EGFR in the skin through topical vitamin K treatment to protect the skin from the systemic effects of EGFR inhibitors 28 .In this patent, the authors reported that 100 μM menadione is the strongest EGFR activator, 1000 μM PK generates 1/10th of the effect of menadione, and MK-4 is ineffectual against human squamous cell carcinoma cells (A431).They subsequently published a research paper based on this patent in Clinical Cancer Research 29 , but it was retracted in 2013.Clinical trials using menadione and PK have been conducted for the prevention and treatment of skin rashes caused by cetuximab 30,31 .Menadione was shown to be ineffective in treating cetuximabinduced skin rashes; however, the efficacy of PK remains an issue of contention.A recent clinical trial (EVITA) showed that the topical application of PK is effective in women 10,32 .There are no clinical trials on the efficacy of menadione and PK in treating EGFR-tyrosine kinase inhibitor (TKI)-related skin rashes.Additionally, the mechanism of action of vitamin K on EGFR inhibitor-induced skin rashes remains to be elucidated.
Dietary PK is converted to menadione in the body, which is then converted to MKH via UBIAD1 [12][13][14] .The findings of our study suggest that patients receiving chronic treatment with EGFR inhibitors have low MKH biosynthesis ability owing to UBIAD1 suppression.Gefitinib and erlotinib reportedly prolong the prothrombin time-international normalized ratio in patients undergoing warfarin treatment; however, the underlying mechanism remains unascertained 33,34 .The suppression of MKH biosynthesis by EGFR inhibitors could be a plausible cause.
Our data also showed that UBIAD1 knockdown, as well as warfarin and siGGCX treatment, upregulated CCL5 mRNA expression, suggesting that MKH delivery and conversion of Glu to Gla in VKDPs are essential for regulating CCL5 expression.VKDPs that exhibit anti-inflammatory effects include protein S, growth-arrest specific gene 6 (Gas6) and Gla-Rich Protein (GRP).Protein S and Gas6 significantly inhibited the expression of pro-inflammatory cytokines such as TNF-α, IL-6, and IL-1β in macrophages following LPS stimulation 35 .GRP suppressed the expression levels of inflammatory markers, including cyclooxygenase 2 (COX2) and matrix metallopeptidase 13 (MMP13), in chondrocytes derived from osteoarthritis and synovial cell lines 36 .These VKDPs may be involved in the regulation of CCL5 expression; however, further studies are needed to clarify this.
For these reasons, we anticipated that efficient MKH delivery would inhibit CCL5 expression.Our data showed that MK-4 and MKH derivatives suppressed CCL5 expression induced by EGFR inhibitors to a greater degree than PK.The ineffectiveness of PK may be attributed to the reduced expression of UBIAD1 caused by EGFR inhibitors, as UBIAD1 plays a crucial role in converting PK to MKH.As a result, the full potential of PK cannot be realized.Furthermore, it is also conceivable that PK exhibits lower cellular uptake and has lower bioavailability than MK-4, contributing to its ineffectiveness on CCL5 expression 18,37 .
CCL5 is an inflammatory chemokine released in the late inflammatory phase that mobilizes leukocytes to inflammatory sites and is involved in maintaining inflammation 20 .Its levels are increased in human skin tissue treated with EGFR inhibitors 19 ; a similar phenomenon has been reported in mouse skin tissue 22,38 .In vitro treatment of human skin cells with EGFR inhibitors has been reported to increase CCL5 levels [21][22][23] .Promotion of CCL5 expression is expected to contribute to the exacerbation of EGFR inhibitor-induced skin rashes.Conversely, CCL5 is also associated with cancer progression and metastasis by promoting the survival, proliferation, and infiltration of tumor cells 38 .Therefore, enhancing the rate of MKH delivery to the skin may also prove efficient in preventing skin cancer metastasis.
Our findings reveal that MKH derivatives produce a CCL5-suppressive effect that is equal to or greater than that exhibited by MK-4.However, MK-4 is easily photodegraded and possesses phototoxic properties, leading to the prohibition of the conventional usage of vitamin K in cosmetics within Europe 39 .Consequently, the effects of light on MK-4 are unavoidable when MK-4 is used as a topical skin treatment.In contrast, our previous research has demonstrated that MKH derivatives are not easily degraded and do not exhibit phototoxicity 18 .Furthermore, MKH derivatives can deliver MKH to cells, even after irradiation with sunlight.Hence, it can be concluded that MKH derivatives are more suitable for topical skin applications than MK-4.
In conclusion, patients using EGFR inhibitors may have decreased MKH biosynthesis ability and increased CCL5 expression together with reduced UBIAD1 expression.MKH treatment via MK-4 and MKH derivatives may contribute to the suppression of CCL5 expression and alleviation of skin damage caused by EGFR inhibitors.Furthermore, MKH derivatives are less sensitive to light and may be more useful as topical skin preparations than MK-4.A limitation of our study is the use of a monolayer culture system, which cannot completely replicate the biological environment.Future analyses using in vivo models are required to obtain more definitive results.We strongly anticipate that our findings will contribute to elucidating the onset and alleviation mechanism of skin rashes.

Cell culture
HaCaT cells obtained from CLS Cell Lines Service GmbH (Eppelheim, Germany) and HSC-1 cells obtained from the Japanese Collection of Research Bioresources Cell Bank were grown in high glucose-containing Dulbecco's modified Eagle's medium (FUJIFILM Wako Pure Chemical) containing 10% fetal bovine serum and 1% penicillin/streptomycin (Thermo Fisher Scientific, Waltham, MA, USA) at 37 °C under a 5% CO 2 environment.

MKH biosynthesis activity of UBIAD1
MKH biosynthesis ability was assessed using a previously reported method 12 .Briefly, HaCaT cells (5.0 × 10 5 cells/well) were seeded in a 10-cm dish and allowed to attach for 72 h.The medium was replaced with fresh medium containing gefitinib, erlotinib, or cetuximab and incubated for 24 h.Cells were washed with cold PBS and collected in 1 mL of PBS using a cell scraper.After ultrasonic homogenization, cells were centrifuged at 10,000×g for 1 h at 4 °C, and S9 fractions were obtained.MK-4 generation reactions were initiated by mixing 1 mM dithiothreitol (DTT; FUJIFILM Wako Pure Chemical), 100 µM menadione (Sigma-Aldrich), and 100 µM geranylgeranyl pyrophosphate (ammonium salt) (GGPP) (Cayman Chemical Company, Ann Arbor, MI, USA) with S9 fraction solutions adjusted to 760 µg protein/mL in PBS.Solutions were incubated for 5 h at 37 °C, and

Figure 3 .
Figure3.Effect of EGFR inhibitors on MK-4 conversion activity in S9 fractions of HaCaT cells.HaCaT cells cultured with 1 µM gefitinib, 1 µM erlotinib, or 100 µg/mL cetuximab for 24 h were harvested and S9 fractions were prepared.Total protein concentration of S9 fractions was normalized in PBS and the suspension was incubated with 100 μM menadione, 100 μM GGPP, and 1 mM DTT for 5 h at 37 °C.Concentrations of MK-4 synthesized over time were determined using LC-MS/MS.Values represent means ± standard deviation (n = 3).**p < 0.01 compared to the control at the same time point.DTT dithiothreitol, EGFR epidermal growth factor receptor, GGPP geranylgeranyl pyrophosphate, LC-MS/MS liquid chromatography with tandem mass spectrometry, MK-4 menaquinone-4.