2022-A novelE-cadherin/SOX9 axis regulates cancer stem cells in multiple myeloma byactivating Akt and MAPK pathways

Samart et al. Experimental Hematology & Experimental Hematology & Oncology (2022) 11:41 Oncology https://doi.org/10.1186/s40164-022-00294-x CORRESPONDENCE Open Access A novel E‑cadherin/SOX9 axis regulates cancer stem cells in multiple myeloma by activating Akt and MAPK pathways Parinya Samart1,2, Yon Rojanasakul3, Surapol Issaragrisil2,4,5 and Sudjit Luanpitpong2*  Abstract  Cancer stem cells (CSCs) have been identified in multiple myeloma (MM) and are widely regarded as a key driver of MM initiation and progression. E-cadherin, in addition to its established role as a marker for epithelial-mesenchymal transition, also plays critical roles in controlling the aggressive behaviors of various tumor cells. Here, we show that depletion of E-cadherin in MM cells remarkably inhibited cell proliferation and cell cycle progression, in part through the decreased prosurvival CD138 and Bcl-2 and the inactivated Akt and MAPK pathways. CSC features, including the ability of the cells to form clonogenic colonies indicative of self-renewal and side population, were greatly suppressed upon the depletion of E-cadherin and subsequent loss of SOX9 stem-cell factor. We further provide evidence that SOX9 is a downstream target of E-cadherin-mediated CSC growth and self-renewal—ectopic re-expression of SOX9 in E-cadherin-depleted cells rescued its inhibitory effects on CSC-like properties and survival signaling. Collectively, our findings unveil a novel regulatory mechanism of MM CSCs via the E-cadherin/SOX9 axis, which could be important in understanding the long-term cell survival and outgrowth that leads to relapsed/refractory MM. Keywords:  Multiple myeloma, E-cadherin, SOX9, Cancer stem cells, Self-renewal To the editor, CDH1) is known to have a pivotal role in the regulation Novel therapies for multiple myeloma (MM), such as of embryonic and normal adult stem cell survival and proteasomal inhibitors, immunomodulatory drugs, self-renewal [3, 4]. In solid tumors, loss of E-cadherin and CAR-T cell therapy, have improved palliation and has traditionally been viewed as a hallmark of the occur- response rates, providing a longer disease-free period; rence of epithelial-to-mesenchymal transition, linking to however, MM inevitably progresses in the vast majority metastasis. The role of E-cadherin in solid tumor growth, of patients [1]. Cancer stem cells (CSCs), also known as however, remains controversial and appears to be cell tumor initiating cells, are believed to be the root cause type- and tumor stage-dependent [5, 6]. E-cadherin pro- of tumor recurrence for most if not all malignancies, tein level is significantly higher in MM tissues compared including MM [2]. Identification of molecular pathways to normal tissues [7], and its increased mRNA expres- that contribute to CSCs is essential to understanding how sion has been correlated with symptomatic MM [8] and MM progression is regulated. E-cadherin (encoded by plasma cell leukemia, an aggressive variant of MM (Addi- tional file 2: Figure S1). We have previously reported the *Correspondence: [email protected] 2 Siriraj Center of Excellence for Stem Cell Research, Faculty of Medicine Siriraj Hospital, Mahidol University, 2 Siriraj Hospital, Bangkoknoi, Bangkok 10700, Thailand Full list of author information is available at the end of the article © The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://c​ reati​ vecom​ mons.o​ rg/​licen​ses/​by/4.​0/. The Creative Commons Public Domain Dedication waiver (http://c​ reat​iveco​ mmons.​org/p​ ubli​cdomai​ n/z​ ero/1.​0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

Samart et al. Experimental Hematology & Oncology (2022) 11:41 Page 2 of 6 decreased E-cadherin level in poorly disseminated MM S3), which could be reactivated by the restoration of cells mediated by hyper-O-GlcNAcylation [9]. E-cadherin (Additional file  2: Figure S4). Additionally, we found that depletion of E-cadherin reduced the side CSC phenotypes include their self-renewal and pro- population (SP) phenotype, a common feature of CSCs liferative properties. To investigate the functional role related to the ABCG2 multidrug efflux transporter of E-cadherin in regulating MM CSCs, we first estab- (Fig.  1G and H; Additional file  2: Figure S5). Profiling lished E-cadherin-depleted cells in human MM-derived of stemness-related genes, i.e., SOX2, SOX9, NANOG, cell lines RPMI 8226 and NCI-H929 using the CRISPR/ and OCT4, pointed out that SOX9 could be a key regu- Cas9 system (Additional file 2: Figure S2) and examined lator of E-cadherin-mediated MM CSCs (Fig.  1I and J; its effects on cell growth and cell cycle. Detailed methods Additional file 2: Figure S6). To first test whether SOX9 can be found in Additional file  1.  Figure  1A − C shows is functionally linked to CSCs, SOX9 was depleted in that both E-cadherin-depleted cells were less prolifera- RPMI 8226 cells using shRNA. Similar to E-cadherin, tive than wild type (WT) control cells, corresponding depletion of SOX9 reduced Akt and MAPK activity, col- to the increased CD138-negative subpopulation and ony-forming capacity, and SP cells and its correspond- decreased prosurvival Bcl-2, but not Mcl-1. Our find- ing ABCG2 when compared to WT cells (Fig.  2A − D; ings were consistent with a previous study reporting the Additional file 2: Figures S7 and S8), indicating the crit- prosurvival effect of CD138 in MM [10]. We also found ical role of SOX9 in MM CSCs. To further validate that that loss of E-cadherin caused either G0/G1 or G2/M SOX9 is downstream of E-cadherin, rescue experiments arrest, depending on the cellular context, by controlling were conducted in which SOX9 plasmid was ectopically its key cell cycle regulators in each phase (Fig.  1D). The overexpressed in E-cadherin-depleted cells. Figure  2E PI3K/Akt and MAPK pathways have been reported to and F shows that the reduced SOX9 and ABCG2 as well regulate the proliferation and survival of MM cells [11]. as the reduced Akt and MAPK signaling in E-cadherin Herein, we showed that E-cadherin activates Akt, p38, depleted cells could be rescued by ectopic SOX9 (see and p44/42 (ERK1/2), but not SAPK/JNK, via protein also Additional file  2: Figures  S9 and S10). This SOX9 phosphorylation (Fig.  1E). Altogether, these results sup- restoration also reversed the inhibitory effects of E-cad- port the positive regulatory role of E-cadherin in MM herin depletion on the colony forming capacity and SP cell growth and survival. cells (Fig. 2G and H; Additional file 2: Figures S11 and S12), thus confirming that E-cadherin mediates MM We hypothesized that E-cadherin may be involved in CSCs via SOX9. We also found that SOX9 is, in turn, CSC self-renewal. To investigate, colony-forming abil- necessary for maintaining E-cadherin level (Additional ity, the potential of a single cell to indefinitely grow and file  2: Figure S13), indicating a positive feedback loop survive [12], was evaluated by clonogenic assay. Fig- that controls MM CSCs. ure 1F shows that depletion of E-cadherin resulted in a reduction in both the number and size of MM colonies when compared to WT cells (Additional file  2: Figure (See figure on next page.) Fig. 1  E-cadherin regulates cell growth and CSC-like phenotypes in human MM-derived cells. E-cadherin was depleted in RPMI 8226 and NCI-H929 cells using the CRISPR/Cas9 system, designated as CDH1-KO RPMI 8226 and CDH1-KD NCI-H929 cells, respectively (Additional file 2: Figure S2). A Cell viability was evaluated by MTT assay to monitor cell proliferation at 24, 48, 72, and 96 h of culture. Data are mean ± SD (n = 3). **p < 0.01, ***p < 0.001, ****p < 0.0001 versus WT control cells; two-tailed Student’s t-test. B Cell surface expression of CD138 was analyzed by flow cytometry. The proportion of CD138-positive ­(CD138+) and CD138-negative ­(CD138−) cells is shown. Data are mean ± SD (n = 3). ***p < 0.001, ****p < 0.0001 versus WT cells; two-tailed Student’s t-test. C Western blot analysis of prosurvival Bcl-2 and Mcl-1 proteins. β-actin was used as a loading control. The significant decrease in Bcl-2, but not Mcl-1, level was detected in CDH1-KO RPMI 8226 and CDH1-KD NCI-H929 cells compared to WT cells (**p < 0.01; two-tailed Student’s t-test). D (upper) Cell cycle analysis based on DNA content was analyzed by flow cytometry using propidium iodide staining. (lower) Quantitative real-time PCR (RT-qPCR) analysis of mRNA expression of cell cycle regulator genes. GAPDH served as the internal control. Data are mean ± SD (n = 3). *p < 0.05, **p < 0.01, ****p < 0.0001 versus WT cells; two-tailed Student’s t-test. E Western blot analysis of Akt and MAPK family proteins. The significant decrease in phosphorylated (p)-Akt, p-p38, and p-p44/42 levels was detected in CDH1-KO RPMI 8226 and CDH1-KD NCI-H929 cells compared to WT cells (*p < 0.05; two-tailed Student’s t-test). F Representative micrographs showing MM colonies under clonogenic assay (see also Additional file 2: Figure S3 for quantitative analysis of colony number and size). Scale bar = 200 μm. G SP subpopulation analysis using flow cytometry based on Hoechst 33342 dye efflux. SP cells (box) were determined by their disappearance in the presence of fumitremorgin C (see also Additional file 2: Figure S5 for quantitative analysis). H Western blot analysis of ALDH1A1 and ABCG2. A significant decrease in ABCG2, but not ALDH1A1, level was detected in CDH1-KO RPMI 8226 and CDH1-KD NCI-H929 cells compared to WT cells (*p < 0.05; two-tailed Student’s t-test). I RT-qPCR analysis of mRNA expression of stemness-regulated genes. Data are mean ± SD (n = 3). ****p < 0.0001 versus WT cells; two-tailed Student’s t-test. J Western blot analysis of SOX9 level in CDH1-KO RPMI 8226 and CDH1-KD NCI-H929 cells (see also Additional file 2: Figure S6 for quantitative analysis)

Samart et al. Experimental Hematology & Oncology (2022) 11:41 Page 3 of 6 A RPMI 8226 NCI-H929 D RPMI 8226 WT-Control #1 CDH1-KO 1000 WT-Control 500 CDH1-KD #1 CDH1-KO 400 800 #2 CDH1-KO 300 24 48 72 Cell survival rate (%) Time (h) WT-Control #2 CDH1-KO 600 Cell survival rate (%) Relative expression*** ****RPMI 8226** S Relative expression 62.3% 50.6% 55.6% G0/G1 G2/M 400 200 23.7% 12.7% 32.5% 15.8% 26.8% 17.3% 200 100 0 0 96 RPMI 8226 **** 0 24 48 72 96 0 WT-Control **** kD 3 #1 CDH1-KO ** Time (h) 26 43 2 #2 CDH1-KO BC 43 **** ** ** kD RPMI 8226 26 43 WT-Control #1 CDH1-KO #2 CDH1-KO 43 1 ******** 0 1.69±0.43 % 15.77±2.34 % 4.57±0.31 % CCND1 *** *** Bcl-2 (Cyclin D1) p65 CDK4 CDKN1A CDKN2A CDKN1B CDKN2B (p21) (p16) (p27) (p15) Mcl-1 -actin NCI-H929 NCI-H929 Bcl-2 WT-Control CDH1-KD NCI-H929 NCI-H929 Mcl-1 WT-Control WT-Control CDH1-KD G0/G1 S 30.0% 38.3% 3 CDH1-KD 1.18±0.38 % 4.36±0.36 % 49 .5% 34.4% 29.7% 2 ** **** G2/M 15.0% CD138-– 1 ** * CD1388++ 0 CCNB1 CDK1 CDKN1A (Cyclin B1) (p21) -actin E F RPMI 8226 NCI-H929 RPMI 8226 NCI-H929 WT-Control #1 CDH1-KO #2 CDH1-KO WT-Control CDH1-KD p-Akt kD 200 m Akt 72 RPMI 8226 NCI-H929 p-p38 MAPK 200 m #1 CDH1-KO p38 MAPK 0.74 % #2 CDH1-KO WT-Control CDH1-KD 72 0.22 % 1.50% 0.17 % G 43 WT-Control 43 3.93 % p-p44/42 MAPK 43 Hoechst Blue-A p44/42 MAPK 43 + Fumitremorgin C + Fumitremorgin C + Fumitremorgin C + Fumitremorgin C + Fumitremorgin C p-SAPK/JNK 55 0.01 % 0.02% 0.00 % 0.09 % 0.08% 43 SAPK/JNK 55 Hoechst Red-A Hoechst Red-A -actin 43 43 J RPMI 8226 H I RPMI 8226 RPMI 8226 NCI-H929 Stemness-related genes 4 #2 CDH1-KO kD SOX2 SOX9 NANOG OCT4 #1 CDH1-KO 55 3 WT-Control kD 72 72 SOX9 43 43 ALDH1A1 **** -actin kD ABCG2 **** 72 -actin 2 43 Fig. 1  (See legend on previous page.) 1 NCI-H929 SOX9 -actin 0.0 0.2 0.4 0.6 0.8 1.0 1.2 Relative expression In summary, we revealed a novel regulatory mecha- to relapsed/refractory MM. Our findings provided a nism of MM CSCs via the E-cadherin/SOX9 axis potential rationale for targeting E-cadherin/SOX9 axis, (Fig.  2I), which could be important in understanding while in  vivo studies are warranted to further validate the long-term cell survival and outgrowth that leads this hypothesis.

Samart et al. Experimental Hematology & Oncology (2022) 11:41 Page 4 of 6 A kD E kD 55 55 SOX9 72 SOX9 72 ABCG2 43 ABCG2 43 -actin kD -actin 55 CDH1-KO kD B 55 F 55 43 55 p-Akt 43 p-Akt 43 Akt 43 Akt 43 43 43 p-p38 MAPK 43 p-p38 MAPK p38 MAPK p38 MAPK 43 shSOX9 43 p-p44/42 MAPK p-p44/42 MAPK p44/42 MAPK p44/42 MAPK SOX9-CDH1-KO -actin -actin C G WT-Control WT-Control 200 m shSOX9 200 m CDH1-KO SOX9-CDH1-KO 0.24 % 2.11% 3.03 % D WT-Control H WT-Control 3.73 % 3.57 % Hoechst Blue-A + Fumitremorgin C + Fumitremorgin C + Fumitremorgin C Hoechst Blue-A 0.57 % 0.46% 0.98 % + Fumitremorgin C + Fumitremorgin C 0.36 % 0.09% Hoechst Red-A Hoechst Red-A I Multiple myeloma cells E-cadherin G2/M G0/G1 Myeloma stem cells Regulating cell cycle Regulating self-renewal capability SOX9 Bcl-2 CD138 Prosurvival proteins Cell proliferation and growth ABCG2 P P Akt ERK1/2 Side population P p38 MAPK SAPK/JNK Stemness markers Activating Akt and MAPK signaling Fig. 2  (See legend on previous page.)

Samart et al. Experimental Hematology & Oncology (2022) 11:41 Page 5 of 6 (See figure on previous page.) Fig. 2  E-cadherin/SOX9 axis regulates CSCs in human MM-derived cells. A–D SOX9 was depleted in RPMI 8226 cells expressing high endogenous SOX9 using lentiviral particles carrying shSOX9 or non-target sequence (WT-control). A Western blot analysis of SOX9 and ABCG2 levels. β-actin was used as a loading control. The significant decrease in SOX9 and ABCG2 levels was detected in shSOX9 cells compared to WT cells (**p < 0.01; two-tailed Student’s t-test). B Western blot analysis of Akt and MAPK family proteins. The significant decrease in p-Akt, p-p38, and p-p44/42 levels was detected in shSOX9 cells compared to WT cells (*p < 0.05; two-tailed Student’s t-test). C Representative micrographs showing MM colonies under clonogenic assay (see also Additional file 2: Figure S7 for quantitative analysis). Scale bar = 200 μm. D SP analysis using flow cytometry based on Hoechst 33342 dye efflux (see also Additional file 2: Figure S8 for quantitative analysis). E–H Rescue experiments were performed in CDH1-KO RPMI 8226 cells by transfection of the cells with SOX9 plasmid. Cells with SOX9 restoration were designated SOX9-CDH1-KO cells. E Western blot analysis of SOX9 and ABCG2 levels. β-actin was used as a loading control. The significant increase in SOX9 and ABCG2 levels was detected in SOX9-CDH1-KO cells compared to CDH1-KO cells (see also Additional file 2: Figure S9 for quantitative analysis). F Western blot analysis of Akt and MAPK family proteins. The significant increase in p-Akt, p-p38, and p-p44/42 levels was detected in SOX9-CDH1-KO cells compared to CDH1-KO cells (see also Additional file 2: Figure S10 for quantitative analysis). G Representative micrographs showing MM colonies under clonogenic assay (see also Additional file 2: Figure S11 for quantitative analysis). Scale bar = 200 μm. H SP analysis using flow cytometry based on Hoechst 33342 dye efflux (see also Additional file 2: Figure S12 for quantitative analysis). I Schematic illustration of how E-cadherin/SOX9 axis governs cell growth and self-renewal of CSCs, in part via Akt and MAPK signaling, in MM cells. It is worth noting that other molecules might be involved in this regulatory axis, which requires further investigation Abbreviations Data availability CSC: Cancer stem cells; MM: Multiple myeloma; p-: Phosphorylated; RT-qPCR: The datasets generated and/or analyzed during the current study are available Quantitative real-time PCR; SP: Side-population; WT: Wild type. from the corresponding author on reasonable request. Additional file informa- tion is available in Additional files 1 and 2. Supplementary Information Declarations The online version contains supplementary material available at https://d​ oi.​ org/1​ 0.1​ 186/s​ 40164-0​ 22-​00294-x. Ethics approval and consent to participate This study was approved by the Siriraj Institutional Review Board (COA No. Additional file 1: Detailed methods. Si 101/2015) and was in accordance with the Helsinki Declaration of 1975. The cell lines used in this study were purchased from American Type Culture Additional file 2: Figure S1. Analysis of CDH1 mRNA expression in Collection (ATCC). clinical samples using publicly available microarray data. Figure S2. Suc- cessful depletion of E-cadherin by the CRISPR/Cas9 system in the human Consent for publication MM– derived cell lines RPMI 8226 and NCI-H929. Figure S3. Depletion of Not applicable. E-cadherin inhibits the clonogenic potential of MM cells. Figure S4. Resto- ration of Ecadherin into E-cadherin-depleted cells rescues the clonogenic Competing interests potential of MM cells. Figure S5. Depletion of E-cadherin decreases the The authors declare no competing interests. proportion of the SP subpopulation in MM cells. Figure S6. Depletion of E-cadherin suppresses SOX9 level in MM cells. Figure S7. Depletion of Author details SOX9 reduces the clonogenic potential of MM cells. Figure S8. Depletion 1 Department of Immunology, Faculty of Medicine Siriraj Hospital, Mahidol of SOX9 decreases the proportion of SP subpopulation in MM cells. Figure University, Bangkok, Thailand. 2 Siriraj Center of Excellence for Stem Cell S9. Re-expression of SOX9 in E-cadherin-depleted MM cells rescues the Research, Faculty of Medicine Siriraj Hospital, Mahidol University, 2 Siriraj Hos- ABCG2 level. Figure S10. Re-expression of SOX9 in E-cadherin-depleted pital, Bangkoknoi, Bangkok 10700, Thailand. 3 WVU Cancer Institute, Depart- MM cells reactivates Akt and MAPK signaling. Figure S11. SOX9 regulates ment of Pharmaceutical Sciences, West Virginia University, Morgantown, WV, E-cadherin-mediated clonogenic growth in MM cells. Figure S12. Re- USA. 4 Division of Hematology, Department of Medicine, Faculty of Medicine expression of SOX9 induces the acquisition of the SP subpopulation in Siriraj Hospital, Mahidol University, Bangkok, Thailand. 5 Bangkok Hematology E-cadherin-depleted MM cells. Figure S13. Depletion of SOX9 suppresses Center, Wattanosoth Hospital, BDMS Center of Excellence for Cancer, Bangkok, E-cadherin level in MM cells. Thailand. Acknowledgements Received: 3 June 2022 Accepted: 6 July 2022 We thank Napachai Rodboon and Sirinart Buasumrit for their assistance. References Author contributions 1. Hernández-Rivas J, Ríos-Tamayo R, Encinas C, Alonso R, Lahuerta JJ. The PS designed research, carried out experiments, analyzed data, and drafted the manuscript. YR participated in the data analysis. SI supervised the project and changing landscape of relapsed and/or refractory multiple myeloma provided resources. SL conceived the study, designed research, participated in (MM): fundamentals and controversies. Biomark Res. 2022;10(1):1. the data analysis, coordinated the project, and drafted and edited the manu- 2. Gao M, Kong Y, Yang G, Gao L, Shi J. Multiple myeloma cancer stem cells. script. All authors read and approved the final manuscript. Oncotarget. 2016;7(23):35466–77. 3. Soncin F, Ward CM. The function of E-cadherin in stem cell pluripotency Funding and self-renewal. Genes. 2011;2(1):229–59. This work was supported by grants from the Royal Golden Jubilee Ph.D. Pro- 4. Karpowicz P, Willaime-Morawek S, Balenci L, DeVeale B, Inoue T, van der gramme (PHD/0085/2558, to PS), Thailand Research Fund (RSA6280103, to SL), Kooy D. E-cadherin regulates neural stem cell self-renewal. J Neurosci. Siriraj Foundation for Stem Cell Research (D003276, to SI), and Specific League 2009;29(12):3885–96. Funds from Mahidol University (to SL).

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