Synthetic Retinoid Kills Drug‐Resistant Cancer Stem Cells via Inducing RARγ‐Translocation‐Mediated Tension Reduction and Chromatin Decondensation

Abstract A recently developed synthetic retinoid abrogates proliferation and induces apoptosis of drug‐resistant malignant‐cancer‐stem‐cell‐like cells. However, the underlying mechanisms of how the synthetic retinoid induces cancer‐stem‐cell‐like cell tumor‐repopulating cell (TRC) apoptosis are elusive. Here, it is shown that although the retinoid and conventional anticancer drugs cisplatin, all‐trans retinoic acid, and tazarotene all inhibit cytoskeletal tension and decondense chromatin prior to inducing TRC apoptosis, half‐maximal inhibitory concentration of the retinoid is 20‐fold lower than those anticancer drugs. The synthetic retinoid induces retinoic acid receptor gamma (RARγ) translocation from the nucleus to the cytoplasm, leading to reduced RARγ binding to Cdc42 promoter and Cdc42 downregulation, which decreases filamentous‐actin (F‐actin) and inhibits cytoskeletal tension. Elevating F‐actin or upregulating histone 3 lysine 9 trimethylation decreases retinoid‐induced DNA damage and apoptosis of TRCs. The combinatorial treatment with a chromatin decondensation molecule and the retinoid inhibits tumor metastasis in mice more effectively than the synthetic retinoid alone. These findings suggest a strategy of lowering cell tension and decondensing chromatin to enhance DNA damage to abrogate metastasis of cancer‐stem‐cell‐like cells with high efficacy.


Introduction
Targeted therapy is one of the frequently used treatments for cancer, [1] but resistance to anticancer drugs by cancer cells hampers their effectiveness in treating many types of malignant tumors. [2] Cancer stem cells (CSCs) or tumor-initiating cells (TICs) are a self-renewing, highly tumorigenic subpopulation of tumor cells [3] that are believed to be responsible for tumor initiation, progression, invasion, and metastasis [4] and are speculated to be key players in cancer relapse after chemotherapy. [4,5] Thus developing novel targeted therapeutic drugs or enhancing inhibitory efficiency of conventional drugs to abrogate CSCs or TICs is essential in cancer research and clinical applications.
We have published a mechanical method for selecting and growing tumorigenic cells from various cancer cell lines and primary cancer cells by culturing single cancer cells in soft fibrin gels. [6] The selected cancer cells display high self-renewal ability and are resistant to chemotherapeutic drugs such as cisplatin and doxorubicin. [6] Remarkably, when injected selected cancer cells into tail veins, as few as 10 of such cells can generate distant metastatic colonization in immune-competent mice [6] and even 5 of such cells are able to generate primary tumors in mice. [7] We thus functionally define these soft-fibrin-gel-selected cancer cells as tumorrepopulating cells (TRCs), which are distinct from CSCs or TICs that are selected by stem cell markers. These TRCs express high levels of self-renewing gene Sox2 and exhibit low levels of histone 3 lysine residue 9 (H3K9) methylation. [8] All-trans-retinoic acid (ATRA) and its retinoic analogs such as tazarotene regulate differentiation, proliferation, and apoptosis of various cancer cells, emerging as one of targeted anticancer strategies. [9] Cisplatin is a platinum-based chemotherapy compound, which has been one of the most active clinical drug classes for the treatment of a variety of solid tumors, [10] but TRCs exhibit resistance to cisplatin that fails to inhibit growth of TRCs even at high concentrations. [6,11] To overcome the drug resistance by TRCs, we have synthesized, screened, and characterized a novel retinoid, named WYC-209 (molecular weight (MW) = 368.1 Da), which effectively inhibits proliferation of TRCs and induces apoptosis of multiple cancer cell lines in culture and inhibits lung metastasis of murine melanoma TRCs in immunecompetent mice with no apparent toxicity. [11] However, the underlying mechanisms of how WYC-209 induces apoptosis remain elusive. Furthermore, physical features of tumors are known to regulate tumor progression [12] but the relationship between mechanical features of TRCs and drug resistance is not understood.
In this study, we show that conventional drugs cisplatin, ATRA, and tazarotene and the novel retinoid WYC-209 all induce TRC apoptosis via decreasing cytoskeleton to decondense chromatin. However, the reason why WYC-209 is more effective than other anticancer drugs is that it can induce retinoic acid receptor gamma (RAR ) translocation from the nucleus to the cytoplasm at a much lower concentration, leading to Cdc42 downregulation and thus reduction of filamentous-actin (F-actin) and tractions. This is accompanied by lowering of H3K9 methylation and elevation of H3K9 acetylation to decondense chromatin of TRCs to increase DNA damage by the synthetic retinoid. The combinatorial treatment of WYC-209 with chaetocin, an inducer of H3K9 demethylation and chromatin decondensation enhances inhibition of lung metastasis of melanoma TRCs in immunecompetent mice.

Retinoid WYC-209 Inhibits F-Actin and Tension Force before Inducing Apoptosis
Cellular traction is a fundamental mechanical function of live cells. Arg-Gly-Asp-coated elastic round microgels (ERMGs) were used to quantify tractions in a 3D tumor cell colony. [13] As the tumor cells attached to the ERMG and generated tractions onto the microgel, they exerted the compressive stresses onto the microgel (Figure 2A,B). Compressive tractions of TRCs substantially decreased 1 h and completely disappeared 6 h after 10 μm WYC-209 treatment ( Figure 2B,C). Furthermore, F-actin in TRCs decreased by ≈20% 1 h and by ≈80% 6 h after WYC-209 treatment ( Figure 2D,E). By contrast, TRCs became apoptotic at later times: no apoptosis was observed 3 h ( Figure 2F,G), 20% of cells became apoptotic 6 h, and over 80% of cells died 12 h after 10 μm WYC-209 treatment ( Figure 2H,I), consistent with time course of expression of apoptosis-associated proteins (Figure 1J,K). The dose-dependent effect of WYC-209 on tractions was observed: 1.0 μm WYC-209 did not decrease TRC tractions, but 5.0 and 10 μm WYC-209 completely abrogated TRC tractions (Figure S1A-C, Supporting Information). F-actin staining data were consistent with the traction results ( Figure S1D,E, Supporting Information). However, 5.0 μm WYC-209 for 24 h only induced apoptosis in ≈20% of TRCs ( Figure S1F,G, Supporting Information) and slight elevation of C-Caspase 3 expression, much lower than 10 μm WYC-209 did ( Figure S1H,I, Supporting Information). In sharp contrast, the conventional anticancer drugs ATRA, tazarotene, and cisplatin at 10 μm for 24 h did not induce F-actin depolymerization ( Figure S2A,B, Supporting Information) nor did they induce apoptosis ( Figure 1B,C). Only after 12 h treatment of 100 μm of these conventional drugs, F-actin began to decrease (by ≈30%) (Figure S2C,D, Supporting Information). Taken together, WYC-209 downregulates TRC tractions before inducing apoptosis and is more efficacious than the conventional anticancer drugs.

Modulating F-Actin and Cdc42 Regulates Retinoid-Induced TRC Apoptosis
It is known that TRCs are undifferentiated cancer-stem-cell-like cells. [6,8] TRCs were softer and generated lower tractions than differentiated melanoma cells cultured on rigid dish ( Figure  S3A,B, Supporting Information), consistent with the published results. [6,8] Compared with control B16-F1 cells cultured on the rigid dish, TRCs exhibited a lower level of F-actin intensity (Figure S3C,D, Supporting Information) and a lower expression of and quantification (E, G, I, K) of relative H2AX, C-PARP, and C-Caspase3 expression of B16 TRCs treated with 100 μm ATRA, 100 μm tazarotene, 100 μm cisplatin, or 10 μm WYC-209 for various durations. Mean ± s.e.m.; three independent experiments. One-way ANOVA testing followed by a Tukey post-hoc test when appropriate was used for statistics. *p < 0.05; **p < 0.01; ***p < 0.001; ns = not significantly different. phosphorylated myosin light-chain 2 (p-MLC2) ( Figure S3E,F, Supporting Information). WYC-209 treatment for 6 h did not induce apoptosis of the differentiated melanoma cells (B16 Ctrs) and only ≈50% of these cells became apoptotic after 12 h treatment ( Figure S3I,J, Supporting Information), thus less sensitive to the retinoid in comparison to TRCs ( Figure 2H,I). Since F-actin and tractions might be important for the differential biological responses between the differentiated melanoma cells and TRCs, we modulated F-actin in TRCs. Increasing F-actin with Jasplakinolide (Jasp) and treating with WYC-209 produced less H2AX and C-PARP in TRCs than with WYC-209 alone (Figure 3A-C). Decreasing traction with myosin light chain kinase inhibitor ML7 and then treating with WYC-209 induced more H2AX and C-PARP in TRCs than with WYC-209 alone ( Figure 3D-F). Similar results were observed with the conventional anticancer drugs using a combination of F-actin or myosin-II modulators and the retinoid ( Figure S4A-D, Supporting Information). Because 5.0 μm WYC-209 resulted in differential effects between TRCs and differentiated melanoma cells, we used this dose in the fol-lowing experiments. Treating with 5.0 μm WYC-209 alone decreased F-actin in TRCs by 80% and treating with RhoA activator lysophosphatidic acid (LPA) increased F-actin ( Figure 3G,H). Pretreating with LPA and then with WYC-209 dampened the effect of WYC-209 in lowering the F-actin ( Figure 3G,H). Importantly, pretreating with LPA then with WYC-209 abrogated the inhibition effect of WYC-209 in TRCs ( Figure 3I,J). We then used a strategy to inhibit F-actin in the differentiated stiff B16 cells that are known to generate higher tractions. [6] Inhibiting F-actin with latrunculin A (Lat A) and then with WYC-209 decreased Factin dramatically ( Figure S5A,B, Supporting Information) and increased apoptosis in these cells, more than with WYC-209 alone ( Figure S5C,D, Supporting Information). Together, these results suggest that modulating F-actin and/or altering myosin-II-mediated traction regulate apoptotic efficiency by the retinoid.
It has been shown that Cdc42 mediates cell stiffening and F-actin accumulation of TRCs. [8] TRCs expressed lower levels of Cdc42 protein than the differentiated melanoma cells (Figure S3G,H, Supporting Information). Depleting Cdc42 together with WYC-209 treatment decreased F-actin and increased apoptosis in the differentiated melanoma cells when compared with the synthetic retinoid alone ( Figure S5E-K, Supporting Information). By contrast, overexpressing Cdc42 together with WYC-209 treatment rescued the cells from the WYC-209 inhibitory effect on F-actin and dramatically decreased apoptosis (both terminal deoxynucleotidyl transferase 2'-deoxyuridine 5'-triphosphate (dUTP) nick end labeling (TUNEL) and C-Caspase 3 assays) of these cells ( Figure 3K-Q). Together these results suggest that WYC-209 induces apoptosis via the Cdc42-dependent F-actin pathway.

WYC-209 Induces RAR Translocation to Decrease Its Binding to Cdc42 Promoters
Next, we examined how WYC-209 decreases Cdc42 levels. It has been reported that WYC-209 binds to RAR with high affinity. [11] The messenger RNA (mRNA) expression levels of RAR were higher than those of RAR and RAR in B16-F1 TRC ( Figure  S6A, Supporting Information). Silencing RAR led to the most dramatic rescuing effect on WYC-209-induced apoptosis of the three isoforms ( Figure S6B-D, Supporting Information). These results suggest that WYC-209 selectively targeted on TRC primar- ily via RAR , consistent with the previous reports that RAR acts as an oncogene in regulating cell fate in melanoma cells. [14,15] Pretreating TRCs with the specific RAR antagonist MM11253 delayed WYC-209-mediated Cdc42 downregulation (Figure 4A-D). Although WYC-209 did not change expressions of RARs ( , , or ) in TRCs within 24 h ( Figure S7, Supporting Information), it induced translocation of RAR from the nucleus to the cytoplasm ( Figure 4E-G). MM11253 delayed RAR translocation ( Figure 4H-J). Molecular dynamics simulation of 3D binding of RAR at the promoter site of Cdc42 revealed the specific tight binding between the two ( Figure 4K). Chromatin immunoprecipitation (ChIP) data revealed that WYC-209 indeed decreased the amount of RAR recruited to Cdc42 promoter sites in a timedependent manner ( Figure 4L). Reporter assays show that RAR increases Cdc42 expression via binding to Cdc42 promoter, which is inhibited by WYC-209 ( Figure S8, Supporting Information), consistent with ChIP data. Meanwhile, the RAR translocation rate of control cells induced by 10 μm WYC-209 was slower than that of TRCs ( Figure S9, Supporting Information). By contrast, 10 μm conventional drugs (ATRA, tazarotene, and cisplatin) did not induce RAR translocation from the nucleus to the cytoplasm ( Figure S10A,B, Supporting Information), consistent with the finding that 10 μm conventional drugs did not inhibit Factin ( Figure S2A,B, Supporting Information). Together, these results suggest that WYC-209 induces apoptosis more efficiently than the conventional anticancer drugs because it induces RAR translocation from the nucleus to the cytoplasm more efficiently, which in turn decreases RAR recruitment to Cdc42 promoters to downregulate Cdc42 expression.

Cdc42 Promotes Chromatin Condensation via Regulating Histone 3 Lysine 9 Trimethylation (H3K9me3) and H3K9 Acetylation (H3K9ac)
Next, we asked whether Cdc42 regulates WYC-209-induced apoptosis by regulating levels of chromatin condensation. Since depleting Cdc42 substantially decreased H3K9me3 and H3K9 methyltransferase (SUV39h1) expression but increased H3K9ac, it decondensed chromatin [16,17] (Figure 5A,B). Conversely, overexpressing Cdc42 substantially increased H3K9me3 and SUV39h1 but decreased H3K9ac ( Figure 5A,C). However, when SUV39h1 was knocked down, H3K9me3 was decreased and H3K9ac was increased; there was no effect in Cdc42 expression ( Figure  S11A,B, Supporting Information). These data suggest that lowering F-actin or cellular tension (i.e., traction) induces chromatin decondensation. Electron microscopy shows that WYC-209 indeed induced chromatin decondensation ( Figure S12, Supporting Information). Together with the results that SUV39h1 de-creased only after 3 h of WYC-209 treatment ( Figure S11C,D, Supporting Information), slower than Cdc42 decreased by WYC-209, these data suggest that WYC-209 induced chromatin decondensation and Cdc42 might be upstream of H3K9me3 and H3K9ac. Indeed overexpressing Cdc42 increased H3K9me3 expression and slowed down the reduction of H3K9me3 expression by WYC-209, whereas knocking down Cdc42 decreased H3K9me3 and accelerated H3K9me3 reduction by WYC-209 ( Figure S13A,B, Supporting Information). By contrast, overexpressing Cdc42 or knocking down Cdc42 led to the opposite effects on H3K9ac ( Figure S13C,D, Supporting Information). Immunofluorescence stain and western blots show that TRCs exhibited lower levels of H3K9me3 and higher levels of H3K9ac in comparison with control B16 cells ( Figure S14, Supporting Information). Treating with LPA to increase F-actin and treating with JIB-04 (a H3K9 demethylase inhibitor) [18] substantially increased H3K9me3 and decreased H3K9ac, but treating with deacetylase inhibitor trichostatin A (TSA) increased H3K9ac and had no effects on H3K9me3 ( Figure 5D-G and Figure  S15A,B (Supporting Information)). Furthermore, treating with Lat A to decrease F-actin or treating with chaetocin (an inhibitor of H3K9me3 methyltransferase SUV39h1) [19] dramatically decreased H3K9me3 and increased H3K9ac in control B16 cells, but treating TSA only increased H3K9ac but had no effects on H3K9me3 ( Figure S16A-D, Supporting Information), suggesting that chaetocin decondenses chromatins of TRCs via lowering H3K9me3 to elevate H3K9ac. Western blots and electron microscopy images show that treating chaetocin alone could only alter levels of H3K9me3 and H3K9ac to decondense chromatin without inducing apoptosis-related proteins expression (Figures S17 and S18, Supporting Information), and MTT assays show that chromatin structure regulators or small interfering RNA (siRNA) treatments do not effect cell viability (  . Mean ± s.e.m.; three independent experiments. K) The crystal structure of RAR was obtained from the protein data bank (PDB) (6FX0) database. The simulated interaction between Cdc42 promoter and RAR was performed by Discovery Studio 2018, exhibiting tight binding between the two. L) ChIP analysis of RAR level binding to Cdc42 promoters of B16-TRCs treated with 10 μm WYC-209 for various time points. Mean ± s.e.m.; three independent experiments. One-way ANOVA testing plus Tukey post-hoc correction when appropriate was used for statistics. *p < 0.05; **p < 0.01; ***p < 0.001; ns = not significantly different.
S20C,D, Supporting Information), after decreasing F-actin (Figure S2C,D, Supporting Information). Moreover, pretreating with chaetocin to downregulate H3K9me3 and then treating with conventional drugs increased expression of cell-apoptosis-associated proteins, demonstrated that decondensing chromatin increased the inducing apoptotic efficiency of conventional drugs, which is consistent with WYC-209 treatment groups combined with chaetocin ( Figure S20E-J, Supporting Information). Taken together, these data suggest that anticancer drugs induce apoptosis via regulating chromatin condensation by regulating H3K9me3 and H3K9ac via Cdc42 and WYC-209 inhibits H3K9me3 better than conventional drugs.
Next, we investigated the underlying mechanism of WYC-209-induced chromatin decondensation as well as TRC apoptosis. As chromatin decondensation is a prerequisite for transcriptional activation, [26] we deduced that apoptosis-related genes such as P21, P27, P53 and DNA damage gene H2AX were altered

Suppressing H3K9me3 Potentiates WYC-209 Inhibition of Tumor Metastasis
To determine whether chromatin decondensation could potentiate WYC-209 inhibition of tumor metastasis in vivo, we injected intravenously via tail veins 100 000 TRCs into immune-competent C57BL/6 mice to form lung metastases. Since it has been reported that as few as 10 TRCs are sufficient to generate metastasis in the lung, [6] the choice of 100 000 TRCs (10 000-fold higher than the cell number needed to generate metastasis) is to overload the mice with malignant tumor cells and to determine if modulation of chromatin condensation levels is able to alter the outcome of the retinoid treatment. Five days after loading the mice with TRCs, WYC-209 was intravenously injected into mice once every two days for 25 days. 0.1% dimethyl sulfoxide (DMSO) was injected as a negative control (Figure 7A). Compared with other groups, none of mice died before 30 days and only one out of 8 mice formed lung metastases when the mice were treated with 0.11 mg kg −1 WYC-209 together with 0.013 mg kg −1 chaetocin, similar to the group treated with 0.22 mg kg −1 WYC-209 ( Figure 7B,C and Figure S23 (Supporting Information)). By contrast, all mice died before or on day 30 from the DMSO group, the chaetocin (H3K9me3 inhibitor that decondenses the chromatin) alone group, or the JIB-04 (H3K9me3 demethylase inhibitor) alone group ( Figure 7C). Hematoxylin and eosin (H&E) staining of liver, stomach, spleen, bone, and brain shows that no metastases were found in any other organs in various groups ( Figure S24, Supporting Information). These results demonstrate that decondensing chromatin with chaetocin substantially potentiates WYC-209 in inhibiting tumor metastasis and increasing the survival rate of the tumor-bearing mouse.

Discussion
Targeted therapy works by aiming at specific genes or proteins that steer cancer growth and survival but few molecules that target cancer metastasis are available. As a synthetic retinoid, WYC-209 exhibits excellent anticancer activities toward TRCs in various cancer cell lines in vitro and inhibits melanoma TRC metastasis in wild-type mice with little toxicity. [11] Based on the experimental evidence from the current study, we propose a working model of how WYC-209 induces cancer cell apoptosis. WYC-209 induces RAR translocation from the nucleus to the cytoplasm, resulting in a decrease of RAR binding to the Cdc42 promoter and hence a reduction in Cdc42 expression. Subsequently, low Cdc42 levels lead to F-actin depolymerization and low contractile forces, which in turn results in chromatin decondensation via decreasing H3K9me3 and increasing H3K9ac, facilitating expression of apoptosis-related genes and DNA damage gene H2AX by the retinoid to induce tumor cell apoptosis. Compared with retinoic acid, the endogenous active metabolic compound that regulates embryonic development, cell differentiation, and apoptosis mainly via RAR transcriptional regulations of downstream genes, WYC-209 is a structurally analog of the third generation retinoid tazarotene. [11] It demonstrates selective and efficient inhibition activities toward cancer-stem-cell-like cells TRCs and is more potent than both ATRA and tazarotene. [11] Conventional anticancer drugs as ATRA, tazarotene, and cisplatin also induce TRC apoptosis by reducing cell traction and promoting chromatin decondensation. However, conventional drugs are much less potent than WYC-209 since 10 μm conventional drugs could not induce RAR translocation as 10 μm WYC-209 does, which is likely the main reason why WYC-209 is more effective than conventional anticancer drugs on inducing TRC apoptosis.
www.advancedsciencenews.com www.advancedscience.com S63845 is a small molecule that inhibits antiapoptosis protein myeloid cell leukemia 1 (MCL1) and potently kills MCL1-dependent cancer cells [27] and synergistically (plus Navitoclax) kills difficult-to-treat melanoma cells. [28] S63845/S64315 (MIK665) are currently under clinical trials (clinical trials.gov; NCT03672695; NCT01989585). Importantly, WYC-209 was much more potent than S63845 in inhibiting melanoma (B16-F1) TRCs ( Figure S25A,B, Supporting Information). IC 50 of WYC-209 was ≈20-fold lower than that of S63845 ( Figure S25C, Supporting Information). Washing out S63845 one day after being treated, colony sizes of TRCs resumed increasing; by contrast, WYC-209-treated tumor colony sizes failed to increase after washout ( Figure S25D, Supporting Information). The much higher potency of WYC-209 than S63845 may be partly attributed to different induction mechanisms of apoptosis. In our study, WYC-209 does not change the total expression levels of RARs (RAR , RAR , and RAR ) in the cells within 24 h, different from the finding of elevation of RAR expressions after retinoic acid treatment. [29][30][31][32] However, how WYC-209 binding to RAR results in RAR translocation is not clear at this time and deserves future study. It is possible that RAR and RAR also play roles in WYC-209-induced tumor cell apoptosis and elucidation of their specific roles will require additional experiments.
TRCs express less Cdc42 than differentiated tumor cells and Cdc42 regulates TRC stiffness, [8] spreading, [33] and softnessdependent extravasation in vivo. [34] In addition, TRCs express low focal adhesion kinase (FAK) and downregulate H3K9 methylation through reduction of Cdc42 and RhoA [35] that promotes TRC growth via Sox2-dependent self-renewal. [8] Currently, it is unclear how the downregulated Cdc42 and lower tractions by WYC-209 induces chromatin decondensation. One hypothesis is that the low tractions as a result of low Cdc42 and low F-actin have direct effects on the chromatin structure to decondense the chromatin. The level of differentiation status of the cancer cells might be a key in controlling endogenous stresses. The results that stem-cell-like cells TRCs exhibit lower tractions and lower levels of H3K9me3 and higher levels of H3K9ac than the differentiated melanoma cells ( Figure S14, Supporting Information) suggest that these TRCs that have low contractile stresses have decondensed chromatins, consistent with this hypothesis. A report shows that inhibition of myosin-II-mediated contractile forces can mitigate large-nuclear-deformation-induced DNA damage, [24] suggesting that high contractile stresses may decondense the chromatin structures. The difference in the effect of contractile forces on chromatin condensation between the published report and the current study is not clear at this time but may be partly because the cells are undifferentiated stem-celllike TRCs that are cultured in 3D soft fibrin gels. Alternatively, Cdc42-mediated F-actin or traction levels indirectly regulate levels of H3K9ac and H3K9me3. Nevertheless, it will be interesting to elucidate the underlying mechanism in the future.
Gene expression depends on chromatin decondensation to open enough space to allow transcription machinery such as RNA polymerase II binding to gene promoter to initiate gene transcription to complete gene expression followed by complete protein synthesis. [36,37] It is logical to enhance apoptosis-inducing efficiency of WYC-209 via decreasing cell traction and chromatin condensation. As such, we pretreated or combined F-actin inhibitor, traction modulators, or chromatin condensation regula-tors with WYC-209. These reagents alone did not have effects on cell viability ( Figure S19, Supporting Information). Chaetocin alone induced chromatin decondensation but not apoptosisrelated gene expression (Figures S17 and S18, Supporting Information), consistent with the mice experiment data that treating with chaetocin alone does not affect mice survival rate. Hence reducing cellular tension (traction) or decreasing chromatin condensation, combined with WYC-209, is a safe way to enhance anticancer efficiency of WYC-209.
It is interesting that 5 μm WYC-209 abolishes cellular traction to a similar level as 10 μm WYC-209 but TRC apoptosis induced by 5 μm WYC-209 is ≈4 times lower than 10 μm WYC-209 ( Figure  S1, Supporting Information). Additional mechanisms might be at play here. Expression of DNA repair factor KU80 of TRCs was higher after 5 μm of treatment than after 10 μm treatment (Figure S26A-D, Supporting Information). [38] In addition, H3K9me3 was higher and H3K9ac was lower in 5 μm WYC-209-treated cells than in 10 μm WYC-209-treated cells, suggesting that the chromatin was less decondensed by the 5 μm treatment ( Figure  S26E,F, Supporting Information). After 24 h treatment by 5 μm WYC-209, the apoptosis was ≈21%, consistent with Figure S1F,G (Supporting Information), and the apoptosis increased to around 60% after 48 h treatment by 5 μm WYC-209 ( Figure S26G,H, Supporting Information). Together, these data suggest that the additional mechanisms of differences in DNA repair factor KU80 and chromatin decondensation exist for the differences in apoptosis induced by 5 and 10 μm WYC-209.
In cancer progression, there is increasing evidence that gene expression is governed by epigenetic changes of histones. [39,40] Histone deacetylases (HDACs) are enzymes involved in the remodeling of the chromatin, which are aberrant in cancer. [41] Several classes of HDAC inhibitors (HDACi) have been found to have potent anticancer activities. [42] In our study, WYC-209 induces melanoma TRC apoptosis via decondensing chromatin and to upregulate apoptotic-related genes and DNA damage gene, similar to the action of HDACi. However, HDACi monotherapy has been largely ineffective in solid tumors. [43] By contrast, WYC-209 shows a strong inhibition of solid tumor metastasis in vivo at very low concentrations. [11,44] Over the last decade or so, a hypothesis that the extracellular matrix stiffens as a result of excessive collagen-1 is the underlying mechanism of cancer progression has been proposed. [45][46][47] A tacit implication of the model is that softening the extracellular matrix by inhibiting collagen-1 may be a strategy of treating cancer patients. However, a recent study utilizing scaling analysis reveals that there is no correlation between collagen-1 and survival time of patients for more than two dozens of solid tumors; [48] importantly, for the only type of cancer (liver cancer) that is associated with collagen-1, the finding is the opposite: more collagen-1 and stiffer extracellular matrix are associated with longer survival time of patients. In fact, decreasing matrix content and lowering tissue stiffness in pancreatic ductal adenocarcinoma mouse models lead to accelerated tumor growth and diminished overall survival. [49] Together with the fact that corticosteroids that are often used to alleviate cancer patients of pain and inflammation can inhibit collagen-1 synthesis but cannot inhibit cancer growth or progression and are potentially a risk factor for failure of solid tumor treatment, [50] it supports a postulate that excessive collagen-1 and the stiffened matrix are a protective response of the body to fight against tumor size expansion. This protective mechanism fails in some cases as tumor cells can break down the stiffened matrix by secreting matrix-degrading enzymes and invade nearby tissues to initiate tumor progression. The finding that a stiffened extracellular matrix in fact induces dormancy of malignant TRCs in mice [7] is consistent with this interpretation. Differentiated tumor cells are stiff, less malignant, and less metastatic than the soft TRCs and stiffening and differentiating TRCs by retinoic acid can inhibit TRC growth. [6,8] By contrast, in this study, we demonstrate that while softening the TRCs and abrogating tractions induce chromatin decondensation of TRCs, they cannot kill the TRCs but can potentiate the efficacy of the synthetic retinoid in inducing TRC apoptosis to inhibit cancer progression.
TRCs are cancer-stem-cell-like cells which are resistant to conventional anticancer drugs such as cisplatin, ATRA, and tazarotene. It is remarkable that WYC-209 is not only much more potent than these anticancer drugs, but also more potent on TRCs than differentiated tumor cells ( Figure S27, Supporting Information). TRCs are soft with decondensed chromatin and WYC-209 induces cell apoptosis accompanied with cellular tension reduction and chromatin decondensation, suggesting that decreasing cellular tension or inducing chromatin decondensation could be an effective strategy for treating TRCs or cancer-stem-cell-like cells.
In summary, we show that the retinoid WYC-209 and other conventional anticancer drugs as ATRA, tazarotene, and cisplatin induce melanoma TRC apoptosis via eliminating tractions to decondense chromatin and upregulate apoptotic-related genes and DNA damage gene. Combining histone demethylation molecule chaetocin with WYC-209 enhances inhibitory capacity of the synthetic retinoid in melanoma metastasis to the lung. Our findings unveil a new mechanism which lowers tension and decondenses chromatin to effectively enhance TRC apoptosis and DNA damage by anticancer drugs. We propose a therapeutic strategy of enhancing cancer inhibition via abolishing cell tension and inducing chromatin decondensation. In the future, it will be interesting to determine if this combined approach could inhibit growth and metastasis of various types of cancer cells in animal models and in patients.

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Fisher's exact test. All other experimental data were analyzed using a twotailed Student's t-test and one-way analysis of variance (ANOVA) test, when multiple comparisons were carried out within a set of experiments, Tukey post-hoc correction was also performed. Graphpad Prism was used to generate IC 50 curves treated with WYC-209 and other compounds in vitro. All data were expressed as mean ± standard error of the mean (s.e.m.) and significance was defined as p ≤ 0.05. Statistical analysis was carried out using Graphpad Prism software.

Supporting Information
Supporting Information is available from the Wiley Online Library or from the author.