Catestatin suppresses melanoma progression and drug resistance through multitargeted modulation of signaling pathways

Catestatin suppresses melanoma progression and drug resistance through multitargeted modulation of signaling pathways | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results Catestatin suppresses melanoma progression and drug resistance through multitargeted modulation of signaling pathways Satadeepa Kal , Suborno Jati , Kechun Tang , Nicholas J.G. Webster , Angelo Corti , View ORCID Profile Sushil K. Mahata doi: https://doi.org/10.1101/2025.10.22.684062 Satadeepa Kal 1 Veterans Medical Research Foundation , San Diego, CA, USA 3 Department of Medicine, University of California , San Diego, CA, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site For correspondence: smahata{at}ucsd.edu skal{at}health.ucsd.edu Suborno Jati 2 Department of Neurosciences, University of California , San Diego, CA, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Kechun Tang 1 Veterans Medical Research Foundation , San Diego, CA, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Nicholas J.G. Webster 3 Department of Medicine, University of California , San Diego, CA, USA 4 VA San Diego Healthcare System , San Diego, CA, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Angelo Corti 5 IRCCS San Raffaele Scientific Institute, San Raffaele Vita-Salute University , Milan, Italy Find this author on Google Scholar Find this author on PubMed Search for this author on this site Sushil K. Mahata 3 Department of Medicine, University of California , San Diego, CA, USA 4 VA San Diego Healthcare System , San Diego, CA, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Sushil K. Mahata For correspondence: smahata{at}ucsd.edu skal{at}health.ucsd.edu Abstract Full Text Info/History Metrics Supplementary material Preview PDF ABSTRACT Background Despite advances in targeted and immune-based therapies, melanoma remains one of the most aggressive and treatment-resistant cancers. Resistance to small-molecule inhibitors and immune checkpoint blockade highlights the need for new mechanistically distinct interventions. Catestatin (CST), a Chromogranin A (CgA)– derived peptide with immunomodulatory and reparative properties, has been implicated in tissue protection, but its role in melanoma remains unknown. Methods CST expression was analyzed across melanoma stages and correlated with disease progression. Functional effects of CST were assessed in patient-derived and established melanoma cell lines, as well as in B16-F10 melanoma–bearing mice. RNA sequencing and pathway analyses were performed to delineate CST-regulated molecular networks. Vemurafenib-resistant A375 cells were used to examine CST’s effects on drug resistance mechanisms. Results CST expression declined with advancing tumor stage. CST treatment inhibited proliferation, migration, and invasion, while inducing apoptosis in melanoma cells but not in normal fibroblasts. In vivo , systemic CST administration significantly reduced tumor volume and mass. Transcriptomic profiling revealed coordinated downregulation of hypoxia-inducible, epithelial–mesenchymal transition (EMT), and collagen-remodeling pathways, alongside suppression of oxidative stress–adaptive signaling. In Vemurafenib-resistant A375 cells, CST restored apoptotic sensitivity and repressed multiple MAPK and PI3K–AKT–linked resistance genes. Conclusions CST acts as a mechanistically distinct peptide modulator that reprograms oncogenic signaling through inhibition of hypoxia, EMT, and survival pathways. These findings identify CST as a promising therapeutic prototype for mitigating melanoma progression and overcoming resistance to targeted therapy. INTRODUCTION Melanoma is one of the most rapidly increasing cancers worldwide, with incidence rates rising faster than any other solid tumor over recent decades 1 , 2 . Although it accounts for a small fraction of all skin cancer cases, melanoma is responsible for more than 80% of skin cancer–related deaths owing to its aggressive metastatic potential and resistance to therapy 2 . The advent of targeted therapies—particularly BRAF and MEK inhibitors—and immune checkpoint blockade has substantially improved outcomes for patients with advanced disease 3 – 5 . Small-molecule inhibitors such as Dabrafenib and Vemurafenib, which selectively target BRAF^V600E mutations, are now standard of care for a majority of melanoma patients 5 , 6 , whereas those harboring KIT mutations benefit from Imatinib 7 . Moreover, the introduction of immune checkpoint inhibitors has revolutionized melanoma management, extending median survival from approximately six months to nearly six years 4 , 8 . Despite these major advances, a significant proportion of patients either fail to respond or acquire resistance during treatment, resulting in relapse and mortality 9 , 10 . These limitations highlight the urgent need for novel therapeutic approaches that engage mechanisms beyond those targeted by current modalities. Chromogranin A (CgA), a prohormone abundantly expressed in neuroendocrine tissues 11 , 12 , undergoes proteolytic processing to generate multiple biologically active peptides. Among them, Catestatin (CST; hCgA 352-372 ) has emerged as a multifunctional regulator of cardiovascular, metabolic, and immune homeostasis 13 – 17 . CST exerts broad protective effects, including anti-inflammatory 18 , 19 , anti-hypertensive 20 – 24 , and anti-diabetic 18 , 25 actions. Mechanistically, it inhibits catecholamine release 26 – 28 , regulates endothelial proliferation and migration 19 , 29 , 30 , and modulates macrophage and lymphocyte phenotypes 18 , collectively supporting its role as a systemic homeostatic peptide with therapeutic potential. CST is also expressed in human skin, where its levels increase following injury 31 , and exerts functional activity in keratinocytes 32 . CST-like immunoreactivity has been reported in rodent skin and sensory ganglia 33 , suggesting a conserved cutaneous role. However, its significance in melanoma pathobiology has not been explored. In the present study, we identify CST as a previously unrecognized regulator of melanoma progression in both human and mouse models. We demonstrate that CST expression decreases with advancing melanoma stage and that exogenous CST markedly suppresses melanoma cell viability, proliferation, and migration across patient-derived and established cell lines. In syngeneic B16-F10 tumor models, CST administration significantly reduces tumor growth and enhances apoptosis, confirming its antitumor efficacy in vivo . Transcriptomic analyses reveal that CST downregulates key pathways associated with extracellular-matrix organization, hypoxia response, and collagen metabolism—hallmarks of melanoma invasion and metastasis. Notably, CST suppresses several resistance-associated genes, including FGFR3, PDGFRB, ID1/2/3, SREBF1, and MITF , in both Vemurafenib-sensitive and Vemurafenib-resistant A375 cells, suggesting a role in overcoming therapeutic resistance. Collectively, our findings establish CST as a novel melanoma-regulatory peptide with multifaceted antitumor activity. By integrating clinical observations, molecular analyses, and in vivo validation, this study positions CST as a promising therapeutic candidate capable of complementing existing targeted and immunotherapies. Given its pleiotropic regulatory properties and favorable safety profile, CST and its optimized analogs may inaugurate a new class of peptide-based therapeutics that modulate immune–metabolic networks to inhibit melanoma progression and resistance. RESULTS 1. Catestatin levels decline with advancing melanoma and exhibit anti-proliferative effects on patient-derived melanoma cells Although CST has been detected in human skin 31 and keratinocytes 32 as well as in rodent skin and ganglia 33 , its presence in melanoma patient samples had not been established. To address this, we analyzed CST protein expression across progressive stages of melanoma using tissue microarray slides ( TissueArray.com ). Immunohistochemical (IHC) analysis revealed a marked decline in CST expression with advancing melanoma stages, whereas normal skin and stage I melanoma tissues retained high CST levels ( Fig. 1A ). Quantitative analysis confirmed a significant reduction in CST staining intensity as melanoma progressed ( Fig. 1B ). Download figure Open in new tab Figure 1. Decline in endogenous CST levels in human melanoma samples and melanoma cell proliferation inhibition upon extraneous CST treatment (A) Representative immunohistochemistry images showing levels of Catestatin (CST) in different melanoma stages (normal, stage I, stage II, stage III, and metastasis) in human samples from TissueArray.com. (B) Quantification of the immunoreactive CST as area fraction as determined by Fiji3 software. ( C) Schematic for experimental setup on melanoma patient derived cells (K06184, 156681, and 128128). ( D&E) Cell viability assay performed on patient derived cell 156681 and K06184 for 120 hours in response to different concentrations of CST (1 µM, 2 µM, 5 µM, and 10 µM as compared to vehicle control (n=4). ( F&H) Live and dead cell imaging of mammalian cells microscopy showing calcein AM (green) stained live cells and EthD-III (red) stained dead cells upon CST treatment versus control in K06184 and 156681 cells. Images were captured in Keyence microscope at a magnification of 10X. Scale bar for imaging is 100 µm. ( G&I) Dead/Live area fraction measured upon CST treatment (5 µM) in K06184 and 56681 cells (n=3). (J&K) Caspase 3/7 assay done in CST-treated and untreated K06184 and 156681 cells. (n=4). Data were presented as Mean ± SEM. Statistical analyses were done using one-way ANOVA (D&E) and Welch’s t-test (B,G,J,I,K). * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001. To determine whether CST administration affects melanoma cell viability, we used patient-derived melanoma cell lines K06184, 156681, 128128 (characteristics mentioned in Supplementary File 1 ) from the NCI-PDMR repository and normal human dermal fibroblasts (CCD1076). A schematic of the experimental plan with the patient derived cell lines are mentioned ( Fig. 1C ). CST treatment 1 µM, 2 µM, 5 µM, 10 µM) for 120 hours caused dose-dependent reduction in melanoma cell viability ( Fig. 1D & E; Supplementary Fig. S1B ), whereas normal fibroblasts remained unaffected even at the highest concentration ( Supplementary Fig. S1A ). Fluorescence-based viability/cytotoxicity assays showed a pronounced increase in dead cells (EthD-III, red) relative to live cells (Calcein-AM, green) following CST treatment ( Fig. 1F-H ; Supplementary Fig. S1C ). Quantification revealed a significantly elevated dead-to-live cell ratio ( Fig. 1G-I , Supplementary Fig. S1D ). Corroboration of these results by increased caspase-3/7 activity not only confirm induction of apoptosis ( Fig. 1J-K , Supplementary Fig. S1E ), but hint at a possible role of CST in curbing melanoma progression. 2. Catestatin reduces viability, proliferation, and migration in melanoma cell lines To further our preliminary observations regarding the role of CST in curbing melanoma, we performed several experiments in established mouse and human melanoma cell lines and their normal counterparts as shown in schematic ( Fig. 2A ). We performed cell viability assay in mouse melanoma cell line B16-F10 and human melanoma cell lines A375, SKMEL28 with increasing concentration of CST (0.5 µM, 1 µM, 2 µM, and 5 µM) for 72 hours. While we recorded a marked decrease in cell viability of the melanoma cell lines ( Fig. 2C, E &F ), there was no significant reduction in cell viability of normal mouse fibroblast or human skin fibroblast cell lines ( Fig. 2B &D ), thus indicating that CST preferentially kills the cancer cells without affecting the normal ones. Based on the maximal effects, a concentration of 2 µM was used for subsequent assays. Download figure Open in new tab Figure. 2. Melanoma progression inhibition upon CST treatment in mouse and human melanoma cell lines (A) Schematic representation of experimental setup on B16F10, A375 and SKMEL28 cell line in control and CST-treated condition. ( B&C) Cell viability of mouse embryonic fibroblast (MEF) and mouse melanoma cell line B16F10 by CCK-8 assay using different concentrations of CST (0.5 µM, 1 µM, 2 µM, and 5 µM) versus vehicle control for 0, 24,48 and 72 hours. ( D-F) Cell viability by CCK-8 assay on normal human skin fibroblast and melanoma cell lines A375,SKMEL28 in response to different concentrations of CST (0.5 µM, 1 µM, 2 µM, and 5 µM) for 0, 24, 48 and 72 hours. For all cck-8 assay (n=4). ( G&H) Colony formation assay and its quantitative analysis in B16F10 mouse melanoma cells. (I&J ) Colony formation and its quantitative analysis in A375 human melanoma cell (n=3). ( K&L ) Transwell migration assay in CST versus vehicle-treated condition and its quantitative analysis in B16F10 cells. ( M-N) Transwell migration assay in CST versus vehicle-treated condition along with quantitative analysis in A375 human melanoma cells (n=3). These bright field images were captured in Keyence microscope at 10X magnification. Scale bar: 100 µm. ( O-Q) Caspase3/7 Assay in CST versus vehicle-treated condition in B16F10, A375 and SKMEL28 cells (n=5). Data were presented as Mean ± SEM. Two-way ANOVA followed by Dunnett’s multiple comparison test was used to analyze cell viability assay data. Welch’s t-test was used to analyze colony formation, transwell migration and Caspase3/7 assay data. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, and **** p ≤ 0.0001. Phase-contrast microscopy revealed increased cell death in A375 cells treated with CST for 24 hours ( Supplementary Fig. S2A ). Colony-formation assays showed that CST markedly suppressed clonogenic potential in both human and mouse melanoma cells ( Fig. 2G–J ; Supplementary Fig. S2B&C ). Because cell migration is a hallmark of metastatic potential, we evaluated migration using transwell and wound-healing assays. CST significantly impaired migration in all melanoma lines compared with controls ( Fig. 2K–N ; Supplementary Fig. S2D–G ). Increased caspase-3/7 activity in CST-treated melanoma cells ( Fig. 2O–Q ) further confirmed apoptotic induction. Collectively, CST inhibits melanoma cell survival, proliferation, and metastatic potential. 3. Catestatin reduces melanoma tumor burden in vivo To assess CST efficacy in vivo , C57BL/6 mice were subcutaneously injected with 5 × 10⁴ B16-F10 melanoma cells and treated intraperitoneally with CST (10 mg/kg, three times per week) ( Fig. 3A ). Our in vitro plasma stability studies revealed the following remaining percentages of CST during the 24 hours of incubation period: 58.9% after 0.125 hour; 20.2% after 8 hours; 0% after 24 hours ( Supplementary Fig. S3A ). We found the following in vivo plasma pharmacokinetic values: C max (maximum concentration): 148 ng/ml; T max (time to reach Cmax): 0.25 hour; t 1/2 (half-life): 1.38 hour ( Supplementary Fig. S3B ). CST treatment significantly suppressed tumor growth compared with vehicle controls ( Fig. 3B ). No major changes in body weight or liver histology were observed ( Supplementary Fig. S3C&D ), indicating systemic tolerability. The tumor volume kinetics revealed marked decrease upon CST treatment and tumor weight was also lower in CST-treated mice ( Fig. 3C &D ). IHC staining revealed reduced Ki-67 expression, indicating diminished proliferation ( Fig. 3E &F ). Hematoxylin and eosin staining showed reduced cellular density ( Fig. 3E ), and TUNEL assay demonstrated increased apoptotic nuclei in CST-treated tumors ( Fig. 3G &H ). Western blot analysis confirmed elevated cleaved caspase-3 levels ( Fig. 3I &J ). Thus, CST treatment significantly curtails melanoma growth in vivo without observable toxicity. Download figure Open in new tab Figure 3. In vivo melanoma tumor is reduced upon CST treatment ( A ) Schematic showing injection of 5*10 ^ 4 B16F10 cells to 7-9 weeks old C57BL/6 mice and administration of CST (10 mg/kg) thrice a week till sacrifice. ( B) Tumor growth kinetics in B16F10 derived tumors treated with CST (10mg/kg) and vehicular control (n=9). ( C) Post-harvesting images of Control and CST-treated tumors. ( D) Bar graph showing changes in tumor weight in response to control or treatment with CST (n=9). ( E) Immunohistochemistry images showing levels of proliferation marker Ki-67 in Control and CST treated mice tumors. Hematoxylin and eosin staining reveal the tissue structures of the tumors. Images were captured in brightfield in Keyence microscope at a magnification of 20X. Scale bar: 50 µm. ( F) Bar graph showing area fraction of Ki-67 staining in control and CST-treated tumors (n=3). ( G) TUNEL assay performed on control and CST treated tumor sections. The TUNEL positive cells stain red and the nuclei stain blue. Images were captured in a Keyence microscope at magnification of 10X. Scale bar: 100 µm. ( H ) The TUNEL-positive area of different tumors are represented in the graph (n=3). ( I) Representative western blot of cleaved caspase3 as compared to actin levels in mice after treatments with control or CST treated tumors. ( J ) Bar graph showing densitometric analysis of I(n=3). Data were presented as Mean ± SEM and analyzed by 2-way ANOVA followed by Tukey’s multiple comparison test (tumor growth kinetics) or by Welch’s t-test. * p ≤ 0.05, ** p ≤ 0.01, and **** p ≤ 0.001. 4. Molecular mechanisms underlying CST-mediated melanoma regression To elucidate the mechanisms of CST-induced tumor suppression, bulk RNA sequencing was performed on control and CST-treated B16-F10 tumors (n = 4 per group). Differential expression analysis revealed widespread transcriptional modulation ( Fig. 4A ) with 184 significantly upregulated and 157 significantly downregulated genes upon CST treatment in vivo . Gene-ontology enrichment upregulated ( Supplementary Fig. S4A&B ) and downregulated transcripts were mapped and when emphasized on the downregulated pathways they were linked to extracellular matrix (ECM) organization , collagen metabolism , response to hypoxia , and epithelial-to-mesenchymal transition (EMT) ( Fig. 4B &C )—processes that facilitate melanoma progression 34 – 36 . Download figure Open in new tab Figure. 4. Molecular mechanisms driving CST function in mouse tumor and human melanoma cell line. ( A ). Volcano plot of differential regulation of genes upon CST treatment. ( B) Heat map showing all downregulated genes with padj <0.05 with emphasis to CCN2 , LOXL2 , DDIT4 , PDGFRB , and FN1 genes (n=4, each group) ( C ) Gene ontology (GO) analysis of enriched downregulated pathways emphasizing on the previously mentioned genes. ( D) Volcano plot of differentially expressed genes upon CST treatment in A375 human melanoma cell line. ( E) Heatmap of downregulated genes with padj <0.05 (n=3, each group) ( F) GO analysis of enriched downregulated pathways upon CST treatment in A375 cells. ( G) Venn Diagram showing common downregulated genes in B16F10 derived mouse tumor and A375 cell line. ( H) List of the 46 common genes in the Venn Diagram with emphasis on CCN2 , LOXL2 , DDIT4 , PDGFRB , and FN1 . ( I-L) Expression levels of CCN2 , DDIT4 , PDGRB and LOXL2 from TCGA database in primary melanoma vs metastasis as analysed in UALCAN database. Similarly, transcriptomic analyses of CST-treated A375 cells showed 2816 upregulated genes and 2909 downregulated genes ( Fig. 4D ) The upregulated ( Supplementary Fig. S4C&D) and downregulated genes were mapped and the downregulated pathways related to cell-substrate adhesion , reactive oxygen species metabolism , and hypoxia response ( Fig. 4E-F ) were also observed in human melanoma A375 cell line. Comparative analysis of the mouse and human cell line datasets revealed 46 commonly downregulated genes ( Fig. 4G &H ), including CCN2 , LOXL2 , DDIT4 , PDGFRB and FN1 , all previously implicated in melanoma aggressiveness 37 – 40 . Consistent with these findings, TCGA-SKCM analyses via UALCAN confirmed elevated expression of these genes in advanced melanoma stages ( Fig. 4I–L ). Together, these results suggest that CST suppresses melanoma by downregulating ECM remodeling, hypoxia, and EMT-associated signaling programs. 5. CST suppresses Vemurafenib-resistant melanoma Resistance to BRAF inhibitors such as Vemurafenib remains a major clinical challenge. Notably, the patient-derived melanoma line 156681 (detailed in Supplementary File 1 ), isolated from a Vemurafenib-treated non-responder, displayed enhanced CST sensitivity with decreased viability and increased cell death as observed previously ( Fig. 1 ). This observation led us to check further the potential of CST as an anti-cancerous agent in Vemurafenib resistant cells. To validate this observation, we generated a Vemurafenib-resistant A375 cell line. The IC₅₀ for resistant cells (1432 nM) was >6-fold higher than that of parental cells (228.9 nM) ( Fig. 5A &B ). CST treatment (0.5 µM, 1 µM, and 2 µM) markedly reduced viability of resistant A375 cells ( Fig. 5C ) and induced extensive cytopathic changes ( Fig. 5D ). Transwell migration assays confirmed diminished migratory capacity following CST exposure ( Fig. 5E ). To further into the molecular mechanism of these observed effects, transcriptomics was performed in control and CST-treated resistant A375 cells. Download figure Open in new tab Figure 5. CST kills Vemurafenib resistant A375 melanoma cells by diminishing the levels of resistance associated genes. (A) IC 50 of Vemurafenib in Vemurafenib-sensitive A375 cell line. ( B) IC 50 of Vemurafenib in Vemurafenib-resistant A375 cell line. ( C) Dose-dependent (0.5 µM, 1 µM, 2 µM, and 5 µM) effects of CST on cell viability of Vemurafenib-resistant A375 cells after 0,24 and 48 hours of treatment (n=6). ( D) Phase contrast image of control versus CST-treated Vemurafenib resistant A375. ( E) Bar graph showing quantitative analysis of Trans well migration assay in Vemurafenib-resistant A375 cells (n=3). ( F ) Volcano plot of differentially expressed genes upon CST treatment in A375 resistant cell ( n=4, each group) (G) Heatmap showing downregulated genes upon CST treatment with padj <0.05. ( H) Heatmap of downregulated oncogenes with known link to resistance towards standard melanoma treatment including WNT5A , KDM5B , PDGFRB , ID1 , ID2 , ID3 , TWIST1 , VAV3 , FGFR3 , SPARC , SOX2 , SOX4 , SREBP1 , and MITF . Cell viability assay graphs were analysed using 2-way ANOVA followed by Dunnett’s multiple comparison test and Welch’s t test for analysis of the transwell migration data. * p ≤ 0.05, ** p ≤ 0.01, and *** p ≤ 0.001. Transcriptomic analysis of these resistant cells treated with CST revealed extensive transcriptional reprogramming ( Fig. 5F &G, Supplementary Fig. S5A&B ) with 1990 upregulated genes and 1991 downregulated genes, including downregulation of WNT5A 41 , PDGFRB 42 , ID1–3 43 – 45 , FGFR3 46 , SREBF1 47 , VAV3 48 , KDM5B 49 – 51 , TWIST1 52 , SOX2 53 , SOX4 54 , MITF 55 , and SPARC 56 ( Fig. 5H )—genes associated with resistance and metastasis. The suppression of these genes that are actively involved in conferring resistance to known treatment regimens underscores the therapeutic potential of CST in overcoming treatment-resistant melanoma. DISCUSSION Melanoma incidence continues to rise worldwide, with an estimated 331,647 new cases and 58,645 deaths reported in 2022 (GLOBOCAN 2022) 57 . In the United States alone, over 104,000 new invasive melanoma cases are projected for 2025 ( https://www.curemelanoma.org/blog/over-104-000-americans-estimated-to-be-diagnosed-with-invasive-melanoma-in-2025 ). Despite major advances in targeted and immune-based therapies, recurrence and therapy resistance remain formidable clinical barriers 58 . This persistent challenge underscores the need for novel, mechanistically distinct therapeutic strategies capable of curbing melanoma progression and overcoming resistance to current regimens. In this study, we introduce CST as a promising therapeutic candidate against melanoma. Peptide-based therapeutics are underexplored in this malignancy 59 – 62 , yet they offer inherent advantages, including biocompatibility, low immunogenicity, and ease of modification. CST, known for its anti-inflammatory 18 , 24 and cardio-metabolic benefits 13 , emerged as a rational candidate owing to its regulatory roles in inflammation, oxidative stress, and metabolic homeostasis—processes intimately linked to oncopathobiology. Our analyses reveal that CST expression declines progressively with melanoma stage, suggesting its loss may facilitate tumor advancement. Restoration of CST in patient-derived melanoma cells elicit marked cytotoxicity and apoptosis in lines with distinct molecular features—K06184 (wild-type BRAF, brain metastasis) and 156681 (BRAF^V600E, Vemurafenib-non-responder) and 128128 (treatment naïve, metastatic melanoma)—highlighting its broad anti-cancer efficacy across genotypes. Mechanistically, CST exerted potent anti-proliferative and anti-migratory effects. In both mouse (B16F10) and human (A375, SKMEL28) melanoma cell lines, CST treatment reduced cell viability, colony formation, and transwell migration, while sparing normal fibroblasts. In vivo , CST administration markedly suppressed tumor growth in B16F10 melanoma-bearing C57BL/6 mice, with concomitant reductions in Ki-67 and elevations in TUNEL and cleaved-caspase-3 signals. The absence of changes in body weight or hepatic histology confirmed that CST is well tolerated and systemically non-toxic. To define CST-driven molecular alterations, transcriptomic analyses identified widespread transcriptional reprogramming. Downregulated gene clusters were enriched in pathways controlling extracellular-matrix organization , collagen metabolism , hypoxia adaptation , and epithelial-to-mesenchymal transition (EMT) —all central to melanoma invasion and metastasis. CST significantly suppressed CCN2, LOXL2, DDIT4, and FN1 , key drivers of ECM remodeling and angiogenesis, indicating that CST disrupts the structural and metabolic axes sustaining tumor progression. Given the high prevalence of therapeutic resistance, particularly to BRAF/MEK inhibitors, we further evaluated any potential role of CST in Vemurafenib-resistant A375 cells. CST markedly reduced cell viability and migration in these resistant cells, accompanied by downregulation of multiple resistance-associated genes—including FGFR3 46 , 63 , ID1/2/3 43 , 64 , PDGFRB , SREBF1 47 , 65 , and MITF 66 . These targets are central mediators of adaptive survival and dedifferentiation in BRAF^V600E melanoma, suggesting that CST not only restrains tumor growth but may re-sensitize resistant cells to existing therapies. Collectively, our findings delineate CST as a multi-targeted anti-melanoma peptide that impairs proliferation, invasion, and resistance mechanisms while maintaining a favorable safety profile. Although CST has been variably reported to promote angiogenesis in other contexts 30 , 67 , our in-vivo and transcriptomic data consistently support an anti-angiogenic and anti-proliferative role in melanoma. Future directions include optimizing CST pharmacodynamics through peptidomimetic modifications to enhance stability, half-life, and tumor bioavailability. Combination therapy paradigms integrating CST with BRAF/MEK inhibitors or immune checkpoint blockade warrant exploration to achieve synergistic efficacy. Moreover, since Chromogranin A yields several bioactive fragments—Pancreastatin 68 , Vasostatin I/II 69 , WE14 70 , and Serpinin 71 —systematic studies of their processing and interactions in melanoma may reveal complementary or cooperative functions. In conclusion, this study provides compelling preclinical evidence that Catestatin represents a novel, safe, and effective therapeutic modality for both treatment-naïve and drug-resistant melanoma. By simultaneously targeting tumor growth, metastatic potential, and resistance pathways, CST holds significant translational promise as the foundation for a new class of peptide-based anti-melanoma therapies. Contribution to work Conceptualization: SKM, SK Methodology: SK, SJ, KT, SKM, AC Investigation: SK Visualization: SK, SJ, SKM, NW Funding acquisition: SKM, NW Project administration: SKM, SK, SJ Supervision: SK, SJ, SKM Writing – original draft: SK, SKM Conflict of Interest SKM is the founder of CgA Therapeuticals, Inc. and co-founders of Siraj Therapeutics. Remaining authors declare no competing interests. MATERIALS AND METHODS Cell Culture Mouse embryonic fibroblast (MEF), human skin fibroblast (CCD1076), human melanoma cell lines (A375, SK-MEL-28), and mouse melanoma cell line (B16-F10) were obtained from the American Type Culture Collection (ATCC). Cells were maintained in DMEM supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 µg/mL streptomycin in a humidified incubator at 37°C with 5% CO₂. Patient-derived melanoma cell lines (K06184, 156681, and 128128) were procured from the NCI Patient-Derived Models Repository (PDMR) and cultured following PDMR standard operating procedures in DMEM/F-12 containing 5% FBS, 0.4 µg/mL hydrocortisone, 0.01 µg/mL epidermal growth factor, 24 µg/mL adenine, 100 µg/mL penicillin-streptomycin, 2 mM L-glutamine, and 10 nM Y-27632 dihydrochloride on Matrigel-coated plates. Human Tissue Samples Commercially available human melanoma tissue microarrays (catalog nos. Me482A and Me551) were purchased from TissueArray.com for immunohistochemical analyses. Peptide Treatment Catestatin ( CST ; sequence SSMKLSFRARAYGFRGPGPQL) was synthesized by GenScript and dissolved in 0.9% saline. Cells were treated with CST at concentrations of 0.5–10 µM as indicated. For in vivo studies, CST was administered intraperitoneally at 10 mg/kg body weight, three times per week (Monday, Wednesday, Friday). Bioinformatic Analyses Expression of CCN2, LOXL2, DDIT4, PDGFRB and FN1 in primary and metastatic skin cutaneous melanoma samples was analyzed using the UALCAN database ( http://ualcan.path.uab.edu ) 72 . Caspase Assay Cells (1 × 10⁴ per well) were seeded in 96-well white plates and treated with or without CST for 48 h (A375, SK-MEL-28, B16-F10) or 120 h (patient-derived lines K06184, 128128, 156681). Caspase-3/7 activity was measured using the Caspase-Glo® 3/7 Assay System (Promega) following the manufacturer’s protocol. Luminescence was recorded using a Varioskan LUX microplate reader (Thermo Fisher Scientific). Live/Dead Viability Assay Cells were treated with CST or vehicle for 120 h, washed with PBS, and incubated with 2 µM calcein AM and 4 µM ethidium homodimer III (Biotium Viability/Cytotoxicity Assay Kit) for 30 min at room temperature in the dark. After washing, cells were imaged under FITC and Texas Red® channels using a Keyence fluorescence microscope (20×). Cell Viability Assay Cells (1 × 10⁴ per well) were plated in 96-well plates and treated with CST (0.5–10 µM) for 72–120 h. Viability was determined using the CCK-8 reagent (APExBIO). Absorbance was measured at 450 nm using a Varioskan LUX reader. Colony Formation Assay A375, SK-MEL-28, and B16-F10 cells (1 × 10³ per well) were treated with or without CST according to the experimental design and cultured for 15 days. Colonies were fixed in methanol for 20 min, stained with 0.1% crystal violet, and photographed. Transwell Migration Assay Cells pretreated with CST or vehicle for 48 h were seeded in serum-free medium in transwell inserts (8 µm pore size). The lower chamber contained complete medium. After 24 h, non-migrated cells were removed, membranes were fixed, stained with crystal violet, and imaged using a Keyence brightfield microscope (20×) as described previously 73 . Wound Healing Assay A375 cells at 40–50% confluency was scratched with a sterile pipette tip, followed by CST or control treatment. Images were captured at 0 and 24 h using Keyence brightfield microscopy (20×). RNA Sequencing Total RNA from mice tumor and A375 cell line was isolated using RNeasy miniprep Kit (Qiagen). RNA was quantified by Nanodrop spectrophotometer and its integrity was evaluated by Tapestation (Agilent). Complementary DNA library preparation was performed using 400ng of RNA using mRNA HyperPrep Kit (KAPA) according to manufacturer’s protocol with Unique Dual-Indexed adapters (KAPA). The cDNA library was amplified and assessed by Qubit2.0 (Thermo Fisher Scientific). The libraries were then pooled and analysis was done in NovaSeq X Plus 10B (Illumina) in UCSD IGM core as described previously 74 . Generation of Vemurafenib-Resistant Cells Vemurafenib-resistant A375 cells were generated following established protocol. Parental cells were treated with 100 nM Vemurafenib for 2 weeks, followed by stepwise doubling of drug concentration every 2 weeks up to 3.2 µM. Surviving cells in maintenance culture containing 2 µM drug was used for downstream studies. Mouse Tumor Model Age-matched male and female C57BL/6 mice (7–9 weeks, n = 9) were subcutaneously injected with 5 × 10⁴ B16-F10 cells in 100 µL PBS. Ten days post-injection, CST (10 mg/kg) was administered intraperitoneally three times weekly. Tumor dimensions were measured with calipers, and volume was calculated using the formula V = (length × width²)/2. Mice were euthanized 22 days post-inoculation. All procedures were approved by the IACUC of UCSD and the VA San Diego Healthcare System and conformed to NIH guidelines. Immunohistochemistry and Histology Tumors were fixed, paraffin-embedded, and sectioned (5 µm). Hematoxylin and eosin staining was performed at the La Jolla Institute for Immunology. Immunohistochemistry was carried out using the Super Sensitive™ M Polymer-HRP Kit (Biogenex). After deparaffinization, antigen retrieval was performed using Histo VT (Nacalai) at 90°C for 20 min. Sections were incubated overnight at 4°C with anti–Ki-67 (Abcam) or anti-CST (gift from Angelo Corti) at 1:100 dilution. Detection was performed with DAB and counterstained with hematoxylin. Slides were dehydrated and mounted in DPX. Images were captured using Keyence brightfield microscopy (20×). TUNEL Assay Apoptotic nuclei were detected in tumor sections using the CF®594 TUNEL Assay Kit (Biotium). After deparaffinization and rehydration, sections were permeabilized with 20 µg/mL Proteinase K for 1 h at 37°C, incubated with TUNEL reaction mix for 2 h, counterstained with Hoechst, and mounted in antifade medium. Fluorescence images were obtained using a Keyence microscope. Phase-Contrast Microscopy Morphological alterations in CST-treated A375 and Vemurafenib-resistant A375 cells were assessed using Keyence phase-contrast microscopy. Western Blotting Tumor lysates were prepared in RIPA buffer containing protease inhibitors (Thermo Fisher). Equal protein amounts (10 µg) were resolved by SDS-PAGE, transferred to PVDF membranes, and incubated with antibodies against cleaved caspase-3 (Proteintech) and β-actin (Cell Signaling Technology). HRP-conjugated secondary antibodies (Cell Signaling Technology) were used, and signals were detected by chemiluminescence. Densitometric analyses were performed using GelQuant.NET ( BiochemLabSolutions.com ). In vitro plasma stability and in vivo plasma pharmacokinetics Plasma stability was determined in 5 µg CST/ml spiked mouse (C57BL/6) plasma (50 µl) and taking aliquots at specific times over a 24-hour period. Each aliquot (50 µl) was mixed with 150 µl methanol, vortexed, spun, removed the supernate and reconstituted the pellet with 150 µl 10% formic acid in water. The sample was vortexed and spun and a 100 µl aliquot was taken, diluted 1:1 with 10 mM ammonium bicarbonate, and analyzed by LC-MS/MS. For in vivo plasma pharmacokinetics, male C57BL/6 mouse received a 5mg/kg intraperitoneal dose of CST. Blood samples (100 µl) were obtained at 0, 0.25, 0.5, 1, 2, 4, 8, and 24 hours via tail vein bleeds. Plasma concentrations of CST was determined by LC-MS/MS methods as described above. Statistical Analyses Data were analyzed using GraphPad Prism 10. Two-tailed Welch’s t -test was used for single-variable comparisons, and two-way ANOVA for multivariate datasets (e.g., concentration and time). 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