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. 2025 Dec 29;15:44864. doi: 10.1038/s41598-025-28984-6

The AKT-p21 phosphorylation signaling axis confers poor prognosis and dacarbazine resistance in melanoma

Gabriela Nana Colaneri 1,#, Ana Carolina Monteiro 1,2,#, Beatriz Cristina Biz Tonin 1,#, Hátylas Azevedo 3,#, Débora Kristina Alves-Fernandes 1,#, Adriana Taveira da Cruz 1, Fabiana Marcelino Meliso 1, Alice Santana Morais 1, Roberta Sessa Stilhano 4, Stefanie Gross 2, Christian Ostalecki 2, Regine Schneider-Stock 2, Sang Won Han 4, Miriam Galvonas Jasiulionis 1,✉
PMCID: PMC12750010  PMID: 41462552

Abstract

Understanding the molecular mechanisms underlying melanoma metastasis and chemoresistance is crucial for predicting patients’ prognoses and developing more effective therapies. In this study, we investigated the role of p21 in melanoma progression and treatment resistance. Our findings reveal that increased gene and protein expression of p21 is a strong predictor of poor prognosis in human melanoma, while reducing p21 makes these cells more responsive to chemotherapy. Immunohistochemistry experiments showed higher levels of total and cytoplasmic p21 in metastatic melanoma compared to primary melanomas and other melanocytic lesions. In silico analysis highlighted the interaction of p21 with proteins based on their cellular localization, identifying AKT1 as a potential regulator of p21. This finding corresponds with the observed positive relationship between p21 and AKT1 levels in both melanoma cell lines and patient samples. Significantly, we observed that increased p21 expression, phosphorylation, and translocation to the cytoplasm are associated with resistance to the chemotherapy drug dacarbazine. Accordingly, the overexpression of p21 in a metastatic melanoma cell line led to a further increase in dacarbazine resistance, whereas downregulation of p21 sensitizes these cells to chemotherapy. In addition, both AKT downregulation and the pharmacological inhibition of PI3K diminished p21 phosphorylation, with the former intervention also sensitizing metastasis-prone cells to chemotherapy. Collectively, these findings highlight the critical role of the PI3K/AKT-p21 signaling in the regulation and subcellular localization of p21 in the context of chemotherapy resistance and melanoma prognosis.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-025-28984-6.

Keywords: P21, PI3K/AKT, Drug resistance, Metastasis, Melanoma, Dacarbazine

Subject terms: Cancer, Cell biology, Oncology

Introduction

Skin Cutaneous Melanoma (SKCM) represents the most aggressive type of skin cancer with increasing incidence worldwide1,2. This tumor arises from the malignant transformation of melanocytes, which are constantly exposed to ultraviolet radiation and high levels of free radicals released during the melanin synthesis3. To survive under this highly mutagenic environment, melanocytes have developed increased resistance to p53-mediated apoptosis after UV light exposure3, which may also provide them with an intrinsic ability to evade apoptotic mechanisms involved in melanoma chemoresistance4.

The tumor-suppressor proteins p53 and p21 are major regulators of the G1/S cell–cycle checkpoint during DNA damage and repair5. The p53 stabilization induces p21 transcription and the subsequent inhibition of cyclin-dependent kinases (CDK)-cyclin complexes by p216. Depending on its subcellular localization, p21 can play both pro- and antitumoral effects in cancer cells7. In the nucleus, p21 has tumor suppressor effects via binding to CDKs and inhibiting the cell cycle, but it can also exert oncogenic roles by inducing a senescence-associated secretory phenotype (SASP) that promotes tumor progression6. In the cytoplasm, p21 also acts as an oncogene by preventing apoptosis, and the cytoplasmic location of p21 is regulated by its phosphorylation6. Both high and low levels of p21 have been correlated with tumor aggressiveness and poor prognosis in different types of cancer, suggesting that dominant pro- or antitumoral effects of p21 could be tumor-specific6. In squamous cell carcinomas, the oncogenic activities of p21 were attributed to its cytoplasmic accumulation promoted by its phosphorylation8. In this condition, p21 can help tumor cells evade apoptosis and thus compromise chemotherapy9.

Dacarbazine (DTIC) is an important chemotherapeutic agent for the treatment of advanced non-surgical and metastatic melanoma10,11. Despite its role in the therapeutic algorithm of the disease, DTIC exhibits low response rates12, which has fostered ongoing research seeking new therapeutic strategies to overcome drug resistance13,14. Notably, repeated exposure of melanoma cells to escalating concentrations of dacarbazine might enhance tumor growth and metastasis in vivo, due to the selection of a more aggressive melanoma phenotype15.

Considering the need to develop new strategies to enhance melanoma responsiveness to treatment and the dual role of p21 in cancer development, which remains underexplored, this study investigated the potential involvement of p21 as a pro-survival molecule contributing to drug resistance in melanoma. We demonstrated that elevated p21 expression is a strong predictor of poor prognosis in human melanoma. Using an in vitro cellular model of melanoma progression16,17, we showed that this increase in p21 expression is associated with its cytoplasmic localization in metastatic melanomas compared to primary melanomas and other melanocytic lesions. Furthermore, a positive association between p21 expression and its upstream regulator AKT was seen in human melanoma samples. Notably, higher expression and phosphorylation of both p21 and AKT, along with cytoplasmic translocation of p21, were linked to dacarbazine resistance in melanoma cells. Overexpression of p21 in a metastatic melanoma cell line further amplified this resistance. Collectively, these findings highlight the role of the AKT-p21 signaling pathway in chemotherapy resistance and underscore its potential as a prognostic marker and therapeutic target in melanoma.

Results

Increased protein and gene expression of p21 predicts poor prognosis in melanoma

Initially, to evaluate the importance of p21 in SKCM progression, the potential prognostic value of p21 (CDKN1A) was compared in several tumor types from The Cancer Genome Atlas (TCGA), using both protein and gene expression data. The p21 protein expression value significantly correlated with poor prognosis in a subset of tumor types from the TCGA (Fig. 1A), such as melanoma (SKCM), renal clear cell carcinoma (KIRC), endometrial carcinoma (UCEC), gastric adenocarcinoma (STAD), head and neck cancer (HNSC), glioblastoma multiforme (GBM), lung squamous carcinoma (LUSC), and thymoma (THYM). In melanoma, the increased p21 protein expression had one of the highest hazard ratios for poor prognosis among the analyzed tumor types (Fig. 1B, HR = 1.61, p = 0.0033), whereas high expression of p27 (CDKN1B) was associated with the lowest hazard ratio (HR = 0.475, p < 0.0001).

Fig. 1.

Fig. 1

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High p21 expression correlates with poor prognosis in human melanoma. (A) Cox regression analysis showing the correlation between p21 gene expression and overall survival in a subset of tumor types* from the TCGA. (B) Cox regression analysis showing the proteins whose expression confers a worse prognosis in SKCM patients. (C) Overall survival of all SKCM patients according to p21 protein expression. Red and blue curves represent, respectively, high (top 50%) and low (bottom 50%) protein expression. (D) Overall survival of melanoma patients presenting NRAS mutation according to their p21 protein expression (high or low). (E) Survival curve of all TCGA SKCM patients and of those with BRAF mutation according to p21 (CDKN1A) gene expression level in samples. (F) Violin plots demonstrating the variability in p21 expression according to SKCM stages. (G–H) Violin plots showing the distribution of overall survival among the subgroups of melanoma patients with high and low p21 protein (G) and mRNA expression (H). The survival curves were generated by Kaplan-Meier Plotter. *Melanoma (SKCM), Renal clear cell carcinoma (KIRC), endometrial carcinoma (UCEC), gastric adenocarcinoma (STAD), head and neck cancer (HNSC), glioblastoma multiforme (GBM), lung squamous carcinoma (LUSC), and thymoma (THYM).

A Kaplan-Meier plot analyzing the expression of p21, categorizing SKCM samples into two groups based on higher or lower p21 expression, showed that high p21 protein levels were significantly associated with worse survival in both all SKCM samples (SKCM total) (Fig. 1C, p = 0.0033) and melanoma tumors with NRAS mutations (Fig. 1D, p = 0.014). A similar trend was observed when analyzing p21 (CDKN1A) gene expression values (Fig. 1E), although the association was not statistically significant.

Further analysis of p21 gene expression revealed that only melanoma tumors with BRAF mutations were significantly associated with worse survival when samples were stratified into high and low p21/CDKN1A expression groups (Fig. 1E).

Additionally, stratifying SKCM samples by stage revealed significant variation in p21 gene expression levels (F = 3.98, p = 0.00351), with higher p21 expression observed in stages II and IV based on TCGA data (Fig. 1F). Violin plots were generated to verify the distribution of overall survival among subgroups with high and low p21 expression. Patients whose tumors exhibited lower p21 expression had significantly better overall survival compared to those with high p21 expression, both at the protein level (median survival of 47.73 vs. 23.81 months, Mann-Whitney test, p = 0.002; Fig. 1G) and at the gene expression level (median survival of 44.84 vs. 27.73 months, Mann-Whitney test, p = 0.0014; Fig. 1H).

The cytoplasmic expression of p21 differentiates metastatic melanomas from other melanocytic lesions representing earlier stages of melanoma

As previously demonstrated, p21 expression is elevated in the later stages of melanoma, which are characterized by a greater propensity for metastasis and poorer overall survival18. To further explore the clinical relevance of p21 levels in melanoma, we investigated whether p21 expression could serve as a potential biomarker for human metastatic melanoma.

Histological sections of non-metastatic and metastatic melanomas were evaluated using immunohistochemistry (IHC) to detect p21 (Fig. 2A). The results showed strong nuclear staining in both primary (non-metastatic) and metastatic tissues, whereas pronounced cytoplasmic p21 staining was observed exclusively in metastatic melanoma tissues (Fig. 2A). Quantification of nuclear and cytoplasmic p21 levels using an immunoreactivity scoring system (IRS) further confirmed the distinct cytoplasmic staining in metastatic melanoma cells (Fig. 2B).

Fig. 2.

Fig. 2

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Human metastatic melanomas express high levels of cytoplasmic p21. (A) Histological sections of primary and metastatic melanoma tissues evaluated by immunohistochemistry for P21 developed by alkaline phosphatase (pink). Inserts show increased magnification. (B) Quantification of the level of nuclear and cytoplasmic p21 staining in primary and metastatic melanoma tissues by the immunoreactivity scoring system (IRS) (n = 5 per group). (C) Histological sections of nevi (nevus and dysplastic nevus) and melanomas (non-metastatic and metastatic) were examined by IHC staining for P21. Error bars represent SEM, and p-values were based on the unpaired t-test. *P < 0.05.

To validate these findings, additional IHC analysis was performed on nevi and dysplastic nevi. This analysis demonstrated that earlier stages of melanoma progression exhibit minimal cytoplasmic p21 levels compared to primary (non-metastatic) and metastatic melanoma tissues (Fig. 2C).

Additionally, to evaluate the expression of p21 in mouse cells, we used an in vitro melanoma model. This model consists of selected melanoma cell lines with different phenotypes16,17,19. Specifically, we used 4C11-, which comprises non-metastatic, slow-growing, and undifferentiated cells, and 4C11+, which are metastatic, differentiated, and highly proliferative cells. The expression and phosphorylation status of p21 were then evaluated in both cell lines to compare different melanoma stages. Increased levels of p21 and p-p21 (Thr145) were observed in 4C11 + when compared with 4C11- cells (Fig. 3A). In accordance with human melanoma cells, the metastatic cell line (4C11+) presented exclusive cytoplasmic localization of phosphorylated p21 levels (Fig. 3B). To further substantiate this observation, an immunofluorescence staining of p21 was performed in 4C11- and 4C11 + cells, confirming distinct patterns of cytoplasmic and nuclear localization in 4C11 + and 4C11- cells, respectively (Fig. 3C).

Fig. 3.

Fig. 3

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Mouse metastatic melanomas express high levels of cytoplasmic p21. (A) The total p21, p-p21 (Thr145) expression was assessed in non-metastatic 4C11- and metastatic 4C11 + melanoma cells by Western blotting using specific antibodies. (B) The presence of p21 was evaluated by Western blot in 4C11 + protein extracts enriched in nuclear (Nucl) and cytoplasmic (Cyt) fractions. (C) p21 localization was assessed by immunofluorescence in 4C11- and 4C11 + cells. Each experiment was carried out three times, and one representative is shown. Hoescht: nucleus *Uncropped figure available on supplementary material (Fig. S1).

This evidence is aligned with the findings from human melanoma samples, in which p21 is localized in the cytoplasm only in metastatic samples. Also, these findings suggest that while total p21 is present in both non-metastatic and metastatic samples, its subcellular localization appears to be more critical than its overall presence. This raises an important question: Does p21 play essential roles in metastasis progression and therapy resistance?

p21 contributes to chemotherapy resistance in metastatic melanoma cells

Our findings so far indicate that increased expression and phosphorylation levels of p21 are associated with aggressive and metastatic melanoma cells, correlating with worse survival outcomes. Building on this insight, we investigated whether this axis could influence the response of melanoma cells to dacarbazine (DTIC) treatment. Initially, both 4C11- (non-metastatic) and 4C11+ (metastatic) cells were treated with different DTIC concentrations to compare their sensitivity to DTIC treatment. The experiment revealed that 4C11- cells were 3-fold more sensitive to DTIC treatment when compared to 4C11 + cells, showing GI50 values of 200 and 800µM, respectively (Fig. 4A).

Fig. 4.

Fig. 4

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P21 overexpression increases Dacarbazine resistance in metastatic melanoma cells, while the P21 downregulation sensitizes these cells to dacarbazine. (A) Cell viability of melanoma cells lines 4C11- and 4C11 + treated with different concentrations of Dacarbazine (0, 50, 100, 200, 400, 800 and 1200 µM). (B) P21 subcellular localization was evaluated by immunofluorescence in the 4C11- and 4C11 + cells after treatment with dacarbazine 800 µM. The data shown are representative of two individual experiments. Hoechst: nucleus. (C–D) Melanoma cell lines 4C11- and 4C11 + were transduced with Lp21SN retrovirus vector to obtain a stable p21 overexpression (p21 OE). (C) The mRNA expression of p21(CDKN1A) was quantified by RT-PCR and β-Actin was used as endogenous control. (D) The overexpression of the p21 protein was evaluated by Western Blotting using whole cell extracts with specific anti-p21 antibodies. PCNA expression was used as a loading control. (E) Cell viability after 48 h of Dacarbazine treatment was verified by MTT. Dacarbazine resistance was determined in overexpression of p21 (shp21 #1 and #2) relative to the GI50 value of the WT 4C11- and 4C11 + lineage (200 and 800 µM, respectively). (F–H) Melanoma cells were transduced with lentiviruses particles containing validated p21 gene (shp21 #1 and shp21 #2) or non-target shRNA sequences to obtain stable knockdown. (F) Quantification of mRNA expression of p21 by RT-qPCR. It is represented relative to the expression of β-Actin. (G) p21 protein expression using whole cell extracts was assessed by western blotting using an anti-p21 specific antibody. PCNA expression was used as a loading control. (H) Cell viability after 48 h of Dacarbazine treatment was verified by MTT. Dacarbazine resistance was determined in knockdown of p21 (shp21 #1 and #2) relative to the GI50 value of the non-target 4C11 + lineage (800 µM). Representative examples of three independent experiments are shown. Error bars represent SEM, and p-values were based on the One-Way ANOVA test followed by the post-hoc Tukey. **p < 0.01; ***p < 0.001.

We also examined the subcellular localization of p21 in cells treated or not with DTIC through immunofluorescence, showing that 4C11 + surviving cells present higher p21 expression, enriched in the cytoplasm. This effect was also observed in the 4C11-surviving cells, although to a lesser degree (Fig. 4B).

To further investigate the potential role of p21 in DTIC resistance, we next overexpressed p21 in both 4C11- and 4C11 + cell lines. Melanoma cells (P21 OE) with greatly overexpressed p21 mRNA (CDKN1A) (Fig. 4C) and protein levels (Fig. 4D) were expanded and used for further experiments. Interestingly, p-p21 expression increases only in 4C11+ (Fig.S1).

Analysis of cell viability revealed that 4C11 + melanoma cells overexpressing p21 exhibited approximately a two-fold increase in resistance to Dacarbazine compared with wild-type (WT) controls. In contrast, no significant effect was observed in 4C11 − melanoma cells (Fig. 4E).

To confirm the importance of p21 for melanoma survival, the downregulation of p21 was induced in 4C11 + cells. Two clones (shp21 #1 and shp21 #2) with remarkably reduced p21 mRNA (Fig. 4F) and protein (Fig. 4G) levels were selected. In agreement with the overexpression experiment, silencing p21 increased their sensitivity to Dacarbazine, as verified by MTT analysis after treatment with Dacarbazine (Fig. 4H). Additionally, the cell cycle activity in 4C11- and 4C11 + cells was evaluated in the context of both p21 overexpression and knockdown. Surprisingly, increased p21 expression did not affect the distribution of cell cycle stages in these cells (Fig.S2A−B).

PI3K/AKT inhibition reduces the phosphorylation and cytoplasmic levels of p21 in metastatic melanoma cells

To elucidate our hypothesis on the potential mechanisms by which cytoplasmic p21 may influence melanoma metastasis and Dacarbazine resistance, the enriched functions of p21-interacting proteins were compared based on their nuclear or cytoplasmic localization. To achieve this, an interactome analysis of p21 was conducted, which was followed by a subsequent functional enrichment analysis of all, and nuclear and cytoplasmic p21-interacting proteins. This analysis revealed that p21-interacting proteins were enriched for KEGG20–22 terms such as phagosome, gap junction, insulin signaling pathway, and neurological diseases (Fig. 5A).

Fig. 5.

Fig. 5

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Interactome revealed association between AKT and p21. (A) Bar plot of functional enrichment of p21-interacting proteins by KEGG terms in cytoplasm and nucleus. (B) Venn diagram displaying genes with prognostic value in the nuclear and cytoplasmic p21-interacting proteins (left) and a list of genes exclusive to the cytoplasmic interactome (right), highlighting their hazard ratio (survival) in SKCM. The red and blue squares indicate a higher and lower HR, respectively, whereas significant p-values are shown as squares with bold lines. (C) Dot plot illustrating the functional enrichment of the 53 prognostic cytoplasmic p21-interacting proteins. (D) Network plot showing interactions between these cytoplasmic proteins and p21, with a focus on the interaction between AKT1 and CDKN1A. Pathway image reproduced with permission from Kanehisa Laboratories.

We also found 66 and 53 p21-interacting proteins in the nuclear and cytoplasmic p21 interactomes, respectively (Fig. 5B). Notably, most of these genes are distributed in both nuclear and cytoplasmic compartments, with only 8 genes exclusively localized in the cytoplasm: EPPK1, FLAD1, OPTN, PSMD2, RPS16, SLC25A11, TXNDC11, and UBE2D1 (Fig. 5B). The higher expression levels of EPPK1, FLAD1, SLC25A11, and PSMD2 are associated with worse melanoma survival while TXNDC11 and OPTN are associated with better outcomes.

A more in-depth analysis of the 53 prognostic cytoplasmic p21-interacting proteins revealed their involvement in critical pathways such as the cell cycle, viral carcinogenesis, cellular senescence, and the p53 signaling pathway (Fig. 5C). Moreover, the interactome network of cytoplasmic p21-interacting proteins highlighted a notable association between p21 and AKT1 (Fig. 5D). Interestingly, the phosphorylation of p21 by AKT has been described as a potential upstream mechanism regulating p21 localization to the cytosol. This is particularly relevant in melanoma given that AKT is constitutively activated in approximately 70% of the cases23.

To further validate the interaction between AKT and p21, an in vitro melanoma cellular model was used. The phosphorylation levels of AKT were also determined in the 4C11- and 4C11 + cells. Interestingly, pS473AKT levels were significantly higher in the metastatic 4C11 + cells (Fig. 6A), cells that presented an exclusive cytoplasmic localization of phosphorylated p21 levels (Fig. 3B). As shown previously, p-p21 expression increases only in 4C11+. Together with the AKT findings, this supports our hypothesis that p21 availability depends on AKT-mediated phosphorylation, given that 4C11 − cells display lower AKT levels.

Fig. 6.

Fig. 6

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The AKT-p21 axis confers Dacarbazine resistance to metastatic melanoma cells. (A) The total AKT, p-AKT (Ser473) expression was assessed in non-metastatic 4C11- and metastatic 4C11 + melanoma cells by Western blotting using specific antibodies. (B) p-p21 expression was evaluated in 4C11 + cells knocked down for AKT with siRNA. (C) The presence of p21 in the cytoplasmic protein fraction was analyzed in 4C11 + cells treated (Wn) or not with the PI3K inhibitor Wortmannin. PCNA and GAPDH were used as internal controls. siScr: scrambled siRNA. (D) Cell viability of 4C11 + cells control (siScr) and AKT-knockdown (siAKT) after treatment with Dacarbazine (800 µM), evaluated by MTT Assay. siScr: scrambled siRNA. (E) Cell viability of 4C11 + cells WT, overexpressing p21 control (siScr) and AKT-knockdown (siAKT) after 24 hours’ treatment with Dacarbazine (800 µM), evaluated by MTT Assay. siScr: scrambled siRNA. The data are presented as the mean ± SD values (n ≥ 3). ***p < 0.001; ****p < 0.0001. Furthermore. silencing AKT with siRNA significantly reduced the GI50 value in 4C11 + cells, (Fig. 6D), indicating that these cells became more sensitive to Dacarbazine treatment and suggesting an enhanced therapeutic response compared with non-treated cells. When AKT was silenced in 4C11 + cells overexpressing p21, an alteration that enhances resistance, this effect was reversed, restoring resistance levels to those observed in 4C11 + WT cells, (Fig. 6E). This finding contrasts with the increased resistance driven by p21 overexpression alone. Collectively, these results indicate that the accumulation of cytoplasmic p21 in metastatic cell lines is mediated by its phosphorylation via the PI3K/AKT pathway.

Considering the increased expression of pAKT, p21 and p-p21 in 4C11 + cells, we next silenced AKT via its downregulation by siRNA (siAKT) (Fig. S3), resulting in a decrease in p21 phosphorylation, when compared to control (siScr) (Fig. 6B)24. We also treated 4C11 + cells with the AKT-upstream PI3K kinase inhibitor Wortmannin (Fig. S4), resulting in significantly reduced cytoplasmic p21 levels (Fig. 6C).

Discussion

Although p21 is widely known for its tumor-suppressor role25, here we demonstrate that high expression of the p21 protein is observed in various cancer types, including melanoma (SKCM), where this overexpression is associated with worse prognosis, independent of NRAS mutation26. A similar result was observed for p21 gene expression (CDKN1A), including in melanomas with BRAF mutations27,28. In melanoma, a few studies have reported increased p21 expression correlating with poor prognosis29–33, and it has been proposed as a predictive marker in these cases29,30,34–36.

Cytoplasmic p21 has been associated with oncogenic activities in some cancers6, while nuclear p21 is generally linked to tumor-suppressive functions. However, these roles are context-dependent, varying with tumor type and cellular conditions. The nuclear localization signal (NLS) of p21 can be altered by truncation37 or phosphorylation at consensus sites Thr145 and Ser14638, which can promote cytoplasmic localization39 and potentially contribute to pro-survival functions6. In this study, we observed a notable presence of p21 in the cytoplasm, specifically in metastatic melanoma tissues and murine cell lines.

We also explored potential pathways regulating p21 and identified an association with AKT1, which is frequently activated in melanoma40. Exploring the interaction between AKT and p21 caught our attention because the activation of AKT can phosphorylate p21 at the Ser146 residue41,42. The phosphorylation of p21 by AKT has been described to drive its stabilization, accumulation, and cytoplasmic localization in HER2-overexpressing cells, suggesting that p21 may acquire an anti-apoptotic function in the cytoplasm39,43–46. Our findings suggest that the PI3K-AKT pathway is an upstream mechanism regulating p-p21 cytoplasm levels in melanoma cells, and these results are aligned with a previous study showing that overexpression of cytoplasmic p21 mediated by AKT could promote mammary tumorigenesis and lung metastasis in mice47.

Notably, AKT downregulation increased the sensitivity of 4C11 + cells to Dacarbazine, highlighting the significant role of this signaling pathway in mediating chemoresistance in metastatic melanoma. These findings are aligned with the lack of response seen upon p21 overexpression in 4C11- cells due to the absence of phosphorylated AKT. The PI3K/AKT pathway has already been implicated in resistance to various drugs in different types of cancer, including Gemcitabine, Irinotecan, Etoposide, and Temozolomide38,48–51.

The mechanism by which p-p21 induces Dacarbazine resistance should be further studied. In normal cells, the cytoplasmic p21 acts as an apoptosis inhibitor by interacting and binding with procaspase-3 or apoptosis signal-regulating kinase 1 (ASK1)52–54. In colorectal cancer, the cytoplasmic p21 was identified as a mediator of 5-Fluorouracil (5FU) apoptosis resistance by inhibiting pro-apoptotic CHK2, a protein activated in response to DNA damage55,56. The Cisplatin and Paclitaxel resistance conferred by cytoplasmic p21 was shown in many cancer types, but in melanoma cells, it remains unknown8,57,58. Our findings unravel the oncogenic role of cytoplasmic p21 in melanoma driven by its phosphorylation by AKT, contributing to dacarbazine resistance. This insight opens new perspectives on therapeutic strategies to enhance melanoma responsiveness to dacarbazine.

Conclusion

In summary, our data provides evidence of an important mechanism linking the expression and cytoplasmic localization of p21 via phosphorylation by the PI3K/AKT pathway to either a worse prognosis in melanoma patients and drug resistance in metastatic cells. Given that constitutive activation of AKT is observed in about 60% of melanomas59 and that several PI3K/AKT inhibitors are currently in clinical trials60, it would be valuable to study their synergistic effects in combination with Dacarbazine therapy for metastatic melanoma. We found that high levels of p21 increase the survival of metastatic melanoma cells upon Dacarbazine treatment. Thus, our results suggest that p21 does not act as a classical tumor suppressor in melanomas but rather functions as an oncogene, preventing chemotherapy-induced cell death. Targeting cytoplasmic p21 could be a promising prognostic marker and a potential therapeutic approach to overcome chemotherapy resistance in metastatic melanomas.

Materials and methods

Murine melanoma cell lines

The murine cells used in this project, 4C11- (slow-growing and non-metastatic mesenchymal-like melanoma cells) and 4C11+ (highly proliferative and metastasis-prone melanoma cells) were previously established in our laboratory by subjecting non-tumorigenic melan-a melanocytes61 to sequential cycles of anchorage blockade, without the use of any chemical carcinogens or oncogene insertion16,62. Non-metastatic 4C11- and metastatic 4C11 + melanoma cell lines were cultured in RPMI 1640 pH 6.9 medium (Gibco, Carlsbad, CA, USA) supplemented with 5% fetal bovine serum (FBS, Gibco) and 1% penicillin-streptomycin (Gibco).

Human tissue samples

Skin biopsies (including nevus, dysplastic nevus, and melanoma) were collected from patients undergoing surgical procedures at the Department of Dermatology, University Hospital Erlangen. Written informed consent was obtained from all participants and/or their legal guardians. The study protocol was approved by the Institutional Ethics Committee of University Hospital Erlangen. Each specimen was cut in half: one half was sent for routine histopathological diagnostics, while the other was forwarded to our research laboratory. The skin samples were placed in transport medium and promptly transferred to the lab. They were first incubated in a washing buffer for 15 min, then placed into a cryomold, embedded in Tissue-Tek® O.C.T.™ Compound, snap-frozen in 2-methylbutane, and stored at − 80 °C. Cryosections of 5 μm thickness were prepared using a cryotome at − 25 °C and mounted on coverslips. Sections were air-dried, fixed in pure acetone for 10 s at room temperature, air-dried again, and further fixed at − 20 °C for 10 min. If not processed immediately, samples were stored at − 80 °C between the two fixation steps.

In vitro treatments

Melanoma cells were treated with different concentrations of dacarbazine (Sigma-Aldrich, Missouri, USA) for 48 h. 4C11 + cells were treated with 2 µM of Wortmannin (Cell Signaling, Darmstadt, Germany) for 24 h.

Bioinformatics analysis

The prognostic assessment of p21 was carried out using the softwares GEPIA2 (Gene Expression Profiling Interactive Analysis 2; http://gepia2.cancer-pku.cn/, accessed in 2025)63 and TRGAted (The Cancer Genome Atlas Transcriptome-Related Gene Analysis Tool; https://www.trgated.org/, accessed in 2025)64, which use gene and protein information collected from the TCGA and The Cancer Proteome Atlas (TCPA, http://tcpaportal.org), respectively65. Functional enrichment analysis was conducted to obtain spatial information on the protein interaction network on a subcellular level and its interaction partners. Data from the compartmentalized protein-protein interaction database (ComPPI, https://comppi.linkgroup.hu/protein_search) were used to capture PPI information for p21 and its subcellular location.

mRNA expression analysis (qRT-PCR)

RNA was isolated from melanoma cell monolayers (4C11- and 4C11+) using TRIzol® (Invitrogen, Carlsbad, CA, USA). cDNA was prepared from 1 µg of RNA using random hexamer primers and OligodT (Superscript III first-strand synthesis system for RT-PCR, Invitrogen, Carlsbad, CA, USA). Quantitative RT-PCR was performed using a Corbett Rotor-Gene 6000 detection system® with a Fast Rotor-Gene SYBR Green PCR Master Mix® (Qiagen, Dusseldorf, Germany). Specific primers were used as follows: mouse p21 (sense: 5′ GTC TTG CAC TCT GGT GTC TGA GC 3′, antisense: 5′ GCA GAA GAC CAA TCT GCG CTT GG 3′) and mouse β-actin (sense: 5’ CGA GGC CCA GAGCAAGAG AG 3′; antisense: 5′ AGG AAG AGG ATG CGG CAG TGG 3′).

Expression constructs and transfection

Murine p21 cDNA was amplified by RT-PCR from the 4C11 + cell line using the following primers: sense (5′ GGG AAT TCT CCA GAC ATT CAG AGC CAC 3′) and antisense (5′ CTT TTG GGA CTT CAC GGG TCA ATT GGG 3′), which contain EcoRI and HpaI restriction sites. The pLXSN66 retroviral vector and the murine p21 amplicon were digested with EcoRI and HpaI (Thermo Scientific, Massachusetts, USA) and ligated to make the pLp21SN. Amphotrophic PT67 packaging cells (Clontech, CA, USA) were seeded in six-well plates, transfected with pLp21SN via electroporation, and selected with 1 mg/ml geneticin (Gibco, Carlsbad, CA, USA) for two weeks. Supernatants containing the viral particles were collected, concentrated, and used to transduce 4C11- and 4C11 + melanoma cell lines in the presence of 8 µg/ml Polybrene (Sigma-Aldrich, Saint Louis, MO, USA) for 24 h, followed by medium replacement. Stable transductants were re-selected in 1 mg/ml geneticin, and p21 expression was validated by RT-PCR and Western blot.

RT-PCR

The PCR was performed with 1 µl of cDNA incubated with 0.5 µl of 10 mM dNTP, 0.75 µl of 50 mM MgCl2, 0.4 µl of each 20 mM primer, 0.1 µl of Platinum Taq enzyme, in a final volume of 25 µl, completed with DEPC water (Invitrogen – California, USA). The samples were kept in the thermocycler under the following conditions: 94 °C for 2 min, 94 °C for 30 s, 50 °C for 30 s, 72 °C for 30 s, 72 °C for 7 min and 4 °C indefinitely; from the 2nd to the 4th stage, there were 25 repetitions and then the subsequent stages continued. DNA sample buffer was added to the PCR products and they were subjected to electrophoresis in a 1% agarose gel in TBE with 0.5 µL of 1 mg/mL ethidium bromide, under an electrical voltage of 100 V in TBE. The gel was observed in a transilluminator, under UV light and photographed.

Western blotting

Total protein extracts were obtained using NP-40 lysis buffer (150 mM NaCl; 1.0% NP-40; 50 mM Tris-HCl pH 8.0) containing Halt™ Protease Inhibitor Cocktail (Thermo Scientific™). Proteins were separated by 12% SDS-PAGE gel and transferred to PVDF membranes. The membranes were blocked with 5% skim milk in TBS for 1 h. The membranes were incubated with primary antibodies specific to each protein p21 (Santa Cruz Biotechnology Cat# sc-6246, RRID: AB_628073), p-p21 (Santa Cruz Biotechnology Cat# sc-20220-R, RRID: AB_2077693), AKT (Santa Cruz Biotechnology Cat# sc-8312, RRID: AB_671714), p-AKT (Cell Signaling Technology Cat# 9018, RRID: AB_2629283) and corresponding secondary antibodies and developed using the SuperSignal West Pico Chemiluminescent Substrate kit (Pierce) with a chemiluminescence imager – UVITEC (Cambridge). The PCNA (Santa Cruz Biotechnology Cat# sc-56, RRID: AB_628110) and β-ACTIN (Santa Cruz Biotechnology Cat# sc-1616, RRID: AB_630836) expression was used as an endogenous control.

RNA interference

4C11 + cells were plated in 6-well plates and maintained in culture for 24 h. Nine uL of lipofectamine® RNAiMAX Reagent (Life Technologies, California, USA) were diluted in 150µL of Optim-MEM® medium (Life Technologies, California, USA). Thirty pmol of AKT-siRNA-pool (Life Technologies, Carlsbad, CA, USA) were diluted in 150µL of Optim-MEM® medium (Life Technologies, Carlsbad, CA USA). The solution with the lipofectamin® and siRNA was mixed and incubated for 5 min at room temperature. After this period, the solution containing the siRNA was added to the wells containing 4C11 + cell line. The cells were maintained in culture for 48 h and were then analyzed for AKT protein expression (Cell Signaling Technology Cat# 9018, RRID: AB_2629283) by Western blot. For the control, the cells were transfected with non-targeting negative control siRNA (Life Technologies) as described above.

shRNA

To obtain stable clones of 4C11 + cell line knocked down for p21, cells were seeded and transduced with the MISSION shRNA Lentiviral Transduction Particles® (Sigma-Aldrich, Saint Louis, MO, USA) containing 5 different sequences, which were previously validated by the company, to knockdown the mouse p21 gene. Cells were incubated in a medium containing 5% FBS, 8 µg/mL Polybrene, and shRNA-p21 lentivectors at a MOI of 0.5 for 6 h. Then, fresh medium containing 5% FBS was added, and 24 h after the beginning of the transduction, the medium was replaced with a fresh one containing 5 µg/mL puromycin (Gibco, California, USA) for selection for 15 days. These experiments were conducted at the Center for Gene Therapy Investigation (CINTERGEN) of the Universidade Federal de São Paulo (Laboratory Biosafety level II).

Immunoblotting

Whole cell extracts were prepared in RIPA buffer (50 mM Tris pH 8.0, 150 mM NaCl, 1% Nonidet P-40, 0.1% SDS, and 0.5% sodium deoxycholate), containing 2 µg/µL aprotinin, leupeptin and pepstatin (Sigma-Aldrich, Saint Louis, MO, USA) and PMSF 10 mM (Amresco, Cleveland, OH, USA). Alternatively, fractionated cell extracts were obtained with Subcellular Protein Fractionation Kit for Cultured Cells® (Thermo Scientific), resulting in cytoplasmic and nuclear proteins. Protein levels were determined by Western blot analysis by following established protocols using the ECL detection system (SuperSignal West Pico Chemiluminescent Substrate/Pierce Chemical – Illinois, USA). The membranes were probed with primary antibodies directed to p21 (C-19) (Santa Cruz Biotechnology Cat# sc-397, RRID: AB_632126), phospho-p21 (Thr145) (Santa Cruz Biotechnology Cat# sc-20220-R, RRID: AB_2077693), PCNA (Santa Cruz Biotechnology Cat# sc-56, RRID: AB_628110), γ- tubulin (Cell Signaling Technology Cat# 5886, RRID: AB_10836184), Anti-Ser473-phosphorylated AKT (Cell Signaling Technology Cat# 9018, RRID: AB_2629283), anti-AKT (Santa Cruz Biotechnology Cat# sc-8312, RRID: AB_671714) and anti-GAPDH (Santa Cruz Biotechnology Cat# sc-47724, RRID: AB_627678). Secondary antibodies were goat anti-mouse (KPL, Cat# 04–18-06) or anti-rabbit IgG coupled to horseradish peroxidase (KPL, Cat# 04–15-06). Western blot imaging was performed with ImageJ software (Version 1.53t, National Institutes of Health, Bethesda, MD, USA; https://imagej.nih.gov/ij/).

Immunofluorescence assay

Cells were seeded on a chambered coverslip (µ-Slide 8 Well ibiTreat - ibidi) and cultured for 48 h. After this time, cells were fixed in paraformaldehyde 4%, permeabilized in 0.1% Triton x-100 and blocked with bovine serum albumin (BSA) 3% in PBS. The cells were then stained with the p21 antibody (Abcam EPR18021) diluted in 3% BSA/PBS for 48 h followed by 1 h incubation with the secondary antibody conjugated to Alexa Fluor® 488(ThermoFisher - #A-11008). The slides were counterstained with Hoechst (Invitrogen) as a nuclear marker and examined under a fluorescence microscope (Nikon eclipse Ti-S, Nikon, Tokyo, Japan) connected to a photographic camera (Retiga 2000R, QImaging, Surrey, Canada). For dacarbazine treatment, cells were seeded, led to adhere for 24 h and then treated as described below. After 48 h of treatment, the standard immunofluorescence protocol was followed.

Immunohistochemistry

Immunohistochemical studies were performed using the avidin-biotin complex immunostaining method and the automated immunohistochemistry slide staining system by Ventana NexES (Ventana Medical System, Strasbourg, France). Paraffin-embedded tissue sections were deparaffinized and dehydrated. For antigen retrieval, pretreatment was performed by microwave heating in 1 mmol/L sodium citrate buffer (30 min, 600 W, pH 6.0). Incubation of each sample with anti-p-p21 was conducted at room temperature for 12 h and followed by PBS-washing. Positive immunohistochemical reactions were revealed using the iVIEW DAB Detection Kit (Ventana, Benchmark XT system, Ventana Medical Systems Inc., Tucson, AZ, USA) as a chromogen. Specimens were counterstained with hematoxylin and mounted with DEPEX. Samples were examined by two independent reviewers blinded to other data. In total, seventeen human tissue samples were analyzed, being 5 nevi, 5 dysplastic nevi, 3 primary melanoma and 4 metastatic melanoma samples.

For immunohistochemistry (IHC) staining, human tissue from nevi and metastatic melanoma (MM) was routinely fixed in neutrally buffered formalin for 24 h and was then dehydrated and embedded in paraffin. FFPE samples were then deparaffinized and antigen retrieval was achieved in Tris-EDTA buffer pH = 9.0 at 100 °C for 30 min. For antigen detection, the LSAB method (Dako REAL Detection Systems) was used, performed by the Dako Autostainer Plus. Before incubation with the primary antibody the specimen was incubated with 5% NGS (Dako, Hamburg, Germany) in PBS (PAA, Pasching, Austria) for 30 min in order to block unspecific binding sites. For the immunohistochemical detection of p21, we used anti-p21 (Abcam, clone: EPR362, ab109520).

The expression of p21 was measured using a semi-quantitative method. The two IHC reaction parameters used were the percentage of cells with a positive reaction and the intensity of p21 reaction. Both parameters were determined for the nucleus and cytoplasm. The immunoreactive score (IRS) was used to evaluate the above parameters. IRS is obtained by multiplying the staining intensity (0 = negative, 1 = weak, 2 = moderate, and 3 = strong) by the percentage of positive cells (0 = negative, 1 = 10% positive, 2 = 11–50% positive, 3 = 51–80% positive, and 4 = > 80%). The ultimate IRS is a product of the multiplication of the above parameters, ranging between 0 and 12 points.

Drug sensitivity assay

To evaluate the effect of dacarbazine treatment and determine melanoma cells growth inhibitory concentration of 50% value (GI50), cells were cultured in 96-well plates, treated with serial dacarbazine concentrations (mouse cells: 50, 100, 200, 400, 800 and 1200 µM; human cells: 400, 800 1200, 1600 and 2000 µM) and incubated at 37 °C in a humidified atmosphere with 5% CO2 for 24 and 48 h. After this period, cell viability was evaluated with a standard methyl thiazol tetrazolium (MTT) assay (Sigma-Aldrich, St. Louis, MO, USA). Cell viability was determined by measuring the optical absorbance of cells at 620 nm wavelength and normalizing the values to the corresponding controls. The values obtained were used to estimate the GI50 value by linear regression using GraphPad Prism software v.5.

Cell cycle analysis

Cells from subconfluent monolayers were harvested, washed in PBS and fixed in 70% ice-cold ethanol overnight. After this period, cells were centrifugated and resuspended in 100 µg/mL Ribonuclease A (Invitrogen, Carlsbad, CA, USA) for 5 min at room temperature, followed by incubation with 50 µg/mL propidium iodide (Sigma-Aldrich, St. Louis, MO, USA) in the dark for 30 min at 4 °C. DNA content in each sample was analyzed by flow cytometry using a Becton Dickinson FACS Calibur (Becton-Dickinson, San Jose, CA, USA). Ten thousand events were evaluated per assay.

Statistical analysis

All tests were conducted in biological triplicate. Data was analyzed in GraphPad Prism software (Version 10.0, GraphPad Software, LLC, San Diego, CA, USA; https://www.graphpad.com/). The level of significance was set at p < 0.05 after a One-Way ANOVA test followed by the post-hoc Tukey.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary Material 1 (49.1KB, tif)
Supplementary Material 2 (154.2KB, tif)
Supplementary Material 3 (151.9KB, tif)
Supplementary Material 4 (1.2MB, tif)
Supplementary Material 5 (1MB, tif)
Supplementary Material 6 (1.9MB, tif)
Supplementary Material 7 (266.9KB, tif)
Supplementary Material 8 (2MB, tif)

Acknowledgements

This work was supported by grants from the Fundação de Amparo à Pesquisa do Estado de São Paulo [2010/08167-3 and 2011/15840-9 to GNC; 2013/04829-0 and 2016/09179-1 to ACM; 2010/18484-6 ATC; 2023/17737-8 BCBT; 2012/08776-5, 2014/13663-0, 2022/00322-7 and 2023/17621-0 to MGJ], Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) [to MGJ], and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) [to MGJ]. We acknowledge the supporting funding by the Emerging Fields Initiative Cell Cycle in Disease and Regeneration (CYDER) of the Friedrich-Alexander University Erlangen-Nürnberg, Germany, to RSS.

Author contributions

Conceived and designed the experiments: G.N.C., A.C.M., A.T.C., S.W.H., R.S.S., M.G.J. Carried out the biological experiments: G.N.C., A.C.M., A.T.C., F.M.M., A.S.M., R.S.S. Conducted the bioinformatics analysis: H.A., M.G.J.. Analyzed the data: G.N.C., A.C.M., H.A., D.K.A.F., A.T.C., R.F., S.G., C.O., R.S.S., S.W.H., M.G.J. Contributed with reagents and materials: C.O., R.S.S., S.W.H., M.G.J. Wrote the paper: GNC, ACM, HA, BCBT, DKAF, MGJ. All authors read and approved the final manuscript.

Data availability

All data generated or analyzed during this study are included in this published article and its supplementary information files. Further information is available from the corresponding author upon reasonable request.

Declarations

Competing interests

The authors declare no competing interests.

Ethical approval

This study involved the analysis of previously collected human tissue samples. All samples were de-identified and obtained in compliance with institutional ethical guidelines. The use of these samples for research purposes was approved by the Ethik-Kommission of the FAU Erlangen-Nürnberg. No new samples were collected specifically for this study, and no animal experiments were performed.

Footnotes

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Gabriela Nana Colaneri, Ana Carolina Monteiro, Beatriz Cristina Biz Tonin, Hátylas Azevedo and Débora Kristina Alves-Fernandes contributed equally to this work.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Material 1 (49.1KB, tif)
Supplementary Material 2 (154.2KB, tif)
Supplementary Material 3 (151.9KB, tif)
Supplementary Material 4 (1.2MB, tif)
Supplementary Material 5 (1MB, tif)
Supplementary Material 6 (1.9MB, tif)
Supplementary Material 7 (266.9KB, tif)
Supplementary Material 8 (2MB, tif)

Data Availability Statement

All data generated or analyzed during this study are included in this published article and its supplementary information files. Further information is available from the corresponding author upon reasonable request.

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