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BRAFV600E: Comparison
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Please note this is a comparison between Version 2 by Dean Liu and Version 1 by Mourad Zerfaoui.

Despite some successes of selective anti-BRAFV600E inhibitors, resistance remains a major challenge. The aim of our study is to determine the role of nuclear BRAFV600E and its newly identified partner, HMOX1, in melanoma aggressiveness and drug resistance. 

  • melanoma
  • HMOX-1
  • nuclear BRAFV600E
  • aggressiveness
  • vemurafenib resistance

[1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20][21][22][23][24][25][26][27][28][29][30][31][32][33][34][35][36][37][38][39][40][41][42]1. Translocation of BRAFV600E into the Nucleus Upregulates HMOX-1 Expression in Melanoma Cell Lines

To determine the differential response of melanoma cells towards PLX-4032 treatment, two BRAFV600E melanoma cell lines, parental and resistant (A375P and A375R) cells, were subjected to 10, 100, 1000, 2000, and 5000 ng of PLX-4032 treatment for 3 days. MTT assay revealed that cell viability starts to decline in A375P at 10 and 100 ng and is drastically reduced at the range of 1–5 μM of the drug. However, A375R showed a substantial resistance up to 2 μM of vemurafenib (Figure 1A). This experiment clearly shows that A375R cells are resistant to vemurafenib treatment. In a previous study, wresearchers demonstrated that the conventional nuclear BRAFV600E localization was associated with melanoma aggressiveness and vemurafenib resistance . In order to explore whether BRAFV600E  has a nuclear localization in A375R cells, weresearcher monitored the cellular localization after 24 h of cell synchronization followed by serum stimulation. The A375R cells presented a pronounced nuclear translocation of the mutant kinase compared to the parental cells after only 3 h of serum stimulation (Figure 1B), confirming a similar finding with a SkMel-28 cell line stimulated with serum or epidermal growth factor (EGF) in our previous study . BRAFV600E  nuclear localization was validated by cell fractionation of the SkMel-28 cells (Figure S8). Taken together, these results suggest that nuclear BRAFV600E localization could be a determinant factor in the resistance to vemurafenib of A375R cells. In our effort to identify the partner(s) of nuclear BRAFV600E  in resistant melanoma cells, we took a proteomics approach. WeResearchers examined the differential expressed proteins in A375P and A375R cells using the Human XL Oncology Array, as recommended by the manufacturer’s protocol (Cat# ARY026; R&D Systems, USA). The full list of the 84 cancer-related proteins of the array is provided in the supplementary material (Figure S4). The screening of the protein lysates of resistant and parental cells identified one downregulated and 17 upregulated specific melanoma-related proteins. Among the 17 upregulated proteins, HMOX-1, a partner to nuclear BRAFV600E, was the most upregulated (10.3-fold) protein (Figure 1C). The high expression level of the HMOX-1 protein in A375R cells compared to A375P cells was confirmed by Western blot analysis (Figure 1D), PCR, and bioinformatic analysis. These data suggest that HMOX-1 may be the most prominent nuclear BRAFV600E partner to reduce the efficiency of vemurafenib treatment and promote cell proliferation.
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Figure 1. Protein expression of HMOX-1 in parental (P) and resistant A375 (R) cell lines. (A) MTT assay was assessed on parental and resistant A375 cell lines after treatment with different concentrations of vemurafenib for 3 days. This experiment was performed in triplicate and independently repeated three times. (B) IF with BRAFV600E antibody. Cells were synchronized and then stimulated with FBS for 3 h. Scale bar = 20 μm (C) Human XL oncology array (relative levels of 84 human cancer-related proteins) incubated with total protein extractions collected from parental and resistant A375 cell lines. (D) HMOX-1 protein levels in parental A375 cells and resistant A375 cells.

2. HMOX-1 Gene Expression Is Associated with an Aggressive Phenotype, a Worse Prognosis, and Resistance to BRAF Inhibitor

HMOX-1 association with aggressive features and survival was studied in 406 TCGA melanoma samples. A higher expression of HMOX1 was correlated with more aggressive lymph node stages (N2 and N3, more than 2 lymph nodes invaded) compared to the absence of lymph node metastasis (N0) (p = 0.005).
Moreover, HMOX-1 overexpression was associated with lower disease-free survival (p = 0.0004), as depicted in (Figure 2A). Samples with a high expression of HMOX-1 were 19% more likely to present a disease relapse at 5 years compared to samples with lower expression. A Cox regression model validated the previous survival analysis, where high HMOX-1 was associated with a higher rate of recurrence (hazard ratio (HR) = 1.55, 95% CI = 1.21–1.99, p = 5.55 × 10−4).
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Figure 2. Transcriptomic analysis on public datasets highlighted the role of HMOX-1 in aggressiveness and BRAF inhibitor resistance in melanoma.
Interestingly, HMOX-1 expression also significantly stratified the patient outcome in BRAFV600E mutant melanoma samples (log-rank p = 0.03), as shown in Figure 2B (HR = 1.54, 95%CI = 1.05–2.27, p = 0.027).
Finally, public transcriptomic databases were used to validate the role of the HMOX-1 gene in response to BRAF inhibitor treatment in melanoma xenograft models and patients.
HMOX-1 expression was collected from a cohort of A375 xenograft mice treated with the BRAF inhibitor. As shown in Figure 2C, HMOX-1 was higher in resistant samples than in sensitive samples (p = 0.005). Furthermore, in an in vitro model of an A375 cell line treated with a BRAF inhibitor, HMOX1 was also found to be upregulated in the resistant clones, harboring a spontaneous NRAS mutation, compared to the sensitive clones (p = 0.08). This observation was confirmed in a patient cohort treated with BRAF inhibitors. HMOX-1 expression was higher in resistant samples than in sensitive samples (p = 0.012) (Figure 2D).

3. Translocation of BRAFV600E to the Nucleus Promotes HMOX-1 Upregulation in a Xenograft Mouse Model of Melanoma

First, we constructed a lentiviral vector in which BRAFV600E was inserted in-frame with a sequence encoding 3 NLS domains followed by DsRed2, which encodes a fluorescent protein. The mutant kinase, NLS-BRAFV600E/DsRed2, was localized exclusively in the nucleus (Figure 3A, right). BRAFV600E was also cloned into the DsRed2 vector without NLS, to be localized exclusively in the cytoplasm, as shown in Figure 3A (left). A vector expressing NLS/DsRed2 was also generated to serve as a control. Melanoma MV3 cells, which express WT BRAF, were transduced with the three different viral vectors. After antibiotic selection, the expression of BRAFV600E was confirmed by immunofluorescence analysis (Figure 3A).
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Figure 3.
 Nuclear BRAF
V600E
and HMOX-1 expression in xenograft mouse and human melanoma tissue cores. 
Next, researchers used a mouse xenograft model of melanoma to examine whether overexpression of nuclear BRAFV600E upregulates HMOX-1 protein expression. Stable clones of melanoma MV3 cells expressing BRAFV600E or NLS-BRAFV600E were subcutaneously inoculated into nude female mice (n = 5 for each group), and the animals were monitored for 5 weeks. Figure 3B and Figure S10 show that tumors generated by the BRAFV600E- and NLS-BRAFV600E-expressing MV3 cells were greater in size and displayed higher HMOX-1 expression compared to the wild-type control, as assessed by immunohistochemistry with a specific antibody to the mutant protein. As expected, Figure 3B shows that the NLS-expressing cells generated the largest size of tumor and much higher HMOX-1 protein. In BRAFV600E-injected mice (n = 5), HMOX-1 was only detected in 2 out of 5 (40%), while in NLS-BRAFV600E-injected mice (n = 5), HMOX-1 was detected in 4 out of 5 mice (80%). This result suggests that HMOX-1 protein expression could be mediated by the nuclear BRAFV600E.

4. The Localization of BRAFV600E and HMOX-1 in Human Melanoma Samples

To test our hypothesis that nuclear BRAFV600E enhances HMOX-1 protein expression, we analyzed 30 tissue cores of human cutaneous and metastatic melanomas (15 cores each) for protein expressions of BRAFV600E and HMOX-1 using the IHC technique (Figure 3C). The expression of nuclear BRAFV600E was 33.3% (5 out of 15 cores) in both cases. Interestingly, 40% of metastatic cores had HMOX-1 expression in the nucleus (6 out of 15 cores) compared to 20% of cutaneous cores (3 out of 15 cores). All metastatic cores with nuclear BRAFV600E staining were positive for nuclear HMOX-1, while only 60% of primary cores with nuclear BRAFV600E localization had nuclear HMOX-1 staining. Researchers next examined whether nuclear localization of BRAFV600E affected the pattern of ERK and Akt phosphorylation in mouse embryonic fibroblasts (MEFs). We used these MEFs because they are BRAF knockout. Figure 3D shows that NLS-BRAFV600E-harboring MEFs compared to control cells displayed higher levels of ERK phosphorylation after 6 h and a persistent pERK up to 12 h of treatment with PLX-4032. Treatment with PLX-4032 of BRAFV600E-harboring MEF cells induced transient feeble phosphorylation of ERK at 12 h. After 0.5 to 12 h, the phosphorylation pattern of Akt in NLS-BRAFV600E-expressing cells remained much higher than those observed in similarly treated BRAFV600E-expressing cells. Interestingly, at 48 h of PLX-4032 treatment, the AKT pathway is reactivated in NLS-BRAFV600E-expressing cells. This is a confirmation of the phosphoERK and phosphoAKT patterns found in our previous study using MV3 cells .

5. Suppression of HMOX-1 Has an Anti-Proliferative Effect in Resistant Melanoma Cells

We first established stable A375R cell lines with HMOX-1 knockdown by using HMOX-1 shRNA lentiviral particles, as recommended by Santa Cruz’s protocol. Next, we selected stable clones that express shRNA via puromycin dihydrochloride. To verify HMOX-1 knockdown and its effect on cell viability, we performed Western blot analyses and an apoptosis assay (Figure 4A). The HMOX-1 knockdown, compared to the parental A375R cells, did not induce apoptosis before the treatment with vemurafenib. In line with a previous study , BRAFV600E knockdown reduced HMOX-1 protein expression in A375R cells (Figure 4A). In a wound-healing assay, we found that the silencing of HMOX-1 reduced cell proliferation by about 3-fold (Figure 4B). Since the number of cells in the denuded area rises either due to immigration of cells from the wound edge or by mitosis of the migrated cells, the contribution of cell migration to the increase of the cell population in the denuded space was determined after inhibiting cell division with an antimitotic agent, Vindoline. No difference was observed before or after Vindoline treatment. Within the 16 h of the wound healing assay, the denuded area was mostly covered up by the migrating cells. Moreover, invasion ability was dramatically reduced in the HMOX-1 knockdown cells and practically eradicated in the BRAF knockdown cells (Figure 4C). The colony formation assay with A375R showed a reduced ability of a single cell to grow into a colony when HMOX-1 was knocked down (Figure 4D). Additionally, drug-sensitive A375 cells expressing NLS-BRAFV600E were more resistant to vemurafenib treatment and were able to grow into colonies compared to control parental cells (Figure 4E). Interestingly, the MTT assay revealed that cell viability in A375R cells was clearly diminished after treatment with the specific HMOX-1 pharmacologic inhibitor OB-24 in combination with vemurafenib, suggesting that HMOX-1 is the predominant mediator of cell aggressiveness in the presence of nuclear BRAFV600E (Figure 4F). These data show that HMOX-1 can promote melanoma cell proliferation. The validation of this finding with a different melanoma cell line was performed with WM983B cells.
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Figure 4.
Suppression of HMOX-1 has an anti-proliferative effect in a resistant A375 (R) cell line. 

6. Suppression of HMOX-1 Reduces the Number and Size of Tumors in a Preclinical Melanoma Model

Furthermore, we explored the role of HMOX-1 in promoting melanoma aggressiveness using a xenograft mouse model. The results show that the number, size, and weight of tumors significantly decreased in mice receiving HMOX-1-knockdown cells.
A375R cells with HMOX-1 knockdown or the parental cells were subcutaneously inoculated into nude female mice. Two groups of mice were used: n = 9 for the A375R cells and n = 11 for the HMOX-1 knockdown cells. Tumors started to develop after 19 days and were monitored twice a week for an extra 14 days. The results show that almost 90% of the A375R-injected mice developed a tumor (8 out of 9) compared to about 20% of the HMOX-1 knockdown mice (2 out of 11), as shown in Figure 5A,B. The growth rate of the tumors generated by HMOX-1 knockdown A375R cells was markedly slower than those expressing the A375R parental cells (Figure 5C). In addition, the average weight of tumors dropped in HMOX-1-knockdown-injected mice from 1.92 to 0.25 g (Figure 5D,E).
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Figure 5. HMOX-1 knockdown reduces tumor growth in a melanoma mouse model. Mice were subcutaneously injected with A375R or A375R with HMOX-1 knockdown (2 × 106 cells/mouse). (A) Mice with visible tumors of each group. (B) Number of tumors per group. (C) Tumor size was monitored twice a week for 4 weeks. (D) Representative tumor images were taken from the HMOX-1 knockdown group and the control group. (E) The tumor weight was recorded at the end of the experiment (day 25).

7. Pathways Analysis Revealed the Functional Role of HMOX1 in the BRAF Inhibitor Resistance Process

KEGG pathways analysis was independently performed on the A375 xenograft database and the melanoma patient database, and 161 signaling pathways were common in both databases (Figure 6A). Interestingly, three pathways contained HMOX-1, “ferroptosis”, “fluid shear stress and atherosclerosis”, and “hepatocellular carcinoma.” Ferroptosis is a newly discovered iron-dependent cell death process. Fluid shear stress and atherosclerosis are diseases characterized by the deregulation of PI3-AKT signaling, focal adhesion, NF-kappa B signaling, and MAPK signaling pathways. Finally, hepatocellular carcinoma is a cancer involving calcium, PI3K/AKT, p53, TGF-beta, Wnt, and MAPK signaling pathways. Interestingly, ferroptosis was the pathway with the highest enrichment score in xenografts and the only one to show a significant correlation with HMOX-1 in both databases (xenografts, rho = 0.79, p = 0.02; patients, rho = 0.39, p = 0.002). These observations suggest that HMOX-1 contributed significantly to the ferroptosis process during BRAF inhibition therapy resistance. From the three pathways containing HMOX-1, 36 genes were identified as significantly deregulated in the resistant models, compared to sensitive samples, in both databases. The 36 genes were enriched in the PI3K-AKT pathway (AKT1, AKT3, PI3KR1, PRKAA1, RPS6KB1, RPS6KB2), the MAPK pathway (KRAS, SOS1, MAPK13, MAPK3, MAPK7, MEF2A), iron metabolism (HMOX1, FTL, ATG5, STEAP3), the chromatin remodeling complex (ARID1B), cell cycle (CDK4, CDKN2A, E2F1, E2F2), the NF-kappa B pathway (CHUK, ITGAV), the WNT pathway (DVL2, FZD2), TGF-beta pathway (SMAD4, TGFBR1), and the antioxidative response (HMOX-1, GSMT3, KEAP1, SQSTM1). A correlation matrix (Figure 6B) identified a cluster of genes strongly positively associated with HMOX-1 (FTL and STEAP3, two ferroptosis genes, FZD2, MAPK13, and SQSTM1; highlighted with the green square on the heatmap) and one gene strongly negatively correlated (CHUK, with a red square on the heatmap). A network analysis emphasized the interdependence of the three pathways involving HMOX-1 (Figure 6C). The highest correlations observed with HMOX-1 in patients with melanoma were with FZD2 (rho = 0.54), KEAP1 (rho = 0.59), MAPK13 (rho = 0.68), the “ferroptosis” gene FTL (rho = 0.58), and CHUK (rho = −0.50).

The data suggest that HMOX-1 actively participates in BRAF inhibitors’ resistance process, linking different signaling pathways that are known to be involved in therapy resistance (PI3K, MAPK, TGF-beta, and Wnt pathways).

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Figure 6. Pathway analysis revealed a key role of HMOX-1 in BRAF inhibitor therapy resistance in the xenograft model and in melanoma patients. (A) Dotplot representing the enrichment score obtained for the common pathways identified in the xenograft model and melanoma patients. (B) Heatmap representing the correlation matrix between the 36 genes involved in the 3 selected pathways (ferroptosis, fluid shear stress and atherosclerosis, and hepatocellular carcinoma) common in the xenograft model and melanoma patients. Values are from the patient dataset. (C) Network representation of the 36 genes (green nodes) involved in the 3 selected pathways (light yellow nodes), with their Spearman correlation as edges. Dark red ellipse, genes belonging to “fluid shear stress and atherosclerosis”; orange, genes belonging to “ferroptosis”; blue ellipse, genes belonging to “hepatocellular carcinoma”; thicker edges, correlation related to HMOX1; blue edges, negative correlation; red edges, positive correlation; edge transparency proportional to correlation value; blue node border, negative correlation with HMOX1; red node border, positive correlation with HMOX1. Correlation values are from the patient dataset.

  1. Discussion

4. Discussion

BRAF is a proto-oncogene (also known as v-raf murine sarcoma viral homolog B1) that belongs to the Raf kinase family [28]. BRAFV600E mutations are linked with several types of tumors, where they activate the MAPK signaling pathway constitutively, resulting in uncontrolled cell proliferation and survival [29]. Transport across cell compartments is required for functional regulation and for diversity in the cell, but aberrant nucleocytoplasmic trafficking has been implicated in cancers such as thyroid, melanoma, and others [30–33] [30][31][32][33][34]. We reported for the first time in our previous study [24] a strong association between nuclear BRAFV600E expression and aggressive clinicopathologic features, including overall stage, tumor stage, lymph node metastasis, depth of invasion, Clark level, mitotic activity, and ulceration. In the same study, we reported that the SkMel-28 cell line (BRAFV600E) displays a dynamic nuclear localization of BRAFV600E, regulated by the removal or addition of FBS or epidermal growth factor (EGF) to the media. In contrast, and as we report in the current study (Figure S11), WT BRAF does not show any nuclear localization by immunofluorescence in the SKMel-202 melanoma cell line. In the literature, two studies mentioned a small fraction of WT BRAF in the nucleus. In the first study, an exogenous truncated BRAF transfected in NIH3T3 cells was used, and this artificial system cannot be generalized or applied to the endogenous BRAF. In the second study, Shin et al. used a non-cancer C2C12 myoblast cell line and found a very tiny fraction of WT BRAF in the nucleus compared to a strong nuclear presence of BRAFV600E [34,35][34][35]. Our hypothesis is that WT BRAF activation requires external growth factors, and its localization is totally cytoplasmic. The constitutively active BRAFV600E is independent of external stimulus, and the nuclear localization may provide the BRAFV600E with a sheltering mechanism against inhibitors, possibly through a novel and unexpected collaboration with overexpressed HMOX1. However, mechanistic studies are required for the validation and explanation of our previous findings. In this study, we report that A375R melanoma vemurafenib-resistant cells display a nuclear localization of BRAFV600E after synchronization and stimulation with the FBS. A 10-fold upregulation of HMOX-1 protein is observed in A375R cells with nuclear localization of BRAFV600E compared to A375 parental cells that have cytoplasmic BRAFV600E. Surprisingly, HMOX1 upregulation in A375R or WM983C cell lines is not the consequence of NRF2 protein overexpression (Figure S7). In another study, it was hypothesized that the existence of a possible HMOX-1 reserve in the form of mRNA to be transformed into protein when necessary [36]. Additionally, we show that HMOX-1 upregulation crucially limits the efficacy of the BRAF inhibitor PLX4032 and is important in promoting cell viability and aggressiveness. Our results provide the first evidence that nuclear BRAFV600E plays a critical role in the regulation of HMOX-1 protein overexpression in resistant melanoma cells. A recent study with a WT BRAF cell line (MeWO) showed neither basal expression nor induction of HMOX-1 after treatment with PLX4032 [37]. HMOX-1 is one of the most important mechanisms of cell adaptation to stress, and chemoand radiotherapy both fundamentally stimulate HMOX-1 expression [38]. However, the transcriptional or post-transcriptional mechanisms controlling HMOX-1 expression in response to cytotoxic stress remain elusive. This study extends our understanding by identifying nuclear BRAFV600E as a potential player in HMOX-1 expression. The nuclear translocation and the molecular mechanism of nuclear BRAFV600E in promoting tumor progression and resistance is still poorly understood. Thus, to the best of our knowledge, the present study provides the first evidence of a possible role of nuclear BRAFV600E/HMOX-1/ AKT in melanoma resistance. Figure 7 is a hypothetical model of how melanoma cells with nuclear BRAF and high HMOX-1 expression can activate the AKT pathway and resist vemurafenib treatment.

Using bioinformatics analysis, we determined a relationship between HMOX-1 upregulation and disease-free survival (DFS). Indeed, patients with high HMOX-1 have a 19% lower chance of survival after five years than patients with lower HMOX-1 expression. The resistance to BRAFV600E inhibitors is also correlated with the high expression of HMOX-1 in a melanoma mouse model and in a melanoma patient database.To further corroborate our bioinformatics results, cells expressing nuclear and cytoplasmic BRAFV600E were subcutaneously injected into nude mice. Nuclear BRAFV600E cells were able to induce more HMOX-1 protein expression and phosphorylated Akt than cells harboring cytoplasmic BRAFV600E. Following the same pattern, the human metastatic melanoma cores expressed more nuclear HMOX-1 when BRAFV600E was detected in the nucleus. This nuclear HMOX-1 expression in nuclear BRAFV600E cores was more prevalent in metastatic malignant melanoma specimens. Remarkably, the intranuclear localization of the kinase raised the resistance of melanoma cells to drug therapy, and this was supported by reactivation of the Akt pathway as an alternative pathway for cell survival. Thus, targeting mediators of Akt activation could be a possible option for intervening with drug resistance in metastatic melanoma [39]. To address whether HMOX-1 reduction could interfere with melanoma aggressiveness, compromised HMOX-1 cell lines have been established and are correlated with melanoma-decelerated cell proliferation and invasion rate along with slower colony formation. Furthermore, an in vivo study showed a significant decrease in the number, size, and weight of tumors after knocking down HMOX-1. Remarkably, the A375 sensitive cells developed resistance when expressing NLS-BRAFV600E. Interestingly, the pathway analysis showed that ferroptosis is highly associated with HMOX-1 expression. Ferroptosis is an iron-dependent cell death process characterized by the accumulation of lipid peroxides and is genetically and biochemically different from apoptosis. It is worth noting that nuclear factor (erythroid-derived 2)-like 2 (NRF2) activation and the subsequent deregulation of iron signaling in cancers have been implicated in cancer development. Constitutive NRF2 activation and NRF2-dependent upregulation of the iron storage protein ferritin (FTL) or HMOX-1 can lead to enhanced proliferation and therapy resistance [40–42][40][41][42].

Figure 7. Hypothetical model of the melanoma resistance to vemurafenib. Melanoma cells with cytoplasmic BRAFV600E and low HMOX-1 expression are more sensitive to vemurafenib treatment than cells with nuclear BRAFV600E, high HMOX-1 expression, and an activated AKT pathway.

  1. Conclusions

5. Conclusions

In conclusion, there is increasing interest in a possible means of inhibiting HMOX-1 expression in order to improve the sensitivity of cancer cells to BRAFV600E inhibitors. In this context, the nuclear BRAFV600E/HMOX-1/AKT axis is associated with melanoma aggressiveness. The cytoplasmic BRAFV600E expression had a modest effect in promoting HMOX-1 overexpression and was not correlated with advanced disease. Therefore, the combination of specific HMOX-1 inhibitors with BRAFV600E inhibitors is anticipated to reduce melanoma aggressiveness and improve BRAFV600E inhibitor-based therapies.

 

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