Overview of Therapies for Melanoma: Analytical Essay

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Abstract

Cancer treatments have been advancing over the previous decade because of the urgent need to identify new therapeutics. Melanoma accounts for only 4% of all dermatologic cancers, but it accounts for almost 80% of the deaths recorded from skin cancer because of its ability of deep invasion and spreading as well as Melanoma tumor cells were able to effectively block chemotherapy which arose the need for new Recent studies has shown that kinases play a crucial role in various types of cancers.

Protein kinases are key regulators of cell function That consist of one of the largest most functionally diverse gene families1. Kinases are enzymes that function by transferring a phosphate group to a protein while it removes another phosphate group from another protein (phosphorylation)2 using this mechanism kinases maintain cellular functions and complex functions such as the cell cycle. It’s crucial role in cell proliferation, survival, and migration when activated as well as many kinase mutations are associated with cancer initiation, promotion, progression, and recurrence. Which made kinase as the key target for developing cancer therapeutics using different kinase inhibitors.

Studies of oncogenic signaling pathways in human cancer has identified molecular targets for cancer therapeutics such as EGFR HER-2/Neu, JAK, Src, Rho, and many other. Kinase Inhibitors has shown a potent effect against cancer proliferation, survival, and metastases. Inhibiting Rho kinase which plays a major role in the organization of actin cytoskeleton and activates myosin II-dependent contraction of actomyosin which regulates cell-to-cell contact and cell migration using AT13148 and CCT12925 reduced metastasis and inhibited cell proliferation3. Inhibiting Src activity using PD180970 and PD166285 Inhibited c-SRC and Stat3 which lead to inhibition of melanoma cell growth and Bcl-XL and Mcl-1 expression4. Using organometallic kinase inhibitor for Mdm2 caused high expression of p53 which induced apoptosis. Using a combined therapy that targets kinases by using multiple kinase inhibitors would stop melanoma cells from spreading and proliferating.

Melanoma frequently metastasizes through lymph nodes4, making it really hard to treat using surgical treatment, chemotherapy, and radiotherapy because melanoma is resistant to systemic treatment. Which made it really important to identify molecular targets that are critical for melanoma cell growth and survival4. One of the most popular oncogenes is Src. Src family kinases (SFK) are involved in regulating a multitude of biological processes, including cell adhesion, migration, proliferation, and survival as well as that it is known to activate other oncogenic pathways. Targeting c-Src or Stat3 leads to inhibition of melanoma cell growth and Bcl-xL and Mcl-1 expression. Targeting Src may allow us to inhibit multiple oncogenic pathways leading to more anti-tumor effects.4

Stat3 signaling is a point of convergence for many tyrosine kinases, activation of Stat3 in tumor cell proliferation, and survival by enhancing expression of anti-apoptotic genes. Inhibiting both Src and Stat3 signaling pathways can lead to apoptosis. Studying Src and Stat3 pathway had shown that Src inhibitors disrupt the DNA binding activity and induce tumor cell death4. That was proven by couple steps first they proved that stat was constitutively activated in human melanoma cell lines a super shift assays were made with Stat1, Stat3, or Stat5 specific antibodies were performed using nuclear extracts of A2058 or JW melanoma cells which showed high activity for STAT3 in melanoma cells as well as tumor tissues showed an elevated Stat DNA-binding activity. To determine which tyrosine kinase signaling pathway is responsible for Stat3 activation in melanoma cells a panel of kinase inhibitors effect was analyzed Stat3 DNA-binding activity in melanoma cell lines. This panel included inhibitors for JAK family kinases (tyrphostin AG490), EGF-R kinase signaling inhibitor (PD158780), and Src inhibitors (PD166285, PD180970). AG490 was used to determine Stat3 DNA-binding which had little or no inhibition effect on Stat3 DNA-binding, as well as PD158780 didn’t show any inhibition. While PD166285 which has broad specificity for various tyrosine kinases including Src4, and PD180970 which is selective for Src and Bcr-Abl kinases showed a great inhibition of Stat3 DNA-binding. To confirm Src activity on Stat3 DNA-binding activity a western blot was performed using c-src antibodies and antibodies to detect phosphorylated src residue tyrosine 419 (Fig1). none of the inhibitors affected c-Src at the protein level but both inhibitors were able (PD166285, PD180970) reduce phosphorylated Src protein which suggests that activated Src kinase mediates constitutive Stat3 activation in human melanoma cells. Figure 1 Src is activated in the melanoma cells and treatment with either of the two Src inhibitors diminished the levels of activated Src in these cells.

Src inhibitors (PD166285, PD180970) blocked growth of human melanoma cells with elevated Stat3 activity, A2058 and JW melanoma cells with activated Stat3 were treated using (PD166285, PD180970) resulting in a dose-dependent growth inhibition and showed reduced Stat3 DNA-binding activity. Those two inhibitors were also able to induce apoptosis because it was able to down-regulate anti-apoptotic genes Bcl-xL, Mcl-1, c-Myc, and cyclin D1, the efficiency of the inhibitors was examined using Annexin V-PE staining analyzed by FACS as an early indicator of apoptosis4 fig2. Figure 2 Inhibiting Src kinase activity, but not EGF-R or JAK tyrosine kinases induces apoptosis of human melanoma cells that display Stat3 activity. (4)

This finding demonstrates that Stat3 is constitutively activated in a majority of melanoma cell lines and tumor tissues. As well as Src but not EGF-R or JAK tyrosine kinases is responsible for Stat3 activation. Concluding that by targeting Src which would disrupt the Stat3-DNA binding activity it would be able to inhibit tumor cells proliferation and metastasis by inducing apoptosis and disrupting signaling pathways.

Dasatinib is potent FDA-approved melanoma treatment, it is a SFK Inhibitor that blocks tumor cells from migrating to normal cells and invading in nmol concentrations without affecting proliferation and survival of cells. It targets majorly focal adhesion kinase and Crk-associated substrate5, down-regulates expression of matrix metalloproteinase-9 (MM-9), and Inhibits EphA2 RTKs which is overexpressed in melanoma cells5.

Dasatinib effect was investigated on eight cell lines that showed SFK activity measured by tyrosyl phosphorylation of their autophosphorylation site. SFKs showed a critical role in regulation of angiogenic factors such as interleukin-8 and vascular endothelial growth factor5. Inhibiting SFK/FAK signaling pathway completely abolished migration and invasion of melanoma cells. That was test by the publisher by first testing if Dasatinib was able to inhibit migration by seeding 1205 lu and A2058 into a 12 well cell culture allowing it to proliferate for 24h then scratching it and treating it with DMSO and Dasatinib, allowing it to rest for 24h then quantifying it by counting the number of cells that migrated into the wound. Cells treated with Dasatinib showed fewer number of migrations. Testing the ability of Dasatinib to down-regulate Expression of Matrix Metalloproteinase-9 (MMP-9has previously been identified as a downstream target of SFK/FAK/p130CAS signaling with a critical role in invasion) collected treated cells after 96 hours of treatment showed down-regulation of MMP-9 by using Biotrak activity assay system to analyze them as well as Dasatinib was able to block enzymatic activity of MMP-9. Dasatinib didn’t show any inhibition for cell proliferation and viability fig3. Western blot was made to analyze SFK and SFK and downstream substrates of SFKs, including FAK and Crk-associated CASsubstrate, p1305 using antibodies for autophosphorylation site in autophosphorylation site in c-Src (Tyr419 in human and Tyr416 in chicken) cross-react with the corresponding autophosphorylation sites in other SFKs. Dasatinib was able to block SFK autophosphorylation and trysol phosphorylation of SFK down Stream. The data presented previously proved that dasatinib it can inhibit signaling pathways that are involved in cell adhesion, migration, and invasion5. Figure 3Dasatinib does not inhibit proliferation and viability of melanoma cells.

Dasatinib and PD180970 inhibited SFK catalytic activity at low concentrations. Dasatinib was able to inhibit cell migration and invasion and PD180970 was able to induce apoptosis by down-regulating anti-apoptotic genes. Using both tyrosine kinase inhibitors as a combined treatment of melanoma might be efficient because of its ability to inhibit proliferation and metastasis at the same time.

Dasatinib was able to inhibit SFK and SFKs downstream but it wasn’t able to inhibit known growth factors and survival pathways for melanoma such as mitogen-activated protein kinases Erk1 and Erk2, AKT, p38, and Stat3 signaling. Inhibiting the Rho family kinases allowed3 inhibiting of Stat3 and other growth factors that inhibited in its role cell migration and was able to decrease cell proliferation in secondary host.

Rho family kinases Rock1and Rock2 belong to the AGC kinase family of serine/ threonine (PKA, AKT/PKB) plays a major role in regulation of cell migration and affects several components of the metastatic process including migration, local invasion, and cell proliferation. Rock signaling plays a significant role in stiffening the extracellular matrix which increases cell proliferation. Activation of Rho phosphorylates several substrates (LIMK1,2, and MLC2)3. Tumor cells can move in either an elongated, protrusive mode or in an amoeboid movement both depend on actomyosin contractile. CCT12954 and AT13148 are multi-AGC ATP-competitive AKT kinase inhibitors. Comparing those two recently designed inhibitors with Y27632, H1152, and CCT129254 showed there efficiency in blocking both the rounded ‘amoeboid’ and elongated, protrusive mode of cell migration suggesting that inhibition of Rock in melanoma could be beneficial in the treatment of metastasis.

[image: ]CCT129254 and AT13148 were tested against a panel of kinases, AT13148 (AGC kinase inhibitor ) had a potent activity against Rock1, Rock2, and AKT 1,2, and 3 while CCT129254 ROCK1 and ROCK2 and AKT2 in nmolar concentrations. Both Inhibitors showed low potency against MRCK (myotonic dystrophy kinase-related Cdc42-binding kinase -a and -b), MLC2 phosphorylation was inhibited as well as AKT downstream signaling as shown by the decrease in Thr246 phosphorylation of the proline-rich Akt-substrate was strongly inhibited. on-target efficiency of the compounds was further confirmed by the dose-dependent decrease in phosphorylation of LIMK-substrate cofilin, a well-established target of ROCK-dependent signaling. AT12148 was stable over a period of 24hwhile CCT129254 was shifted toward higher potency after 24h. Potency was determined by Rock inhibition and determining their 24-hour-EC50s. Inhibition of ROCK actomyosin contractility (essential for the maintenance of cell shape) by CCT129254 and AT13148 induced cytoskeletal collapse which reduces cell movement which mimic the action of complete inhibition of myosin II by blebbistatin. All those results shows that inhibition of ROCK using CCT129254 and AT13148 decrease melanoma cell motility. AT13148 was then rejected because of its high toxicity. CCT129254 treatment led to a strong decrease in the number of metastases, with a marginal [image: ]effect on the growth of the primary tumor (Fig. 4). it also affected the colonization of the lung parenchyma, survival in the bloodstream, and their extravasation that was tested by using luciferase-expressing 4599 melanoma cells that, after 40 hours of pretreatment with the compounds, were injected into the tail vein of athymic mice and their Lumi- nescence measured 24 days after the injection. (Fig. 5) shows that lungs injected with the CCT129254-treated cells exhibit a consistently lower luminescence count than the control or CCT130293-treated cell. Figure 4CCT129254 inhibits spontaneous melanoma metastasis, number of metastases into the lung after treatment

Figure 5Rock inhibition decrease growth of melanoma metastases when treated with CCT11299254

Since Dasatinib only affected metastases without showing any effect on tumor proliferation, I think it would be more efficient to use CCT129254 as a combined therapy with PD180970. PD180970 was proven to induce apoptosis by down-regulating anti-apoptotic genes in the primary tumor combining that with CCT129254 which decrease metastases and reduces the ability of the melanoma cells to efficiently grow in the lung. This combined therapy would have a great impact on primary tumor, metastases, and secondary host.

Inhibiting of MAP/extracellular signal-regulated kinase (ERK) kinase (MEK) sensitized melanoma cells to apoptosis induced by tumor necrosis factor-related apoptosis-inducing ligand as well as inhibition of MEK induces apoptosis of melanoma cells by mitochondrial [image: ]apoptotic pathway6 which is independent of other pathways and controlled by the up-regulation of proapoptotic BH3-only proteins PUMA and Bim and the down-regulation of the antiapoptotic Bcl-2 family member Mcl-16. U0126 was used to inhibit MEK which induced apoptosis, this was studied using a panel of melanoma cell lines (fresh melanoma isolates and [image: ]melanocytes) then treated with U0126 for 48h fig6 we can see that by inhibiting MEK6 using U0126 had induced apoptosis specifically in SK-MEL-28. To confirm the effect of U0126 MEK1 was silenced in two cell lines SK-MEL-28, and Mel-RM by transfecting the cells with MEK1 siRNA FIG7 which inhibited MEK1 when switched to culture and induced apoptosis but at the same time it wasn’t effective in multiple other melanoma cell lines shown in fig6. PD166285 and PD180970 were both proven to induce apoptosis by down-regulating anti-apoptotic genes. But didn’t show any [image: ]dramatic effects on the growth of SK-Mel-28 cells4 fig4. While inhibiting MEK using U0126 induced apoptosis in SK-Mel-28 cells6. Using U0126 combined with PD166285 and PD180970 would allow it to induce apoptosis in bigger range of melanoma cell types. Figure 6 induction of apoptosis by U0126 in a panel of melanoma cell lines, fresh melanoma isolates, and melanocytes.

Figure 7 Knockdown of MEK1 bysiRNA induces apoptosis of melanoma cells. a) western blot analysis of MEK1, pERK1/2, and ERK1/2 B)% of apoptotic cells after treatment

Figure 8 Photomicrographs of SK-MEl-28, A2058, JW after treatment for 24h with inhibitors

Treatment of metastatic melanoma by conventional chemotherapeutic and biological agents remains unsatisfactory. The recent cancer treatment development is focusing on targeting kinases. Kinases are directly related to cancer initiation and progression which make them a perfect target for cancer therapies. Using kinase inhibitors in cancer treatment showed a promising future and couple kinase inhibitors received FDA approval. Inhibiting FSK, STAT3, Rho family, and MEK as discussed previously have shown that they can be used in targeting cancer but inhibiting each one by itself had its limitations which we can overcome by using combined therapies of different kinase inhibitors.

References:

Protein Kinases: Introduction | CST https://www.cellsignal.com/contents/science/protein-kinases-introduction/kinases
Bhullar, K. S.; Lagarón, N. O.; McGowan, E. M.; Parmar, I.; Jha, A.; Hubbard, B. P.; Rupasinghe, H. P. V. Kinase-Targeted Cancer Therapies: Progress, Challenges and Future Directions. Mol Cancer 2018, 17 (1), 48. https://doi.org/10.1186/s12943-018-0804-2.
Sadok, A.; McCarthy, A.; Caldwell, J.; Collins, I.; Garrett, M. D.; Yeo, M.; Hooper, S.; Sahai, E.; Kuemper, S.; Mardakheh, F. K.; et al. Rho Kinase Inhibitors Block Melanoma Cell Migration and Inhibit Metastasis. Cancer Res 2015, 75 (11), 2272–2284. https://doi.org/10.1158/0008-5472.CAN-14-2156.
Niu, G.; Bowman, T.; Huang, M.; Shivers, S.; Reintgen, D.; Daud, A.; Chang, A.; Kraker, A.; Jove, R.; Yu, H. Roles of Activated Src and Stat3 Signaling in Melanoma Tumor Cell Growth. Oncogene 2002, 21 (46), 7001–7010. https://doi.org/10.1038/sj.onc.1205859.
Buettner, R.; Mesa, T.; Vultur, A.; Lee, F.; Jove, R. Inhibition of Src Family Kinases with Dasatinib Blocks Migration and Invasion of Human Melanoma Cells. Molecular Cancer Research 2008, 6 (11), 1766–1774. https://doi.org/10.1158/1541-7786.MCR-08-0169.
Wang, Y. F.; Jiang, C. C.; Kiejda, K. A.; Gillespie, S.; Zhang, X. D.; Hersey, P. Apoptosis Induction in Human Melanoma Cells by Inhibition of MEK Is Caspase-Independent and Mediated by the Bcl-2 Family Members PUMA, Bim, and Mcl-1. Clinical Cancer Research 2007, 13 (16), 4934–4942. https://doi.org/10.1158/1078-0432.CCR-07-0665.
Panka, D. J. Targeting the Mitogen-Activated Protein Kinase Pathway in the Treatment of Malignant Melanoma. Clinical Cancer Research 2006, 12 (7), 2371s–2375s. https://doi.org/10.1158/1078-0432.CCR-05-2539.

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Overview of Therapies for Melanoma: Analytical Essay

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