MAPK and PI3K pathways

BRAF targets in melanoma. Biological mechanisms, resistance, and drug discovery. Cancer drug discovery and development. Volume 82. Ed. Ryan J. Sullivan. Springer (2015)

Oncogenic driver mutations have been identified in key signaling and developmental pathways that are involved in survival and proliferation of melanocytes. The most frequently observed recurrent mutations in melanoma occur within the mitogen-activated protein kinase (MAPK) signaling pathway, which promotes cell survival, cell cycle progression, and transformation (Fig. 2.1). In nonmalignant cells, the MAPK pathway is only activated in response to ligand binding to receptor tyrosine kinases or cytokine receptors. Stimulation of receptors leads to activation of RAS family members, monomeric G proteins that act as GTPase switch proteins. RAS-GTP promotes formation of signal-transduction complexes and activates a cascade of serine/threonine kinases culminating in activation of ERK, also known as MAPK. ERK is a serine/threonine kinase that can phosphorylate many targets such as transcription factors.

MAPK signaling is constitutively activated in almost all melanomas. The vraf murine sarcoma viral oncogene homolog B1 (BRAF) is the dominant genetic target in this pathway, with 40–50% of melanomas carrying a somatic mutation [19–22]. To a lesser extent, BRAF mutations are also observed in other cancers [23, 24]. BRAF is a serine/threonine kinase directly activated by RAS and is highly expressed in melanocytes, neuronal tissues, testis, and haematopoietic cells. Unlike CRAF, which can participate in signaling events outside the MAPK pathway, BRAF’s only known substrate is MEK/MAP2K. Phosphorylated MEK activates ERK by phosphorylation, leading to pro-growth and transforming effects that are critical in melanoma pathogenesis.

BRAF Targets in Melanoma_ Biological Mechanisms, Resistance, and Drug Discovery-Springer-Verlag New York (2015) 2.1

Fig. 2.1. RAS signaling. RAS family members are monomeric G proteins that are activated by receptor tyrosine kinases and signal through direct interaction with effector enzymes including phosphoinositide (PI) 3-kinases, RAF kinases, and Ral-guanine nucleotide exchange factors (?RalGEFs). Although RAS mutations are less common in melanoma than other solid tumors, NRAS activating mutations are found in 10–20% of melanomas. Mitogen-activated protein (MAP) kinase signaling in response to RAF kinase activity promotes cell growth and survival, and the MAPK pathway is constitutively activated in almost all melanomas. BRAF is the most frequently mutated gene in melanoma, with activating lesions found in 40–50% of tumors. Melanoma oncogenes and tumor suppressors are labeled in red. Dotted lines represent omitted pathway components. NF1 neurofibromatosis 1, PTEN phosphatase and tensin homolog, PIP3 phosphatidylinositol-3,4,5-triphosphate, mTOR mammalian target of rapamycin, GSK-3β glycogen synthase kinase-3β, RSK ribosomal S6 kinase, Mnk1 MAP kinase-interacting kinase 1, Cdc42 cell division control protein 42 homolog

The most common BRAF mutation in melanoma, accounting for 90% of variants, is a valine to glutamic acid substitution at codon 600 (V600E) in exon 15 [25].

This mutation constitutively activates the kinase domain. Other oncogenic BRAF mutations are found elsewhere in exon 15 or in exon 11, and most of the over 100 rare non-V600E mutations described occur in the glycine-rich loop and activation segment of the kinase domain. Mutations in these regions indirectly activate BRAF by disrupting the normal intramolecular interactions which hold BRAF in an inactive conformation [26].

An alternate oncogenic mechanism in melanoma involves rare BRAF mutants with low kinase activity. Although no CRAF activating mutations have been reported in melanoma [25, 27, 28], mutations such as G469E and D594G produce a BRAF that directly activates CRAF but minimally phosphorylates MEK. Melanoma lines with these low-activity BRAF mutations are dependent on CRAF for survival [29].

BRAF(V600E) mutations are observed much more frequently in melanomas arising in intermittently sun-exposed skin regions than acral or mucosal melanomas, suggesting that BRAF mutations may be linked to sun exposure. However, the thymidine to adenine (T > A) transversion at position 1799 that is responsible for the V600E substitution is not a typical UV-signature DNA mutation. It is possible that the transversion could result from a “non-classic” UV-induced DNA lesion or from secondary effects of UVR exposure such as generation of reactive oxygen species [30].

Mutations that increase RAS activity also promote cell proliferation. In comparison to other solid tumors, RAS mutations occur with relatively low frequency in melanomas. Only 10–20% of melanomas, most often amelanotic nodular subtypes, carry an activating RAS mutation. NRAS is the most commonly affected RAS family member in melanoma [31, 32], and NRAS activating mutations [33, 34] primarily involve glycine 12, glycine 13, and glutamine 61 and trap NRAS in its active, GTP-bound conformation. While BRAF mutations activate only MAPK signaling, NRAS activating mutations simultaneously activate the MAPK and phosphatidylinositide 3-kinase (PI3K) pathways.

Although oncogenic mutations are usually not stand-alone events in melanoma, some are thought to be mutually exclusive. For example, NRAS and BRAF mutations almost never occur concomitantly [35, 36], suggesting that NRAS and BRAF have overlapping oncogenic activities and either is sufficient for constitutive activation of the MAPK pathway. Both BRAF and NRAS mutations are associated with poorer clinical prognosis. In the rare cases when both BRAF and NRAS mutations are present in melanoma, the BRAF mutation is not the classic V600E substitution [37]. “Acquired” (or selected) NRAS mutations have also been observed simultaneously with BRAF(V600E) in the context of melanomas which initially responded to BRAF-targeted therapy but subsequently became resistant [38].

PI3K signaling results in increased activation of the serine/threonine kinase AKT (also known as protein kinase B), which is a major mediator of cell survival through activation of targets such as mammalian target of rapamycin (mTOR) and inhibition of pro-apoptotic signals. While PI3K itself is rarely mutated in melanoma [39], constitutive activation of NRAS, amplification of AKT3, or loss of the phosphatase and tensin homolog (PTEN) tumor suppressor can lead to dysregulation of the PI3K pathway. PTEN encodes a lipid and protein phosphatase that negatively regulates signaling pathways which use the cytosolic second messenger phosphatidylinositol-3,4,5-triphosphate (PIP3), such as the PI3K pathway. Lower levels of intracellular PIP3 result in less downstream activating phosphorylation of AKT. Thus, loss of PTEN protein or function eliminates a mechanism of negative regulation of AKT  and cell survival. Increased phospho-AKT levels are associated with poor melanoma prognosis [40].

PTEN can be lost upon chromosome 10q deletion. 50–60% of melanomas contain hemizygous deletions or point mutations in 10q, while 10% contain homozygous deletion [41]. Epigenetic silencing of PTEN has also been described [36, 42, 43]. Hemizygous PTEN deletions tend to occur with BRAF mutation [42, 44], suggesting that BRAF and PTEN can cooperate in melanomagenesis. This idea is supported by studies of a murine model of melanoma in the setting of BRAF(V600E) and PTEN inactivation [45].

Neoplastic transformation of melanocytes can give rise to benign nevi as well as malignant melanoma, and activating mutations in BRAF and NRAS are implicated in both. Activating BRAF mutations are found in 70–80% of dysplastic nevi [22, 46–48], while NRAS mutations are rare in dysplastic nevi [49, 50] but present in most congenital nevi [50]. Mutation of HRAS is associated with Spitz nevi [51]. The BRAF(V600E) mutation induces nevus formation, involving initial cell proliferation followed by oncogene-induced senescence likely due in part to accumulation of p16INK4A [52]. Mutation of p16INK4A in addition to BRAF leads to transformation of cells in vitro, and deletion of PTEN or p16INK4A results in the formation of invasive melanoma in BRAF(V600E) mice [53]. In zebrafish, concomitant BRAF(V600E) mutation and deletion of TP53 leads to the formation of invasive and metastatic melanoma [54].

Given the high incidence of BRAF mutations in nevi, mutation of BRAF was traditionally thought to be a founder event that preceded all other oncogenic events in BRAF mutant melanoma [46]. In this model, senescence induced by BRAF activation is overcome by cooperating genetic lesions such as loss of p16INK4A or PTEN. However, other evidence suggests that the order of melanocytic lesions and relationship between nevi and tumor may be more complex. Although BRAF mutations are found in most nevi and half of vertical growth and metastatic melanomas, they are rare in initial malignant lesions: only 10% of radial growth phase melanomas and 6% of in situ melanomas have mutant BRAF. In addition, many nevi and primary melanomas are polyclonal (contain both BRAF wild type and BRAF mutant cells) while metastatic melanomas are not polyclonal, suggesting that BRAF mutation might occur at later stages of melanomagenesis [25, 46, 55, 56]. In addition, recent data have suggested that a stereotypical mutation in the promoter of the enzyme telomerase reverse transcriptase (TERT) is found in both BRAF mutant or NRAS mutant melanomas, suggesting that it may be an earlier mutation event [57]. This mutation in the TERT promoter occurs at a frequency of approximately 70% in melanomas and is also found in many non-melanoma cancers [57]. Regardless of when BRAF lesions occur, activation of BRAF in invasive melanoma promotes cell growth and dependence on the MAPK signaling pathway [58].

Dysregulation of MAPK signaling in melanoma can alternatively be caused by overexpression or hyperactivation of growth factor receptors such as c-Met, KIT, and epidermal growth factor receptor (EGFR) [59–61]. Mutations in the tumor suppressor neurofibromatosis 1 (NF1), a negative regulator of Ras, were identified in 5 out of 21 tumors without BRAF or NRAS mutations [62]. In the context of BRAF(V600E), NF1 mutations dysregulate the MAPK and PI3K pathways, ultimately suppressing mutant BRAF-induced senescence and promoting melanoma development and proliferation [63]. In some melanomas, inactivating mutations have also been identified in the tumor suppressor neurofibromatosis 2 (NF2) [64]. Germline mutations in NF1 and NF2 are associated with hereditary neurofibromatosis. Recent whole-exome sequencing approaches have identified somatic mutations in downstream MAPK effectors such as MAP3K5, MAP3K9, MEK1, and MEK2 in melanomas [65, 66].

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