We performed a functional genomic study of the RTK c-Kit gene (KIT) using 17 sporadic ACC tumor specimens. We found that two ACC cases had distinct missense mutations in KIT (). We then explored the functional consequences of these mutations. Surprisingly, they were inactive, and we observed a significant reduction of MAPK activity in tumor cells harboring them. In addition, we observed that two ACC tumors without KIT mutations had missense mutations in the downstream effectors KRAS or BRAF, causing kinase-inactive mutant forms. This is the first time that a study has shown that MAPK activity from RTK signaling is inhibited by gene mutations during tumor development. Our results suggest that ACC is able to proliferate despite inactivation of the RTK c-Kit signaling pathway. This mechanism may explain why no objective responses were observed in the phase 2 clinical trial of imatinib for 27 ACC patients.
Figure 4 Cell signaling from the RTK c-Kit is disrupted during tumor development in a subset of salivary gland ACCs. C-Kit regulates cell survival and growth control through the PI3K/Akt and MAPK signaling pathways. We found inactivating mutations in KIT, KRAS (more ...)
Tumors generally have a number of genomic aberrations, and no single genetic alteration is thought to be enough to cause tumor progression. Some mutations are necessary for initiating and/or developing tumors but are not necessary for late-stage tumor maintenance [39
Only a few of the numerous defective proteins responsible for tumor maintenance might be attractive targets for drug development [19
]. In addition, anticancer drugs, which are usually low-molecular weight organic compounds, inhibit biochemical functions rather than enhance them. Thus, target molecules should be activated in cancer. Recent data from mouse models of cancer have shown that even the brief interruptions of the activities of a single oncogene such as H-Ras, K-Ras, c-Myc, and EGFR can reverse cancer [39
]. There have been no reports of reversibility of c-Kit-induced cancer in mice. Activation of c-Kit might play an important role at some stage of ACC tumor progression. However, its expression is probably not necessary for ACC tumor maintenance at the late stage because at least a subset of ACC cells can proliferate despite inhibition of the c-Kit signaling pathway.
Thus, c-Kit does not seem to be a suitable therapeutic target in ACC. Partial or complete loss of c-Kit function is associated with other diseases. For example, piebaldism (also known as piebald trait; OMIM 172800) is an autosomal-dominant genetic disorder of melanocyte development. It is characterized by white patches of skin and hair [40
]. Piebaldism is a disorder of haploinsufficiency, in which there is loss of function in one allele of the c-Kit proto-oncogene. Some subsets of piebaldism, however, are attributed to distinct mutations in the zinc finger transcription factor SNAI2 [41
A disorder similar to human piebaldism also occurs in mice [42,43
]. Spontaneous mutations at the dominant white (W
) locus of the laboratory mouse are associated with defects of pigmentation (albinism; white spotting of the skin), hematopoiesis (anemia), and germ cell development (sterility). Affected mice have partial or complete loss of c-Kit function. This fact highlights a significant function of c-Kit in normal stem and progenitor cells for hematopoiesis and pigmentation.
In this study, we examined the biochemical functions of two ACC c-Kit mutants: G664R and R796G. Interestingly, both mutations have been reported in patients with piebaldism [40,44
]. In particular, a patient harboring the R796G c-Kit mutation had severe piebaldism and profound sensorineural deafness [44
]. The symptom seemed much worse than in other cases of piebaldism, suggesting that the R796G mutant must completely abolish c-Kit function and that the added severity may be attributed to a dominant-negative effect [6
]. This observation is in accordance with our findings. The R796G c-Kit could not be activated by SCF stimulation. Piebaldism patients do not have a higher incidence of ACC, even if they have completely lost c-Kit function through a R796G mutation. Thus, loss-of-function mutations in c-Kit are probably not sufficient to cause ACC.
Gain-of-function mutations of c-Kit are frequently found in neoplasms, including GIST (OMIM 606764). GISTs are mesenchymal tumors found in the gastrointestinal tract. They originate from the interstitial cells of Cajal in the intestines [25
]. Approximately 50% to 90% of GISTs are caused by mutations in KIT
, and 10% to 35% of GIST subsets arise due to the mutations in PDGFRA
]. Mutations in KIT
are mutually exclusive. Familial cases of GIST have germ line mutations, whereas patients with sporadic disease have somatic mutations in tumor tissue. In contrast to piebaldism, familial GIST patients often present with hyperpigmentation and mast cell hyperplasia [46
GISTs are sensitive to c-Kit-targeted therapy with imatinib. Most GISTs, however, develop resistance to this compound. After 2.5 years of initial treatments, ~75% of tumors did not respond to further treatment with the drug [19,47
]. This fact suggests that long-term therapy with c-Kit targeted therapy may not be effective. To date, there have been no reports of familial ACC or other salivary gland tumors in GIST patients, indicating that gain-of-function mutations in c-Kit play little role in ACC. In fact, our ACC c-Kit mutations demonstrate loss of function.
The mechanism of c-Kit overexpression in ACC is not yet elucidated. In the melanocyte, c-Kit is transcriptionally regulated by the basic-helix-loop-helix-leucine zipper protein family of transcription factor MITF [48
]. We speculate that MITF might contribute to c-Kit expression in ACC and could play a role for tumor progression in a similar way in pigment precursor cells. The MITF is encoded at microphthalmia (mi
) locus in mice [49
]. Mutant mice have small eyes, show loss of pigmentation in the eye, inner ear, and skin, and early-onset sensorineural deafness (OMIM 156845). The phenotype apparently associates mi
mutant animals with W
locus-deletion mice. The association of MITF with c-Kit is mutual. MITF must be phosphorylated at serine 73 and serine 409 to get full transcriptional activity in melanocytes. This phosphorylation is mediated by an upstream signaling pathway from the RTK c-Kit [50
]. Despite their strong correlation in the progenitor cell melanocyte and frequent MITF locus amplification particularly in advanced melanomas, c-Kit expression is often lost in malignant melanoma [51
]. Further studies are necessary, but c-Kit V600E B-Raf mutations and p16Ink4a
inactivation may reduce the c-Kit requirement for MITF activation in melanoma cells. Otherwise, c-Kit may have the properties of a tumor-suppressor gene product in some contexts [52
We conclude that c-Kit must be dispensable for tumor maintenance in ACC and that selective c-Kit inhibition may not be a promising strategy for ACC therapeutic development. C-Kit activity may be necessary for tumor initiation and at some stage of tumor progression, although further studies are required to answer this question.