We have demonstrated that mice completely deficient for Mob1
have the most severe phenotype among strains lacking a Hippo signaling component. Mob1a/1b
double-homozygous–null mutant mice die at gastrulation, much earlier than mice lacking Mst1/2
), or Nf2
). Our data further indicate that MOB1 is essential for embryogenesis and that functions of MOB1A and MOB1B overlap. In addition, the tumor spectrum observed in heterozygous Mob1
-deficient mice is the broadest among mutants lacking Hippo components. These findings suggest both that MOB1 is the key molecule in the Hippo signaling pathway and that MOB1 may have molecular target(s) other than the Hippo pathway. This notion is consistent with the reduced expression or mutation of MOB1 frequently observed in a variety of human cancers (17
), and with MOB1’s reported binding to a range of molecules, including TSSC1, NUP98, HDAC3, and DIPA (CCDD85B) (41
). Our results also imply that MOB1A may be more important than MOB1B, at least for embryogenesis and liver homeostasis, because Mob1b
heterozygotes lacking Mob1a
show partial embryonic lethality (Supplemental Table 1) and develop liver cancers (Table ), whereas Mob1a
heterozygotes lacking Mob1b
are all viable and free of liver tumors.
The lethality of Mob1
-deficient mice may stem from their failure to form primitive endoderm. The endodermal markers Pdgfra
were markedly reduced in Mob1
-deficient cells, whereas the primitive ectoderm markers Nanog
, and Pax6
were normal. Our mutant showed abnormal YAP1 activation, and activated YAP1 normally activates the transcription factor TEAD2. TEAD2 regulates primitive endoderm-specific genes such that sustained inhibition of TEAD2 enhances primitive endoderm-specific gene expression (43
). YAP1 and TEADS reportedly increase the expression of pluripotency genes such as Oct3/4
), as well as the trophoblast gene Cdx2
). However, levels of Oct3/4, SOX2, and CDX2 proteins were normal in our Mob1
-deficient embryos. Thus, although we found YAP1 to be activated in the mutant ICM, a much stronger alteration of YAP1 activity may be necessary to induce a detectable effect on these genes.
We have shown that, in contrast to MST1/2 and LATS1/2, MOB1 is a potent tumor suppressor in a range of tissue types that includes the skin. All cancers examined in Mob1aΔ/+1btr/tr and Mob1aΔ/Δ1btr/+ mice had lost the WT Mob1 allele, suggesting increased genetic instability. Mob1a/1b mRNAs and proteins are frequently cosuppressed in tumor cell lines (data not shown), and the MOB1A and MOB1B amino acid sequences are 95% identical. We are currently clarifying whether a common mechanism regulates these 2 genes.
In our Mob1aΔ/+1btr/tr
mice, the most frequent tumors were malignant outer root sheath tumors resembling trichilemmal carcinomas (Figure A). Histologically, these malignancies were not BCCs because they lacked the cellular palisading typical of BCCs (4
). Moreover, cultured Mob1a/1b
double-mutant keratinocytes did not show the SHH pathway activation important for BCC onset (Figure C). Benign trichilemmomas are frequently observed in Cowden disease patients with hereditary PTEN mutations (47
), but these tumors seldom become malignant. The trichilemmal growths in our Mob1
-deficient mutants were clearly cancerous but showed no activation of the PTEN effector AKT (Figure C). Importantly, like our mutant mouse tissues, our human trichilemmal carcinoma samples exhibited frequent MOB1A/1B inactivation and YAP1 activation (Figure G and Table ). These findings suggest that impaired Hippo signaling may drive trichilemmal carcinoma onset in humans.
-deficient keratinocytes exhibit enhanced proliferation, apoptotic resistance, impaired contact inhibition, increased centrosomes, accelerated mitotic exit, and enhanced progenitor self-renewal. In addition, polarity must be defective without MOB1 because (a) KRT15+
cells were scattered inside the IFE and not localized to the basal layer; (b) CD34+
bulge stem cells were not localized in the bulge; and (c) hair bundles in the organ of Corti were disorganized (Supplemental Figure 2B). To date, 2 transgenic mouse strains overexpressing Yap1
in the skin have been described (23
). Like our Mob1
transgenic mice show hyperplastic IFE. However, these latter animals also have a severe defect in HF formation. This difference may account for the development of IFE-derived squamous cell carcinomas in Yap1
transgenic mice, but HF-derived trichilemmal carcinomas in Mob1
At the biochemical level, Mob1
-deficient keratinocytes exhibited reductions in not only phospho-LATS1/2, but also total LATS1/2 proteins. Lats1/2
mRNAs were comparable in control and mutant keratinocytes (data not shown), indicating that this surprising decrease in LATS1/2 proteins occurs posttranscriptionally. Like MOB1, SAV1 is a scaffolding protein in the Hippo pathway, but unlike Mob1
deficiency decreases phospho-MST, but does not affect total MST or LATS1/2 proteins (20
). Another interesting biochemical observation was the activation of ERK in our Mob1
-deficient keratinocytes: no connection between MOB1 and ERK has been reported to date. With respect to Hippo signaling, ERK can be activated by YAP1 (49
) or suppressed by NF2 (50
) or MST2 (51
). Since NF2 and phospho-MST1/2 were not decreased in our Mob1
-deficient keratinocytes (data not shown), we speculate that the increased YAP1 in these cells may have triggered their abnormal ERK activation.
Because all of our Mob1a/1b-deficient mice spontaneously developed tumors (and especially trichilemmal carcinomas), we believe that the loss of MOB1A/1B helps to both initiate and promote carcinoma onset. With respect to tumor initiation, the observed increase in the number of centrosomes and/or micronuclei in our mutant cells may have introduced detrimental alterations into their DNA. With respect to cancer promotion, the enhanced proliferation, apoptotic resistance, impaired contact inhibition, and increased progenitor self renewal associated with loss of MOB1A/1Bmay support the growth and progression of cells that have undergone tumor initiation events.
The very rare occurrence of trichilemmal carcinomas in humans has slowed the identification of genes involved in their development. Expression levels of MOB1A, LATS1/2, and SAV1 are more frequently reduced in the advanced stages of colon (33
), breast (52
), and renal cancers (53
) than in the early stages of these malignancies. In lung cancers, the reverse is true, since MOB1A levels are frequently lower in the early pT1 stage of non–small-cell lung cancer as compared with later stages (34
). These observations suggest that the loss of Hippo signaling molecules can be an important driver of cancer progression in humans. Further study of alterations to gene or protein expression or functions of Hippo signaling components in a broad range of human malignancies may increase our understanding of their involvement in tumorigenesis.
In conclusion, our results demonstrate that (a) MOB1 is a broadly-acting tumor suppressor in mice and (b) Hippo signaling drives trichilemmal carcinoma onset in the skin. Therapeutic strategies to control Hippo signaling or MOB1 expression might therefore benefit many cancer patients, particularly those with HF-derived cancers.