Eleanor Y. M. Sum

Genetic analysis of Ras signalling pathways in cell proliferation, migration and survival

We have used mouse embryonic fibroblasts (MEFs) devoid of Ras proteins to illustrate that they are essential for proliferation and migration, but not for survival, at least in these cells. These properties are unique to the Ras subfamily of proteins because ectopic expression of other Ras‐like small GTPases, even when constitutively active, could not compensate for the absence of Ras proteins. Only constitutive activation of components of the Raf/Mek/Erk pathway was sufficient to sustain normal proliferation and migration of MEFs devoid of Ras proteins. Activation of the phosphatidylinositol 3‐kinase (PI3K)/PTEN/Akt and Ral guanine exchange factor (RalGEF)/Ral pathways, either alone or in combination, failed to induce proliferation or migration of Rasless cells, although they cooperated with Raf/Mek/Erk signalling to reproduce the full response mediated by Ras signalling. In contrast to current hypotheses, Ras signalling did not induce proliferation by inducing expression of D‐type Cyclins. Rasless MEFs had normal levels of Cyclin D1/Cdk4 and Cyclin E/Cdk2. However, these complexes were inactive. Inactivation of the pocket proteins or knock down of pRb relieved MEFs from their dependence on Ras signalling to proliferate.

The LIM domain gene LMO4 inhibits differentiation of mammary epithelial cells in vitro and is overexpressed in breast cancer

LMO4 belongs to a family of LIM-only transcriptional regulators, the first two members of which are oncoproteins in acute T cell leukemia. We have explored a role for LMO4, initially described as a human breast tumor autoantigen, in developing mammary epithelium and breast oncogenesis. Lmo4 was expressed predominantly in the lobuloalveoli of the mammary gland during pregnancy. Consistent with a role in proliferation, forced expression of this gene inhibited differentiation of mammary epithelial cells. Overexpression of LMO4 mRNA was observed in 5 of 10 human breast cancer cell lines. Moreover, in situ hybridization analysis of 177 primary invasive breast carcinomas revealed overexpression of LMO4 in 56% of specimens. Immunohistochemistry confirmed overexpression in a high percentage (62%) of tumors. These studies imply a role for LMO4 in maintaining proliferation of mammary epithelium and suggest that deregulation of this gene may contribute to breast tumorigenesis.

Defective Neural Tube Closure and Anteroposterior Patterning in Mice Lacking the LIM Protein LMO4 or Its Interacting Partner Deaf-1

ABSTRACT LMO4 belongs to a family of transcriptional regulators that comprises two zinc-binding LIM domains. LIM-only (LMO) proteins appear to function as docking sites for other factors, leading to the assembly of multiprotein complexes. The transcription factor Deaf-1/NUDR has been identified as one partner protein of LMO4. We have disrupted the Lmo4 and Deaf-1 genes in mice to define their biological function in vivo. All Lmo4 mutants died shortly after birth and showed defects within the presphenoid bone, with 50% of mice also exhibiting exencephaly. Homeotic transformations were observed in Lmo4-null embryos and newborn mice, but with incomplete penetrance. These included skeletal defects in cervical vertebrae and the rib cage. Furthermore, fusions of cranial nerves IX and X and defects in cranial nerve V were apparent in some Lmo4−/− and Lmo4+/− mice. Remarkably, Deaf-1 mutants displayed phenotypic abnormalities similar to those observed in Lmo4 mutants. These included exencephaly, transformation of cervical segments, and rib cage abnormalities. In contrast to Lmo4 nullizygous mice, nonexencephalic Deaf-1 mutants remained healthy. No defects in the sphenoid bone or cranial nerves were apparent. Thus, Lmo4 and Deaf-1 mutant mice exhibit overlapping as well as distinct phenotypes. Our data indicate an important role for these two transcriptional regulators in pathways affecting neural tube closure and skeletal patterning, most likely reflecting their presence in a functional complex in vivo.

Structural basis for the recognition of ldb1 by the N-terminal LIM domains of LMO2 and LMO4

LMO2 and LMO4 are members of a small family of nuclear transcriptional regulators that are important for both normal development and disease processes. LMO2 is essential for hemopoiesis and angiogenesis, and inappropriate overexpression of this protein leads to T‐cell leukemias. LMO4 is developmentally regulated in the mammary gland and has been implicated in breast oncogenesis. Both proteins comprise two tandemly repeated LIM domains. LMO2 and LMO4 interact with the ubiquitous nuclear adaptor protein ldb1/NLI/CLIM2, which associates with the LIM domains of LMO and LIM homeodomain proteins via its LIM interaction domain (ldb1‐LID). We report the solution structures of two LMO:ldb1 complexes (b: 1M3V and 1J2O) and show that ldb1‐LID binds to the N‐terminal LIM domain (LIM1) of LMO2 and LMO4 in an extended conformation, contributing a third strand to a β‐hairpin in LIM1 domains. These findings constitute the first molecular definition of LIM‐mediated protein–protein interactions and suggest a mechanism by which ldb1 can bind a variety of LIM domains that share low sequence homology.

The LIM Domain Protein Lmo4 Is Highly Expressed in Proliferating Mouse Epithelial Tissues

LMO4 belongs to the LIM-only family of zinc finger proteins that have been implicated in oncogenesis. The LMO4 gene is overexpressed in breast cancer and oral cavity carcinomas, and high levels of this protein inhibit mammary epithelial differentiation. Targeted deletion of Lmo4 in mice leads to complex phenotypic abnormalities and perinatal lethality. To further understand the role of LMO4, we have characterized Lmo4 expression in adult mouse tissues by immunohistochemical staining using monoclonal anti-Lmo4 antibodies. Lmo4 was highly expressed within specific cell types in diverse tissues. Expression was prevalent in epithelial-derived tissues, including the mammary gland, tongue, skin, small intestine, lung, and brain. High levels of Lmo4 were frequently observed in proliferating cells, such as the crypt cells of the small intestine and the basal cells of the skin and tongue. Lmo4 was highly expressed in the proliferative cap cell layer of the terminal end buds in the peripubertal mammary gland and in the lobuloalveolar units during pregnancy. The expression profile of Lmo4 suggests that this cofactor is an important regulator of epithelial proliferation and has implications for its role in the pathogenicity of cancer.

Loss of p53 induces cell proliferation via Ras-independent activation of the Raf/Mek/Erk signaling pathway

Significance In spite of the large number of biochemical studies contributing to the analysis of Ras-mediated cell cycle regulation, we do not know how Ras signaling controls the cell cycle. Here, we used an unbiased genetic approach to unveil essential components of Ras-mediated proliferation. Our results reveal that Ras signaling induces cell proliferation by a mechanism that requires inactivation of the p53/p21 Cdk-interacting protein 1 (Cip1) axis by preventing acetylation of specific p53 lysine residues. More importantly, loss of p53 or p21Cip1 can sustain cell proliferation in the absence of Ras proteins via Ras-independent activation of the Raf/Mek/Erk cascade. These results may have important implications for tumor growth and treatment, because activation of Ras oncogenes and inactivation of p53 are frequent events in human cancer. The Ras family of small GTPases constitutes a central node in the transmission of mitogenic stimuli to the cell cycle machinery. The ultimate receptor of these mitogenic signals is the retinoblastoma (Rb) family of pocket proteins, whose inactivation is a required step to license cell proliferation. However, little is known regarding the molecular events that connect Ras signaling with the cell cycle. Here, we provide genetic evidence to illustrate that the p53/p21 Cdk-interacting protein 1 (Cip1)/Rb axis is an essential component of the Ras signaling pathway. Indeed, knockdown of p53, p21Cip1, or Rb restores proliferative properties in cells arrested by ablation of the three Ras loci, H-, N- and K-Ras. Ras signaling selectively inactivates p53-mediated induction of p21Cip1 expression by inhibiting acetylation of specific lysine residues in the p53 DNA binding domain. Proliferation of cells lacking both Ras proteins and p53 can be prevented by reexpression of the human p53 ortholog, provided that it retains an active DNA binding domain and an intact lysine residue at position 164. These results unveil a previously unidentified role for p53 in preventing cell proliferation under unfavorable mitogenic conditions. Moreover, we provide evidence that cells lacking Ras and p53 proteins owe their proliferative properties to the unexpected retroactivation of the Raf/Mek/Erk cascade by a Ras-independent mechanism.

Loss of the LIM domain protein Lmo4 in the mammary gland during pregnancy impedes lobuloalveolar development

LMO4, a member of the LIM-only family of zinc-finger proteins, is overexpressed in a significant proportion of breast carcinomas and acts as a negative regulator of mammary epithelial differentiation. To delineate cell types within the developing mouse mammary gland that express Lmo4, we analysed different stages of mammopoiesis by immunohistochemistry. Lmo4 was found to be highly expressed in the proliferating cap cells of the terminal end bud and in the ductal and alveolar luminal cells of the mature mammary gland but was negligible or low in myoepithelial cells. To assess the physiological role of Lmo4 in the mammary gland, we generated conditionally targeted mice lacking Lmo4 in the mammary epithelium during pregnancy. Acute loss of Lmo4 in late pregnancy impaired lobuloalveolar development, accompanied by a two-fold reduction in the percentage of BrdU-positive cells. In contrast, germline loss of Lmo4 did not alter lobuloalveolar development arising from transplanted mammary anlagen, implying the existence of a compensatory mechanism in these knockout mice. Thus, the use of a conditional targeting strategy has revealed that Lmo4 is required for proper development of the mammary gland during pregnancy and indicated that Lmo4 acts as a positive regulator of alveolar epithelial proliferation.

Expression of LMO4 and outcome in pancreatic ductal adenocarcinoma

Identification of a biomarker of prognosis and response to therapy that can be assessed preoperatively would significantly improve overall outcomes for patients with pancreatic cancer. In this study, patients whose tumours exhibited high LMO4 expression had a significant survival advantage following operative resection, whereas the survival of those patients whose tumours had low or no LMO4 expression was not significantly different when resection was compared with operative biopsy alone.

Mutational analysis of the LMO4 gene, encoding a BRCA1-interacting protein, in breast carcinomas

The LIM domain‐only genes LMO1 and LMO2 are translocated in acute T cell leukemia (T‐ALL) and have been shown to be oncogenes in T lymphoid cells. LMO4, the fourth member of this family, is overexpressed in more than 50% of sporadic breast cancers, suggesting a role in breast oncogenesis. We recently found that LMO4 interacts with the breast/ovarian tumor suppressor BRCA1 and that LMO4 can repress its transcriptional activity. Since proto‐oncogene deregulation can result from activating mutations in their coding or regulatory sequences, we explored whether the LMO4 gene undergoes somatic mutagenesis in breast cancer. Mutation analysis of the coding and 3′ untranslated regions of the LMO4 gene was performed on 82 primary breast and 22 tumor cell lines. A somatic mutation was detected in one primary breast cancer, at the 3′ end of exon 2, but was not present in normal DNA derived from the same patient. This mutation causes a frame‐shift and potentially results in a truncated LMO4 polypeptide, LIM1mut, lacking the second LIM domain. This mutant protein could still bind Ldb1 but no longer associated with CtIP or BRCA1. Our results show that somatic mutations within the LMO4 gene do occur in breast cancer but at a very low frequency. Thus, the primary mechanism by which LMO4 is deregulated in breast cancers appears to reflect overexpression of the gene rather than the acquisition of activating genetic mutations.

Using cells devoid of RAS proteins as tools for drug discovery

Mutational activation of RAS proteins occurs in nearly 30% of all human tumors. To date direct pharmacological inhibition of RAS oncoproteins has not been possible. As a consequence, current strategies are focusing on the development of inhibitors that target those kinases acting downstream of RAS proteins, including those of the RAF/MEK/ERK and PI3K/AKT pathways. Most of these inhibitors have undesired off‐target effects that mask the potential therapeutic effect of blocking their targeted kinases. To facilitate the screening of selective inhibitors, we have generated lines of mouse embryonic fibroblasts that lack endogenous Ras proteins. These cells proliferate due to ectopic expression of either Ras oncoproteins that selectively activate the Raf/Mek/Erk pathway such as H‐RasG12V/D38E or constitutively active kinases such as B‐Raf and Mek1. These cell lines were exposed to inhibitors against the RAF, MEK, and AKT kinases as well as inhibitors of other kinases known to crosstalk with RAS signaling such as JNK and p38. Amongst all compounds tested, only the MEK inhibitors U0126 and PD0325901, showed the expected specificity pattern. Yet, PD0325901, but not U0126, was able to inhibit a cell line lacking Ras proteins that owed its proliferative properties to loss of p53. Thus, suggesting unexpected off‐target activities for this compound. The use of cell lines whose proliferative properties exclusively depend on selective targets provide a novel strategy to analyze the specificity of selective inhibitors designed against molecular targets implicated in human cancer.