Alberto Martín

Cyclin-dependent kinase 2 is essential for meiosis but not for mitotic cell division in mice

We targeted the locus encoding the cyclin-dependent kinase 2 (CDK2) by homologous recombination in mouse embryonic stem (ES) cells. Embryonic fibroblasts lacking CDK2 proliferate normally and become immortal after continuous passage in culture. Elimination of a conditional Cdk2 allele in immortal cells does not have a significant effect on proliferation. Cdk2−/− mice are viable and survive for up to two years, indicating that CDK2 is also dispensable for proliferation and survival of most cell types. But CDK2 is essential for completion of prophase I during meiotic cell division in male and female germ cells, an unforeseen role for this cell cycle kinase.

A Synthetic Lethal Interaction between K-Ras Oncogenes and Cdk4 Unveils a Therapeutic Strategy for Non-small Cell Lung Carcinoma

We have unveiled a synthetic lethal interaction between K-Ras oncogenes and Cdk4 in a mouse tumor model that closely recapitulates human non-small cell lung carcinoma (NSCLC). Ablation of Cdk4, but not Cdk2 or Cdk6, induces an immediate senescence response only in lung cells that express an endogenous K-Ras oncogene. No such response occurs in lungs expressing a single Cdk4 allele or in other K-Ras-expressing tissues. More importantly, targeting Cdk4 alleles in advanced tumors detectable by computed tomography scanning also induces senescence and prevents tumor progression. These observations suggest that robust and selective pharmacological inhibition of Cdk4 may provide therapeutic benefit for NSCLC patients carrying K-RAS oncogenes.

Lysyl oxidase-like 2 (LOXL2), a new regulator of cell polarity required for metastatic dissemination of basal-like breast carcinomas

Basal‐like breast carcinoma is characterized by the expression of basal/myoepithelial markers, undifferentiated phenotype, highly aggressive behaviour and frequent triple negative status (ESR−, PR−, Her2neu−). We have previously shown that epithelial–mesenchymal transition (EMT) occurs in basal‐like breast tumours and identified Lysyl‐oxidase‐like 2 (LOXL2) as an EMT player and poor prognosis marker in squamous cell carcinomas. We now show that LOXL2 mRNA is overexpressed in basal‐like human breast carcinomas. Breast carcinoma cell lines with basal‐like phenotype show a specific cytoplasmic/perinuclear LOXL2 expression, and this subcellular distribution is significantly associated with distant metastatic incidence in basal‐like breast carcinomas. LOXL2 silencing in basal‐like carcinoma cells induces a mesenchymal‐epithelial transition (MET) associated with a decrease of tumourigenicity and suppression of metastatic potential. Mechanistic studies indicate that LOXL2 maintains the mesenchymal phenotype of basal‐like carcinoma cells by a novel mechanism involving transcriptional downregulation of Lgl2 and claudin1 and disorganization of cell polarity and tight junction complexes. Therefore, intracellular LOXL2 is a new candidate marker of basal‐like carcinomas and a target to block metastatic dissemination of this aggressive breast tumour subtype.

Mice thrive without Cdk4 and Cdk2.

Mammalian cell division is thought to be driven by sequential activation of several Cyclin‐dependent kinases (Cdk), mainly Cdk4, Cdk6, Cdk2 and Cdk1. Since mice lacking Cdk4, Cdk6 or Cdk2 are viable, it has been proposed that they play compensatory roles. We report here that mice lacking Cdk4 and Cdk2 complete embryonic development to die shortly thereafter presumably due to heart failure. However, conditional ablation of Cdk2 in adult mice lacking Cdk4 does not result in obvious abnormalities. Moreover, these double mutant mice recover normally after partial hepatectomy. In culture, Cdk4−/−;Cdk2−/−embryonic fibroblasts become immortal, display robust pRb phosphorylation and have normal S phase kinetics. These observations indicate that Cdk4 and Cdk2 are dispensable for the mammalian cell cycle and for adult homeostasis.

Tumorigenesis: Twist1 links EMT to self-renewal

The epithelial–mesenchymal transition (EMT) occurs in pre-metastatic cancer cells and is also associated with the acquisition of stem-cell-like characteristics. A molecular link between EMT and stemness now emerges with the finding that Bmi1, a polycomb protein that promotes self-renewal of certain stem-cell populations, is a direct transcriptional target of the EMT inducer, Twist1.

Driving the Cell Cycle to Cancer

Cell cycle progression requires the co-ordinated activation of several kinases, some of which are activated upon the binding of a cyclin subunit. At least four of these so-called cyclin-dependent kinases, namely Cdk4, Cdk6, Cdk2 and Cdk1, have specific roles at particular stages of the cell cycle, including passage through the various cell cycle transitions and the response to specific checkpoints. Not surprisingly, most human tumors carry mutations that deregulate at least one of these kinases. To analyze their specific role in vivo, we are generating strains of gene-targeted mice carrying either activated or defective alleles of these Cdks. As an example, Cdk4 expression appears to be expendable in most cell types since mice lacking Cdk4 are viable. Yet, Cdk4 mutant mice are smaller in size and infertile (only partial infertility in males). In addition, Cdk4 defective mice develop insulin dependent diabetes early in life. However, the importance of these Cdks in tumor cell cycles is underscored by the phenotype of knock in mice where the normal Cdk4 gene has been replaced by a Cdk4 R24C (insensitive to INK inhibitors) mutant. These animals develop a wide spectrum of spontaneous tumors and are highly susceptible to specific carcinogenic treatments. These models are being used now to understand how deregulation of these Cdks leads to cancer development and will be a valuable tool to design and validate new therapeutic strategies against tumour development.

Lysyl oxidase‐like 2 represses Notch1 expression in the skin to promote squamous cell carcinoma progression

Lysyl oxidase‐like 2 (LOXL2) is involved in a wide range of physiological and pathological processes, including fibrosis and tumor progression, implicating intracellular and extracellular functions. To explore the specific in vivo role of LOXL2 in physiological and tumor contexts, we generated conditional gain‐ and loss‐of‐function mouse models. Germ‐line deletion of Loxl2 promotes lethality in half of newborn mice mainly associated to congenital heart defects, while Loxl2 overexpression triggers male sterility due to epididymal dysfunction caused by epithelial disorganization, fibrosis and acute inflammation. Remarkably, when challenged to chemical skin carcinogenesis, Loxl2‐overexpressing mice increased tumor burden and malignant progression, while Loxl2‐deficient mice exhibit the opposite phenotypes. Loxl2 levels in premalignant tumors negatively correlate with expression of epidermal differentiation markers and components of the Notch1 pathway. We show that LOXL2 is a direct repressor of NOTCH1. Additionally, we identify an exclusive expression pattern between LOXL2 and members of the canonical NOTCH1 pathway in human HNSCC. Our data identify for the first time novel LOXL2 roles in tissue homeostasis and support it as a target for SCC therapy.

Lysyl Oxidase–like Protein LOXL2 Promotes Lung Metastasis of Breast Cancer

The lysyl oxidase-like protein LOXL2 has been suggested to contribute to tumor progression and metastasis, but in vivo evidence has been lacking. Here we provide functional evidence that LOXL2 is a key driver of breast cancer metastasis in two conditional transgenic mouse models of PyMT-induced breast cancer. LOXL2 ablation in mammary tumor cells dramatically decreased lung metastasis, whereas LOXL2 overexpression promoted metastatic tumor growth. LOXL2 depletion or overexpression in tumor cells does not affect extracellular matrix stiffness or organization in primary and metastatic tumors, implying a function for LOXL2 independent of its conventional role in extracellular matrix remodeling. In support of this likelihood, cellular and molecular analyses revealed an association of LOXL2 action with elevated levels of the EMT regulatory transcription factor Snail1 and expression of several cytokines that promote premetastatic niche formation. Taken together, our findings established a pathophysiologic role and new function for LOXL2 in breast cancer metastasis. Cancer Res; 77(21); 5846-59. ©2017 AACR.

Heteronuclear and heterovalent diphenylphosphido-bridged complexes of rhodium, iridium and ruthenium. Crystal structure of [(C5Me5)ClRh(μ-Cl)(μ-PPh2)Rh(COD)]•Et2O (COD = 1,5-cyclooctadiene)

Mononuclear complexes of formulae [LMCl2(HPRR′)], [LCMl(HPR2)2]BF4 and [LM(acac)(HPR2)]BF4 (M = Rh, Ir; L = C5Me5. M = Ru; L = p-Cymene; R = Ph, H; R′ = H; Hacac = acetylacetone, but not in all possible combinations) have been prepared from the corresponding [{(LMCl}2(μ-Cl)2] or [LMCl(acac)] and HPRR′. Complexes [LMCl2(HPPh2)] [L = C5Me5; M = Rh (I), Ir (IV), L = p-Cymene; M = Ru (VII)] react with [M′(μ-OMe)(diolefin)]2 (M′ = Rh; diolefin = tetrafluorobenzobarrelene (TFB), 1,5-cyclooctadiene (COD). M′ = Ir; diolefin = COD) to yield diphenylphosphido-bridged complexes of formula [LMCl2(μ-PPh2)M′(diolefin)]. The structure of [(C5Me5)ClRh(μ-Cl)(μ-PPh2)Rh(COD)]·12Et2O has been determined by X-ray diffraction methods. The complex consists of two moieties. (C5Me5)RhCl and Rh(COD), doubly-bridged by a diphenylphosphido group and a chlorine atom. The two rhodium atoms are separated by 3.3657(6) A.