Phosphoproteomic analysis of neoadjuvant breast cancer suggests that increased sensitivity to paclitaxel is driven by CDK4 and filamin A.

Precision oncology research is challenging outside the contexts of oncogenic addiction and/or targeted therapies. We previously showed that phosphoproteomics is a powerful approach to reveal patient subsets of interest characterized by the activity of a few kinases where the underlying genomics is complex. Here, we conduct a phosphoproteomic screening of samples from HER2-negative female breast cancer receiving neoadjuvant paclitaxel (N = 130), aiming to find candidate biomarkers of paclitaxel sensitivity. Filtering 11 candidate biomarkers through 2 independent patient sets (N = 218) allowed the identification of a subgroup of patients characterized by high levels of CDK4 and filamin-A who had a 90% chance of achieving a pCR in response to paclitaxel. Mechanistically, CDK4 regulates filamin-A transcription, which in turn forms a complex with tubulin and CLIP-170, which elicits increased binding of paclitaxel to microtubules, microtubule acetylation and stabilization, and mitotic catastrophe. Thus, phosphoproteomics allows the identification of explainable factors for predicting response to paclitaxel.

Mielopatía por déficit de cobre: la gran simuladora / Myelopathy secondary to copper deficiency: The great imitator

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An essential role for Ink4 and Cip/Kip cell-cycle inhibitors in preventing replicative stress.

Cell-cycle inhibitors of the Ink4 and Cip/Kip families are involved in cellular senescence and tumor suppression. These inhibitors are individually dispensable for the cell cycle and inactivation of specific family members results in increased proliferation and enhanced susceptibility to tumor development. We have now analyzed the consequences of eliminating a substantial part of the cell-cycle inhibitory activity in the cell by generating a mouse model, which combines the absence of both p21(Cip1) and p27(Kip1) proteins with the endogenous expression of a Cdk4 R24C mutant insensitive to Ink4 inhibitors. Pairwise combination of Cdk4 R24C, p21-null and p27-null alleles results in frequent hyperplasias and tumors, mainly in cells of endocrine origin such as pituitary cells and in mesenchymal tissues. Interestingly, complete abrogation of p21(Cip1) and p27(Kip1) in Cdk4 R24C mutant mice results in a different phenotype characterized by perinatal death accompanied by general hypoplasia in most tissues. This phenotype correlates with increased replicative stress in developing tissues such as the nervous system and subsequent apoptotic cell death. Partial inhibition of Cdk4/6 rescues replicative stress signaling as well as p53 induction in the absence of cell-cycle inhibitors. We conclude that one of the major physiological activities of cell-cycle inhibitors is to prevent replicative stress during development.

Killing cells by targeting mitosis.

Cell cycle deregulation is a common feature of human cancer. Tumor cells accumulate mutations that result in unscheduled proliferation, genomic instability and chromosomal instability. Several therapeutic strategies have been proposed for targeting the cell division cycle in cancer. Whereas inhibiting the initial phases of the cell cycle is likely to generate viable quiescent cells, targeting mitosis offers several possibilities for killing cancer cells. Microtubule poisons have proved efficacy in the clinic against a broad range of malignancies, and novel targeted strategies are now evaluating the inhibition of critical activities, such as cyclin-dependent kinase 1, Aurora or Polo kinases or spindle kinesins. Abrogation of the mitotic checkpoint or targeting the energetic or proteotoxic stress of aneuploid or chromosomally instable cells may also provide further benefits by inducing lethal levels of instability. Although cancer cells may display different responses to these treatments, recent data suggest that targeting mitotic exit by inhibiting the anaphase-promoting complex generates metaphase cells that invariably die in mitosis. As the efficacy of cell-cycle targeting approaches has been limited so far, further understanding of the molecular pathways modulating mitotic cell death will be required to move forward these new proposals to the clinic.

Genetic cooperation between p21Cip1 and INK4 inhibitors in cellular senescence and tumor suppression.

Cell-cycle inhibitors of the Cip/Kip and INK4 families are involved in cellular senescence and tumor suppression. Some of these proteins, p21(Cip1), p16(INK4a) and p15(INK4b), are coexpressed in response to antiproliferative signals such as cellular senescence resulting in cell-cycle arrest. To understand the roles of these inhibitors and their synergistic effect, we have characterized the growth properties and senescent behavior of primary cells deficient in p21(Cip1) and expressing an endogenous Cdk4(R24C) (cyclin-dependent kinase) mutant (Cdk4(R24C) knock-in cells) insensitive to INK4 proteins. Inactivation of both p21(Cip1) and INK4 pathways strongly cooperate in suppressing cellular senescence in vitro. These double mutant cells behavior as immortal cultures and display high sensitivity to cellular transformation by oncogenes. Moreover, mice double mutant in the INK4 and p21(Cip1) pathways (Cdk4(R24C); p21(Cip1)-null mice) display an increased incidence of specific sarcomas, suggesting a significant cooperation between these two families of cell-cycle inhibitors in senescence responses and tumor suppression in vivo.

Cyclin-dependent kinase 2 is dispensable for normal centrosome duplication but required for oncogene-induced centrosome overduplication.

Cyclin-dependent kinase 2 (CDK2) has been proposed to function as a master regulator of centrosome duplication. Using mouse embryonic fibroblasts (MEFs) in which Cdk2 has been genetically deleted, we show here that CDK2 is not required for normal centrosome duplication, maturation and bipolar mitotic spindle formation. In contrast, Cdk2 deficiency completely abrogates aberrant centrosome duplication induced by a viral oncogene. Mechanistically, centrosome overduplication in MEFs wild-type for Cdk2 involves the formation of supernumerary immature centrosomes. These results indicate that normal and abnormal centrosome duplication have significantly different requirements for CDK2 activity and point to a role of CDK2 in licensing centrosomes for aberrant duplication. Furthermore, our findings suggest that CDK2 may be a suitable therapeutic target to inhibit centrosome-mediated chromosomal instability in tumor cells.

Cell cycle and cancer: genetic analysis of the role of cyclin-dependent kinases.

Most human tumors harbor mutations that misregulate the early phases of the cell cycle. Here, we summarize genetic evidence, mostly obtained in our laboratory using strains of gene-targeted mice, that provides direct experimental support for a role of Cdk4 in tumor development. Moreover, these genetic studies challenge some well-established concepts regarding the role of Cdks during the early phases of the cell cycle. For instance, they have illustrated that Cdk4 and Cdk6 are not essential for cell division during embryonic development except in the hematopoietic system. More surprisingly, mice lacking Cdk2 survive for over 2 years without detectable abnormalities except in their germ cells, indicating that Cdk2 is essential for meiosis but dispensable for the normal mitotic cell cycle. Cdk2 is also dispensable for cell cycle inhibition and tumor suppression by the Cip/Kip inhibitors, p21(Cip1) and p27(Kip1). These observations have important implications not only to understand cell cycle regulation, but also to validate Cdks as potential targets for the development of therapeutic strategies to block proliferation of tumor cells.

Wide spectrum of tumors in knock-in mice carrying a Cdk4 protein insensitive to INK4 inhibitors.

We have introduced a point mutation in the first coding exon of the locus encoding the cyclin-dependent kinase 4 (Cdk4) by homologous recombination in embryonic stem cells. This mutation (replacement of Arg24 by Cys) was first found in patients with hereditary melanoma and renders Cdk4 insensitive to INK4 inhibitors. Here, we report that primary embryonic fibroblasts expressing the mutant Cdk4R24C kinase are immortal and susceptible to transformation by Ras oncogenes. Moreover, homozygous Cdk4(R24C/R24C) mutant mice develop multiple tumors with almost complete penetrance. The most common neoplasia (endocrine tumors and hemangiosarcomas) are similar to those found in pRb(+/-) and p53(-/-) mice. This Cdk4 mutation cooperates with p53 and p27(Kip1) deficiencies in decreasing tumor latency and favoring development of specific tumor types. These results provide experimental evidence for a central role of Cdk4 regulation in cancer and provide a valuable model for testing the potential anti-tumor effect of Cdk4 inhibitors in vivo.

Invasive melanoma in Cdk4-targeted mice.

Many human tumors harbor mutations that result in deregulation of Cdk4 activity. Most of these mutations involve overexpression of D-type cyclins and inactivation of INK4 inhibitors. In addition, a mutation in the Cdk4 protein has been described in patients with familial melanoma (Wolfel, T., Hauer, M., Schneider, J., Serrano, M., Wolfel, C., et al. (1995) Science 269, 1281-1284; Zuo, L., Weger, J., Yang, Q., Goldstein, A. M., Tucker, M. A., et al. (1996) Nat. Genet. 12, 97-99). This mutation, R24C, renders the Cdk4 protein insensitive to inhibition by INK4 proteins including p16(INK4a), a major candidate for the melanoma susceptibility locus. Here we show that knock-in mice expressing a Cdk4 R24C allele are highly susceptible to melanoma development after specific carcinogenic treatments. These tumors do not have mutations in the p19(ARF)/p53 pathway, suggesting a specific involvement of the p16(INK4a)/Cdk4/Rb pathway in melanoma development. Moreover, by using targeted mice deficient for other INK4 inhibitors, we show that deletion of p18(INK4c) but not of p15(INK4b) confers proliferative advantage to melanocytic tumor growth. These results provide an experimental scenario to study the role of Cdk4 regulation in melanoma and to develop novel therapeutic approaches to control melanoma progression.

Targeted genomic disruption of H-ras and N-ras, individually or in combination, reveals the dispensability of both loci for mouse growth and development.

Mammalian cells harbor three highly homologous and widely expressed members of the ras family (H-ras, N-ras, and K-ras), but it remains unclear whether they play specific or overlapping cellular roles. To gain insight into such functional roles, here we generated and analyzed H-ras null mutant mice, which were then also bred with N-ras knockout animals to ascertain the viability and properties of potential double null mutations in both loci. Mating among heterozygous H-ras(+/-) mice produced H-ras(-/-) offspring with a normal Mendelian pattern of inheritance, indicating that the loss of H-ras did not interfere with embryonic and fetal viability in the uterus. Homozygous mutant H-ras(-/-) mice reached sexual maturity at the same age as their littermates, and both males and females were fertile. Characterization of lymphocyte subsets in the spleen and thymus showed no significant differences between wild-type and H-ras(-/-) mice. Analysis of neuronal markers in the brains of knockout and wild-type H-ras mice showed that disruption of this locus did not impair or alter neuronal development. Breeding between our H-ras mutant animals and previously available N-ras null mutants gave rise to viable double knockout (H-ras(-/-)/N-ras(-/-)) offspring expressing only K-ras genes which grew normally, were fertile, and did not show any obvious phenotype. Interestingly, however, lower-than-expected numbers of adult, double knockout animals were consistently obtained in Mendelian crosses between heterozygous N-ras/H-ras mice. Our results indicate that, as for N-ras, H-ras gene function is dispensable for normal mouse development, growth, fertility, and neuronal development. Additionally, of the three ras genes, K-ras appears to be not only essential but also sufficient for normal mouse development.

To cycle or not to cycle: a critical decision in cancer.

Tumour cells undergo uncontrolled proliferation, yet tumours most often originate from adult tissues, in which most cells are quiescent. So, the proliferative advantage of tumour cells arises from their ability to bypass quiescence. This can be due to increased mitogenic signalling and/or alterations that lower the threshold required for cell-cycle commitment. Understanding the molecular mechanisms that underlie this commitment should provide important insights into how normal cells become tumorigenic and how new anticancer strategies can be devised.

Genetic analysis of mammalian cyclin-dependent kinases and their inhibitors.

Entry into the cell cycle, in particular the G1/S transition, is a tightly regulated process that involves a combination of mitogenic signaling pathways and cell cycle checkpoints. Some of the key regulators of this process are frequently altered in human cancer. Although the proteins that control the G1/S transition have been extensively studied at the biochemical level, little is known regarding their physiological role in vivo. During the last few years, a series of mouse strains carrying gene targeted mutations in key regulators of the G1/S transition have been generated. They include the Rb family of proteins and some of their downstream and upstream regulators. The latter include the regulatory (cyclin) and catalytic (Cdk) subunits of some of the kinases responsible for Rb inactivation as well as all the members of two families of cell cycle inhibitors, the INK4 and the Cip/Kip proteins. In this review, we summarize the most relevant information derived from the characterization of these strains of mice and attempt to integrate it within a functional framework of cell cycle regulation in vivo.

Limited overlapping roles of P15(INK4b) and P18(INK4c) cell cycle inhibitors in proliferation and tumorigenesis.

Entry of quiescent cells into the cell cycle is driven by the cyclin D-dependent kinases Cdk4 and Cdk6. These kinases are negatively regulated by the INK4 cell cycle inhibitors. We report the generation of mice defective in P15(INK4b) and P18(INK4c). Ablation of these genes, either alone or in combination, does not abrogate cell contact inhibition or senescence of mouse embryo fibroblasts in culture. However, loss of P15(INK4b), but not of P18(INK4c), confers proliferative advantage to these cells and makes them more sensitive to transformation by H-ras oncogenes. In vivo, ablation of P15(INK4b) and P18(INK4c) genes results in lymphoproliferative disorders and tumor formation. Mice lacking P18(INK4c) have deregulated epithelial cell growth leading to the formation of cysts, mostly in the cortical region of the kidneys and the mammary epithelium. Loss of both P15(INK4b) and P18(INK4c) does not result in significantly distinct phenotypic manifestations except for the appearance of cysts in additional tissues. These results indicate that P15(INK4b) and P18(IKN4c) are tumor suppressor proteins that act in different cellular lineages and/or pathways with limited compensatory roles.

The Rgr oncogene (homologous to RalGDS) induces transformation and gene expression by activating Ras, Ral and Rho mediated pathways.

The effects of the 5'-truncated Rgr oncogene, a previously shown specific guanine exchange factor for Ral in vitro, in stimulating proliferation, cell transformation and gene expression were investigated. We have established TetRgr cell lines in which expression of Rgr can be inhibited by the presence of tetracycline in the medium. Using this system, we show that Rgr overexpressing cells are morphologically transformed and grow in a disorganized manner. At the transcriptional level, Rgr enhances the activity of the serum response element and c-Jun. Rgr induces phosphorylation of ERKs, p38 and JNK kinases, and increases the levels of the GTP-bound forms of Ral and Ras. Ras activation could account for the broad spectra of effects displayed by Rgr. The important role of these pathways is confirmed by experiments in which the transcriptional activation events can be blocked by dominant negative versions of Ras, Ral and Rho. Among all the Rgr-induced pathways, the Ras-Raf-MEK-ERK cascade is essential for the transforming properties of Rgr. Additional analysis has shown that the activation of this pathway by Rgr is not due to a feed back mechanism mediated by the Grb2 adaptor protein. Oncogene (2000).

Characterization of the murine p19(ARF) promoter CpG island and its methylation pattern in primary lymphomas.

The INK4a/ARF locus encodes two different proteins involved in cell cycle control. Both molecules, p16(INK4a) and p19(ARF), inhibit cell cycle progression and have been shown to act as tumor suppressors in a variety of models. Their expression is controlled by separate promoters responding to different stimuli and they therefore show independent transcriptional regulation. We have cloned and characterized a 2.5 kb region upstream of the murine p19(ARF) gene to determine the role of DNA methylation in suppressing p19(ARF) transcription in a wide panel of murine primary T cell lymphomas. This region contains a DNA fragment with the characteristics of a CpG island similar to those described for the murine p16(INK4a) and p15(INK4b) genes. Expression of p19(ARF) is decreased in a significant number (20%) of the murine lymphomas analyzed. Overexpression of the p19(ARF) transcript is also frequent, suggesting alterations in molecules of the retinoblastoma or p53 pathways that are involved in p19(ARF) regulation. Although hypermethylation of the INK4a and INK4b promoters is frequently involved in murine lymphomas, the p19(ARF) CpG island is infrequently methylated in the murine primary lymphomas studied in this work. Since loss of p19(ARF) expression cannot be explained as the result of homozygous deletions or hypermethylation of the ARF gene, other regulatory mechanisms seem to be altered in these malignancies.

Cellular response to oncogenic ras involves induction of the Cdk4 and Cdk6 inhibitor p15(INK4b).

The cell cycle inhibitor p15(INK4b) is frequently inactivated by homozygous deletion together with p16(INK4a) and p19(ARF) in some types of tumors. Although the tumor suppressor capability of p15(INK4b) is still questioned, it has been found to be specifically inactivated by hypermethylation in hematopoietic malignancies in the absence of p16(INK4a) alterations. Here we show that, in vitro, p15(INK4b) is a strong inhibitor of cellular transformation by Ras. Surprisingly, p15(INK4b) is induced in cultured cells by oncogenic Ras to an extent similar to that of p16(INK4a), and their expression is associated with premature G(1) arrest and senescence. Ras-dependent induction of these two INK4 genes is mediated mainly by the Raf-Mek-Erk pathway. Studies with activated and dominant negative forms of Ras effectors indicate that the Raf-Mek-Erk pathway is essential for induction of both the p15(INK4b) and p16(INK4a) promoters, although other Ras effector pathways can collaborate, giving rise to a stronger response. Our results indicate that p15(INK4b), by itself, is able to stop cell transformation by Ras and other oncogenes such as Rgr (a new oncogene member of the Ral-GDS family, whose action is mediated through Ras). In fact, embryonic fibroblasts isolated from p15(INK4b) knockout mice are susceptible to transformation by the Ras or Rgr oncogene whereas wild-type embryonic fibroblasts are not. Similarly, p15(INK4b)-deficient mouse embryo fibroblasts are more sensitive than wild-type cells to transformation by a combination of the Rgr and E1A oncogenes. The cell cycle inhibitor p15(INK4b) is therefore involved, at least in some cell types, in the tumor suppressor activity triggered after inappropriate oncogenic Ras activation in the cell.

Targeted gene transfer system using a streptavidin-transforming growth factor-alpha chimeric protein.

The previously reported streptavidin-TGFalpha chimeric protein-based delivery system (Ohno and Meruelo, DNA Cell Biol. 15:401-406, 1996) could efficiently transfer protein molecules into A431 cells via the epidermal growth factor (EGF) receptor. We have modified this delivery system for the transfer of DNA. For this purpose, we have linked the chimeric protein ST-TGFalpha to DNA through biotinylated polylysine molecules. We show with this system, in the presence of the endosome-destabilizing reagent chloroquine, an average of 50-fold increase in reporter gene expression in comparison with polylysine DNA complexes alone. This gene expression is specific for EGF receptor-expressing cells and is blocked by EGF-binding molecules. These results suggest that the ST-TGFalpha biotinylated polylysine system could be used to deliver DNA to targeted cells.