Mutations in SIPA1L3 cause eye defects through disruption of cell polarity and cytoskeleton organization.

Correct morphogenesis and differentiation are critical in development and maintenance of the lens, which is a classic model system for epithelial development and disease. Through germline genomic analyses in patients with lens and eye abnormalities, we discovered functional mutations in the Signal Induced Proliferation Associated 1 Like 3 (SIPA1L3) gene, which encodes a previously uncharacterized member of the Signal Induced Proliferation Associated 1 (SIPA1 or SPA1) family, with a role in Rap1 signalling. Patient 1, with a de novo balanced translocation, 46,XY,t(2;19)(q37.3;q13.1), had lens and ocular anterior segment abnormalities. Breakpoint mapping revealed transection of SIPA1L3 at 19q13.1 and reduced SIPA1L3 expression in patient lymphoblasts. SIPA1L3 downregulation in 3D cell culture revealed morphogenetic and cell polarity abnormalities. Decreased expression of Sipa1l3 in zebrafish and mouse caused severe lens and eye abnormalities. Sipa1l3(-/-) mice showed disrupted epithelial cell organization and polarity and, notably, abnormal epithelial to mesenchymal transition in the lens. Patient 2 with cataracts was heterozygous for a missense variant in SIPA1L3, c.442G>T, p.Asp148Tyr. Examination of the p.Asp148Tyr mutation in an epithelial cell line showed abnormal clustering of actin stress fibres and decreased formation of adherens junctions. Our findings show that abnormalities of SIPA1L3 in human, zebrafish and mouse contribute to lens and eye defects, and we identify a critical role for SIPA1L3 in epithelial cell morphogenesis, polarity, adhesion and cytoskeletal organization.

The role of DNA damage responses in p53 biology.

The tumour suppressor p53 is a central player in cellular DNA damage responses. P53 is upregulated and activated by genotoxic stress and induces a transcriptional programme with effectors promoting apoptosis, cell cycle arrest, senescence and DNA repair. For the best part of the last three decades, these DNA damage-related programmes triggered by p53 were unequivocally regarded as the major if not sole mechanism by which p53 exerts its tumour suppressor function. However, this interpretation has been challenged by a number of recent in vivo studies, demonstrating that mice which are defective in inducing p53-dependent apoptosis, cell cycle arrest and senescence suppress thymic lymphoma as well as wild-type p53 expressing animals. Consequently, the importance of DNA damage responses for p53-mediated tumour suppression has been questioned. In this review, I summarize current knowledge on p53-controlled DNA damage responses and argue that these activities, while their role has certainly changed, remain an important feature of p53 biology with relevance for cancer therapy and tumour suppression.

Resistance acquisition to MDM2 inhibitors.

Various experimental strategies aim to (re)activate p53 signalling in cancer cells. The most advanced clinically are small-molecule inhibitors of the autoregulatory interaction between p53 and MDM2 (murine double minute 2). Different MDM2 inhibitors are currently under investigation in clinical trials. As for other targeted anti-cancer therapy approaches, relatively rapid resistance acquisition may limit the clinical efficacy of MDM2 inhibitors. In particular, MDM2 inhibitors were shown to induce p53 mutations in experimental systems. In the present article, we summarize what is known about MDM2 inhibitors as anti-cancer drugs with a focus on the acquisition of resistance to these compounds.

Transcription-independent pro-apoptotic functions of p53.

Induction of apoptosis is one of the central activities by which p53 exerts its tumor-suppressing function. Aside from its primary function as a transcription factor, it can promote apoptosis independent of transcription. Recent studies have started to define the mechanisms of non-transcriptional pro-apoptotic p53 activities operating within the intrinsic mitochondria-mediated pathway of apoptosis. So far, two different mechanisms have been described, each of which was assigned to a specific localization of the p53 protein, either in the cytosol or directly at the mitochondria. Although mechanistically different, both transcription-independent modes of apoptosis induction converge, as they both initiate permeabilization of the outer mitochondrial membrane via activation of the pro-apoptotic Bcl-2 family members Bax or Bak.

Mutant p53(R270H) gain of function phenotype in a mouse model for oncogene-induced mammary carcinogenesis.

In human breast cancer, mutations in the p53 gene are associated with poor prognosis. However, analysis of patient data so far did not clarify, whether missense point mutations in the p53 gene, in addition to causing loss of wild-type p53 function, also confer a gain of function phenotype to the encoded mutant p53. As heterogeneity of patient material and data might obscure a clear answer, we studied the effects of a coexpressed mutant p53(R270H) in transgenic mice in which SV40 early proteins initiate the development of mammary adenocarcinoma (WAP-T mice). In such tumors the endogenous wild-type p53 is functionally compromised by complex formation with SV40 T-antigen, thereby constituting a loss of wild-type p53 function situation that allowed analysis of the postulated gain of function effects of mutant p53(R270H). We found that mutant p53(R270H) in bi-transgenic mice enhanced the transition from intraepithelial neoplasia to invasive carcinoma, resulting in a higher frequency of invasive carcinoma per gland and per mouse, a more severe tumor phenotype, and more frequent pulmonary metastasis. Surprisingly, mutant p53(R270H) in this system does not increase genomic instability. Therefore, other postulated gain of function activities of mutant p53 must be responsible for the effects described here.

RUNX1 Upregulation by Cytotoxic Drugs Promotes Apoptosis.

Mutations in the RUNX1 gene have been associated with chemotherapy resistance and poor prognosis in patients with acute myeloid leukemia (AML), T-cell acute lymphoblastic leukemia, and myelodysplastic syndromes. However, the underlying mechanisms connecting RUNX1 to the success of therapy remain elusive. Here we explore the hypothesis that RUNX1 is directly involved in the response of hematopoietic cells to cytotoxic agents. RUNX1 was upregulated posttranscriptionally by cytotoxic agents in C57BL/6 mice in vivo and hematopoietic cell lines. Upregulation was also seen in primary human AML cells after treatment with cytarabine in vitro Upon overexpression, RUNX1 restricted proliferation, promoted apoptosis, and augmented the DNA damage response. This unknown activity of RUNX1 required an intact runt homology domain (RHD), a domain where most leukemia-associated point mutations cluster. Consistent with this, two RHD-defective RUNX1 proteins lacked any antiproliferative or apoptotic activity, and RHD-defective (K83N, N109D) mutant RUNX1 conferred resistance to ionizing radiation when overexpressed in Ba/F3 cells under certain conditions. Our experiments reveal a novel function of RUNX1 and offer an explanation for the link between RUNX1 mutations and chemotherapy and radiation resistance. Moreover, these data suggest that pharmacologic modulation of RUNX1 might be an attractive new approach to treat hematologic malignancies. Cancer Res; 77(24); 6818-24. ©2017 AACR.

Inhibition of casein kinase I delta alters mitotic spindle formation and induces apoptosis in trophoblast cells.

The serine/threonine-specific casein kinase I delta (CKIdelta) is ubiquitously expressed in all tissues, is p53 dependently induced in stress situations and plays an important role in various cellular processes. Our immunohistochemical analysis of the human placenta revealed strongest expression of CKIdelta in extravillous trophoblast cells and in choriocarcinomas. Investigation of the functional role of CKIdelta in an extravillous trophoblast hybrid cell line revealed that CKIdelta was constitutively localized at the centrosomes and the mitotic spindle. Inhibition of CKIdelta with the CKI-specific inhibitor IC261 led to structural alterations of the centrosomes, the formation of multipolar spindles, the inhibition of mitosis and, in contrast to other cell lines, the induction of apoptosis. Our findings indicate that CKIdelta plays an important role in the mitotic progression and in the survival of cells of trophoblast origin. Therefore, IC261 could provide a new tool in treating choriocarcinomas.

High-resolution cell cycle and DNA ploidy analysis in tissue samples.

This unit describes an easy, rapid, and universal procedure to process fresh and nitrogen-frozen tissue specimens for high-resolution cell cycle and DNA ploidy analysis. Unlike other protocols, this procedure does not require treating tissues with enzymes, detergents, or other plasma membrane-lysing chemicals, but it achieves tissue dispersion by a simple two-step mechanical process that can be performed in ∼5 min. Resulting single-cell suspensions are fixed with ethanol, stained with propidium iodide, and subjected to flow cytometric DNA content analysis. The method can be applied without any alterations to all tissue types (except bones) derived from several species and results in highly reproducible cell cycle profiles of excellent resolution. The described protocol can be used to reliably and accurately detect subtle cell cycle and ploidy alterations in tissue specimens, including cell cycle arrest, aneuploidy, and apoptosis/necrosis-associated DNA fragmentation.

Transcription-independent p53 apoptosis: an alternative route to death.

Apoptosis induced by p53 is firmly established as a central mechanism of tumour suppression. In addition to its complex functions as a nuclear transcription factor, p53 can act in the cytosol and mitochondria to promote apoptosis through transcription-independent mechanisms. Recent studies have shown that physical and functional interactions of p53 with various members of the Bcl-2 family provide the basis for this alternative route of p53-mediated cell death. However, different models of how these interactions promote apoptosis have been proposed. This review focuses on the mechanisms, regulation and physiological roles of transcription-independent p53 activities and highlights recent findings suggesting that the utilisation of these activities provides a promising alternative strategy for p53-based cancer therapy.

A rapid and optimization-free procedure allows the in vivo detection of subtle cell cycle and ploidy alterations in tissues by flow cytometry.

Cell cycle alterations are fundamental to many physiological processes but their detection has proven difficult when cells are in the context of a tissue structure. Here we describe an easy, rapid and optimization-free procedure for obtaining high resolution cell cycle profiles from nearly all tissue types derived from mouse, human and sheep. Using a standardized and non-enzymatic procedure that is universally suitable for soft, solid and epithelial tissues alike, we reproducibly obtain cell cycle profiles of highest quality with half peak coefficients of variation below 2.0. We are able to reduce preparation-derived debris to almost zero and efficiently exclude doublets, but retain multinucleated cells and apoptotic subG1-fragments. Applying this technique, we determine DNA-indices as small as 1.09 in tumor samples containing large necrotic areas and follow ploidy changes within different sections of individual tumors. Moreover, we examine tissue-specific cell cycle arrest and apoptosis as an in vivo stress response caused by radiation of mice. This method significantly improves the quality of DNA content analysis in tissues and extends the spectrum of applications. It allows assessing changes in ploidy, cell cycle distribution and apoptosis/necrosis in vivo and should be instrumental in all research that involves experimental animal models and/or patient biopsies.

Chloroquine activates the p53 pathway and induces apoptosis in human glioma cells.

Glioblastoma is the most common malignant brain tumor in adults. The currently available treatments offer only a palliative survival advantage and the need for effective treatments remains an urgent priority. Activation of the p53 growth suppression/apoptotic pathway is one of the promising strategies in targeting glioma cells. We show that the quinoline derivative chloroquine activates the p53 pathway and suppresses growth of glioma cells in vitro and in vivo in an orthotopic (U87MG) human glioblastoma mouse model. Induction of apoptosis is one of the mechanisms underlying the effects of chloroquine on suppressing glioma cell growth and viability. siRNA-mediated downregulation of p53 in wild-type but not mutant p53 glioblastoma cells substantially impaired chloroquine-induced apoptosis. In addition to its p53-activating effects, chloroquine may also inhibit glioma cell growth via p53-independent mechanisms. Our results clarify the mechanistic basis underlying the antineoplastic effect of chloroquine and reveal its therapeutic potential as an adjunct to glioma chemotherapy.

HOXB4 inhibits cell growth in a dose-dependent manner and sensitizes cells towards extrinsic cues.

Ectopic expression of the homeodomain transcription factor HOXB4 expands hematopoietic stem and progenitor cells in vivo and in vitro, making HOXB4 a highly interesting candidate for therapeutic stem cell expansion. However, when expressed at high levels, HOXB4 concomitantly perturbs differentiation and thus likely predisposes the manipulated cells for leukemogenesis. We therefore asked whether the expression level of HOXB4 may be a critical parameter that influences the growth and transformation properties of transduced cells. Using a set of retroviral vectors which covered a 40-fold range of expression levels, we studied the consequences of HOXB4 expression at different levels in the well established Rat-1 fibroblast cell system. HOXB4 transformed Rat-1 fibroblasts beyond a certain threshold level of expression. Further escalation of HOXB4 expression, however, did not enhance transformation. Instead, HOXB4 mediated a dose dependent anti-proliferative effect on Rat-1 and NIH3T3 fibroblasts. This effect was aggravated under reduced serum concentrations and was, at least partially, due to an enhanced sensitivity of HOXB4 overexpressing cells to induction of apoptosis. Based on these results we propose that HOXB4 affects cell growth in a dose-dependent manner by sensitizing cells towards extrinsic signals.