Cellular senescence | EPFL Graph Search

Cellular senescence is a phenomenon characterized by the cessation of cell division. In their experiments during the early 1960s, Leonard Hayflick and Paul Moorhead found that normal human fetal fibroblasts in culture reach a maximum of approximately 50 cell population doublings before becoming senescent. This process is known as "replicative senescence", or the Hayflick limit. Hayflick's discovery of mortal cells paved the path for the discovery and understanding of cellular aging molecular pathways. Cellular senescence can be initiated by a wide variety of stress inducing factors. These stress factors include both environmental and internal damaging events, abnormal cellular growth, oxidative stress, autophagy factors, among many other things. The physiological importance for cell senescence has been attributed to prevention of carcinogenesis, and more recently, aging, development, and tissue repair. Senescent cells contribute to the aging phenotype, including frailty syndrome, s

Identification and functional characterization of protein kinases that modulate Notch signaling under physiological conditions and in cancer

Chhavi Jain

The Notch signaling pathway is a key regulator of cell fate decisions in embryonic development and in adult tissue homeostasis. Mounting evidence suggests that Notch signaling is frequently deregulated in human neoplasms, where depending upon the cellular context it can function both as an oncogene or a tumor suppressor. A plethora of studies shed light on how Notch crosstalks with signaling pathways downstream to manifest either its oncogenic or tumor suppressive activity. However, regulatory networks upstream of the Notch signaling pathway are still poorly understood. Since protein kinases are well-known regulators of a majority of cell signaling pathways, the aim of the current study was to identify novel positive and negative regulatory kinases that modulate Notch signaling. Using a HeLa cell based co-culture assay to read Notch-dependent luciferase activity, a small interference RNA (siRNA) library against the human kinase genome was previously tested in a high-throughput manner. Validation of top candidate kinases from the screening using both siRNA as well as pharmacological inhibitors led to further elimination of false positives and identification of Protein Kinase B (AKT2) and Calmodulin Kinase II (CaMKII) as key positive while Janus kinases (JAKs) as key negative regulators of the Notch signaling pathway. In the first part of the study, we analyzed the positive regulation of AKT2 and CaMKII kinases on Notch signaling in the T-cell acute lymphoblastic leukemia (T-ALL) cells where Notch is oncogenic. It was shown that pharmacological inhibition of either AKT2 or CaMKII kinase in T-ALL cell lines in vitro abrogated the expression of active Notch1 intracellular domain (N1-ICD) as well as of Notch target genes. Moreover, inhibition of these kinases blocked proliferation of T-ALL cells. In the second part of the study, we analyzed the negative regulation of JAK kinases on Notch signaling using a distinct physiological context where Notch is tumor suppressive. It was shown that pharmacological inhibition of JAK kinases led to an induction of Notch receptor transcription and of Notch target genes in the human primary epidermal keratinocytes (HPEK) as well as in the skin squamous cell carcinoma (SCC) cell lines. In addition, the pharmacological inhibition of JAK kinases induced a block in proliferation, cell cycle arrest and differentiation in HPEKs and skin SCCs by increased expression of early differentiation markers. Suppression of Notch signaling in primary keratinocytes counteracted the differentiation-inducing effect of JAK inhibition. Since JAK inhibition affected Notch transcription, global gene expression profiling using RNA sequencing was done to identify transcription factors involved in an indirect regulation of JAK kinases on the Notch cascade. EGR1 and EGR2 belonging to the early growth response family of transcription factors were significantly modulated downstream of JAK inhibition. In vivo, topical inhibition of JAK kinases provided marked resistance against chemically induced cutaneous carcinogenesis.Taken together, our data reveals a key role of AKT2 and CaMKII kinases in positive regulation while of JAK kinases in the negative regulation of Notch signaling, of potential relevance for combinatory approaches for cancer therapy. Pharmacological inhibitors against these kinases could be tested for their ability to modulate Notch signaling and may prove highly beneficial in the therapy of Notch-driven cancers.

EPFL2016

Muscle stem cell's in vitro bipolar niche

Adrien de Tonnac

In the domain of regenerative medicine research there are many fronts aimed at increasing our understanding of induced pluripotent, embryonic and adult stem cells. Recently a new front has appeared, stem cell niches, the microenvironment that is a crucial component in the regulation of stem cells. New in vitro platforms are continually being created to answer the multitude of biological questions that surround stem cell niches. The transition from in vitro to in vivo is a heavy setback in cellular research due to a huge gap between the two conditions, which can only be reduced if the in vitro conditions better mimic those found in vivo. This project has reduced the gap and has the potential to continue minimizing the differences between the two conditions in the field of muscle stem cell research. Through a combination of the forefront techniques in biological research, we were able to create a new innovative bioengineered platform, a bipolar hydrogel microenvironment. First, we took advantage of polyethylene glycol (PEG) hydrogels, which are highly hydrophilic polymer networks that have properties that mimic physiologic tissue, including high water content and low Young's Modulus. Microfabrication techniques allowed us to topographically pattern the hydrogel at micro-dimensions resembling the native cell environment. Utilizing microfluidics and micro-printing techniques, the three-dimensional hydrogel surface was also patterned with tethered niche proteins found in the in vivo muscle stem cell niche. The swelling properties of the PEG hydrogel were considered as an advantage and controlled to form niche gaps with physical properties that were tuned to correspond to those found in vivo. This new complex and controlled surface for cells to be cultured on is a tool, which will allow biologists to probe new questions and gain insight into the regulatory networks that control cell fate

2009

Inhibition of Notch pathway arrests PTEN-deficient advanced prostate cancer by triggering p27-driven cellular senescence

Jingjing Chen, Mitko Dimitrov, Patrick Fraering, Hermeto Gerber, Maria Luna Perciato, Andrea Rinaldi

Activation of NOTCH signalling is associated with advanced prostate cancer and treatment resistance in prostate cancer patients. However, the mechanism that drives NOTCH activation in prostate cancer remains still elusive. Moreover, preclinical evidence of the therapeutic efficacy of NOTCH inhibitors in prostate cancer is lacking. Here, we provide evidence that PTEN loss in prostate tumours upregulates the expression of ADAM17, thereby activating NOTCH signalling. Using prostate conditional inactivation of both Pten and Notch1 along with preclinical trials carried out in Pten-null prostate conditional mouse models, we demonstrate that Pten-deficient prostate tumours are addicted to the NOTCH signalling. Importantly, we find that pharmacological inhibition of gamma-secretase promotes growth arrest in both Pten-null and Pten/Trp53-null prostate tumours by triggering cellular senescence. Altogether, our findings describe a novel pro-tumorigenic network that links PTEN loss to ADAM17 and NOTCH signalling, thus providing the rational for the use of gamma-secretase inhibitors in advanced prostate cancer patients.

Nature Publishing Group2016

Identification and functional characterization of protein kinases that modulate Notch signaling under physiological conditions and in cancer

Chhavi Jain

EPFL2016