The cell cycle revisited: DNA replication past S phase preserves genome integrity.

Accurate and complete DNA duplication is critical for maintaining genome integrity. Multiple mechanisms regulate when and where DNA replication takes place, to ensure that the entire genome is duplicated once and only once per cell cycle. Although the bulk of the genome is copied during the S phase of the cell cycle, increasing evidence suggests that parts of the genome are replicated in G2 or mitosis, in a last attempt to secure that daughter cells inherit an accurate copy of parental DNA. Remaining unreplicated gaps may be passed down to progeny and replicated in the next G1 or S phase. These findings challenge the long-established view that genome duplication occurs strictly during the S phase, bridging DNA replication to DNA repair and providing novel therapeutic strategies for cancer treatment.

Ras suppressor-1 (RSU1) exerts a tumor suppressive role with prognostic significance in lung adenocarcinoma.

Ras suppressor-1 (RSU1), originally described as a suppressor of Ras oncogenic transformation, localizes to focal adhesions interacting with the ILK-PINCH-PARVIN (IPP) complex that exerts a well-established oncogenic role in cancer. However, RSU1 implication in lung cancer is currently unknown. Our study aims to address the role of RSU1 in lung adenocarcinoma (LUADC). We here show that RSU1 protein expression by immunohistochemistry is downregulated in LUADC human tissue samples and represents a significant prognostic indicator. In silico analysis of gene chip and RNA seq data validated our findings. Depletion of RSU1 by siRNA in lung cancer cells promotes anchorage-independent cell growth, cell motility and epithelial to mesenchymal transition (EMT). Silencing of RSU1 also alters IPP complex expression in lung cancer cells. The p29 RSU1 truncated isoform is detected in lung cancer cells, and its expression is downregulated upon RSU1 silencing, whereas it is overexpressed upon ILK overexpression. These findings suggest that RSU1 exerts a tumor suppressive role with prognostic significance in LUADC.

Focal adhesion proteins in hepatocellular carcinoma: RSU1 a novel tumour suppressor with prognostic significance.

AIM: Hepatocellular carcinoma (HCC) is a common cause a cancer-related death. Focal adhesions (FAs) represent multiprotein complexes at integrin-mediated cell-extracellular matrix adhesion sites that orchestrate vital cellular functions. The heterotrimeric ILK-PINCH-PARVB (IPP) complex, RSU1, a PINCH binding protein and CTEN, a member of the tensin family of proteins exert a critical role in FAs, where they regulate important cancer related functions such as cell adhesion, migration, proliferation and survival. Previous studies implicate these FA proteins in liver pathophysiology but their detailed role in human HCC is not fully understood. Here in we investigated expression and function of IPP, RSU1 and CTEN in human HCC. METHODS: The expression of focal adhesion proteins was studied in human HCC by immunohistochemistry in relation to clinicopathological parameters, previous studied genomic instability markers and patient's survival. Effects on cell proliferation and FA proteins expression upon ILK inhibition and RSU1 silencing were also investigated in HCC in vitro. RESULTS: IPP complex and CTEN proteins are overexpressed while RSU1 expression is decreased in human HCC. CTEN expression correlates with reduced patients' survival while RSU1 represents an independent favorable prognostic indicator in human HCC. Nuclear ILK expression correlates with markers of genomic instability. Pharmacological targeting of ILK suppresses, while RSU1 silencing promotes cell growth of HCC cells in vitro, while in both experimental conditions expression and/or localization of focal adhesion proteins is deregulated. CONCLUSION: Our results suggest that FA signaling is implicated in hepatocellular carcinogenesis with prognostic significance. RSU1 seems to exert tumor suppressive functions in HCC and represents a novel favorable prognostic indicator.

Integrin-Linked-Kinase Overexpression Is Implicated in Mechanisms of Genomic Instability in Human Colorectal Cancer.

BACKGROUND: Genomic instability is a hallmark of cancer cells contributing to tumor development and progression. Integrin-linked kinase (ILK) is a focal adhesion protein with well-established role in carcinogenesis. We have previously shown that ILK overexpression is critically implicated in human colorectal cancer (CRC) progression. In light of the recent findings that ILK regulates centrosomes and mitotic spindle formation, we aimed to determine its implication in mechanisms of genomic instability in human CRC. METHODS: Association of ILK expression with markers of genomic instability (micronuclei formation, nucleus size, and intensity) was investigated in diploid human colon cancer cells HCT116 upon ectopic ILK overexpression, by immunofluorescence and in human CRC samples by Feulgen staining. We also evaluated the role of ILK in mitotic spindle formation, by immunofluorescence, in HCT116 cells upon inhibition and overexpression of ILK. Finally, we evaluated association of ILK overexpression with markers of DNA damage (p-H2AX, p-ATM/ATR) in human CRC tissue samples by immunohistochemistry and in ILK-overexpressing cells by immunofluorescence. RESULTS: We showed that ILK overexpression is associated with genomic instability markers in human colon cancer cells and tissues samples. Aberrant mitotic spindles were observed in cells treated with specific ILK inhibitor (QLT0267), while ILK-overexpressing cells failed to undergo nocodazole-induced mitotic arrest. ILK overexpression was also associated with markers of DNA damage in HCT116 cells and human CRC tissue samples. CONCLUSIONS: The above findings indicate that overexpression of ILK is implicated in mechanisms of genomic instability in CRC suggesting a novel role of this protein in cancer.

Integrin-linked kinase (ILK) regulates KRAS, IPP complex and Ras suppressor-1 (RSU1) promoting lung adenocarcinoma progression and poor survival.

Integrin-linked kinase (ILK) forms a heterotrimeric protein complex with PINCH and PARVIN (IPP) in Focal Adhesions (FAs) that acts as a signaling platform between the cell and its microenvironment regulating important cancer-related functions. We aimed to elucidate the role of ILK in lung adenocarcinoma (LUADC) focusing on a possible link with KRAS oncogene. We used immunohistochemistry on human tissue samples and KRAS-driven LUADC in mice, analysis of large scale publicly available RNA sequencing data, ILK overexpression and pharmacological inhibition as well as knockdown of KRAS in lung cancer cells. ILK, PINCH1 and PARVB (IPP) proteins are overexpressed in human LUADC and KRAS-driven LUADC in mice representing poor prognostic indicators. Genes implicated in ILK signaling are significantly enriched in KRAS-driven LUADC. Silencing of KRAS, as well as, overexpression and pharmacological inhibition of ILK in lung cancer cells provide evidence of a two-way association between ILK and KRAS. Upregulation of PINCH, PARVB and Ras suppressor-1 (RSU1) expression was demonstrated in ILK overexpressing lung cancer cells in addition to a significant positive correlation between these factors in tissue samples, while KRAS silencing downregulates IPP and RSU1. Pharmacological inhibition of ILK in KRAS mutant lung cancer cells suppresses cell growth, migration, EMT and increases sensitivity to platinum-based chemotherapy. ILK promotes an aggressive lung cancer phenotype with prognostic and therapeutic value through functions that involve KRAS, IPP complex and RSU1, rendering ILK a promising biomarker and therapeutic target in lung adenocarcinoma.

GemC1 governs multiciliogenesis through direct interaction with and transcriptional regulation of p73.

A distinct combination of transcription factors elicits the acquisition of a specific fate and the initiation of a differentiation program. Multiciliated cells (MCCs) are a specialized type of epithelial cells that possess dozens of motile cilia on their apical surface. Defects in cilia function have been associated with ciliopathies that affect many organs, including brain and airway epithelium. Here we show that the geminin coiled-coil domain-containing protein 1 GemC1 (also known as Lynkeas) regulates the transcriptional activation of p73, a transcription factor central to multiciliogenesis. Moreover, we show that GemC1 acts in a trimeric complex with transcription factor E2F5 and tumor protein p73 (officially known as TP73), and that this complex is important for the activation of the p73 promoter. We also provide in vivo evidence that GemC1 is necessary for p73 expression in different multiciliated epithelia. We further show that GemC1 regulates multiciliogenesis through the control of chromatin organization, and the epigenetic marks/tags of p73 and Foxj1. Our results highlight novel signaling cues involved in the commitment program of MCCs across species and tissues.This article has an associated First Person interview with the first author of the paper.

Controlling centriole numbers: Geminin family members as master regulators of centriole amplification and multiciliogenesis.

To ensure that the genetic material is accurately passed down to daughter cells during mitosis, dividing cells must duplicate their chromosomes and centrosomes once and only once per cell cycle. The same key steps-licensing, duplication, and segregation-control both the chromosome and the centrosome cycle, which must occur in concert to safeguard genome integrity. Aberrations in genome content or centrosome numbers lead to genomic instability and are linked to tumorigenesis. Such aberrations, however, can also be part of the normal life cycle of specific cell types. Multiciliated cells best exemplify the deviation from a normal centrosome cycle. They are post-mitotic cells which massively amplify their centrioles, bypassing the rule for once-per-cell-cycle centriole duplication. Hundreds of centrioles dock to the apical cell surface and generate motile cilia, whose concerted movement ensures fluid flow across epithelia. The early steps that control the generation of multiciliated cells have lately started to be elucidated. Geminin and the vertebrate-specific GemC1 and McIdas are distantly related coiled-coil proteins, initially identified as cell cycle regulators associated with the chromosome cycle. Geminin is required to ensure once-per-cell-cycle genome replication, while McIdas and GemC1 bind to Geminin and are implicated in DNA replication control. Recent findings highlight Geminin family members as early regulators of multiciliogenesis. GemC1 and McIdas specify the multiciliate cell fate by forming complexes with the E2F4/5 transcription factors to switch on a gene expression program leading to centriole amplification and cilia formation. Positive and negative interactions among Geminin family members may link cell cycle control to centriole amplification and multiciliogenesis, acting close to the point of transition from proliferation to differentiation. We review key steps of centrosome duplication and amplification, present the role of Geminin family members in the centrosome and chromosome cycle, and discuss links with disease.

Mcidas and GemC1 are key regulators for the generation of multiciliated ependymal cells in the adult neurogenic niche.

Multiciliated cells are abundant in the epithelial surface of different tissues, including cells lining the walls of the lateral ventricles in the brain and the airway epithelium. Their main role is to control fluid flow and defects in their differentiation are implicated in many human disorders, such as hydrocephalus, accompanied by defects in adult neurogenesis and mucociliary disorder in the airway system. Here we show that Mcidas, which is mutated in human mucociliary clearance disorder, and GemC1 (Gmnc or Lynkeas), previously implicated in cell cycle progression, are key regulators of multiciliated ependymal cell generation in the mouse brain. Overexpression and knockdown experiments show that Mcidas and GemC1 are sufficient and necessary for cell fate commitment and differentiation of radial glial cells to multiciliated ependymal cells. Furthermore, we show that GemC1 and Mcidas operate in hierarchical order, upstream of Foxj1 and c-Myb transcription factors, which are known regulators of ependymal cell generation, and that Notch signaling inhibits GemC1 and Mcidas function. Our results suggest that Mcidas and GemC1 are key players in the generation of multiciliated ependymal cells of the adult neurogenic niche.