Nicotinamide in Combination with EGFR-TKIs for the Treatment of Stage IV Lung Adenocarcinoma with EGFR Mutations: A Randomized Double-Blind (Phase IIb) Trial.

PURPOSE: RUNX3 is a tumor suppressor gene, which is inactivated in approximately 70% of lung adenocarcinomas. Nicotinamide, a sirtuin inhibitor, has demonstrated potential in re-activating epigenetically silenced RUNX3 in cancer cells. This study assessed the therapeutic benefits of combining nicotinamide with first-generation EGFR-tyrosine kinase inhibitors (TKI) for patients with stage IV lung cancer carrying EGFR mutations. PATIENTS AND METHODS: We assessed the impact of nicotinamide on carcinogen-induced lung adenocarcinomas in mice and observed that nicotinamide increased RUNX3 levels and inhibited lung cancer growth. Subsequently, 110 consecutive patients with stage IV lung cancer who had EGFR mutations were recruited: 70 females (63.6%) and 84 never-smokers (76.4%). The patients were randomly assigned to receive either nicotinamide (1 g/day, n = 55) or placebo (n = 55). The primary and secondary endpoints were progression-free survival (PFS) and overall survival (OS), respectively. RESULTS: After a median follow-up of 54.3 months, the nicotinamide group exhibited a median PFS of 12.7 months [95% confidence interval (CI), 10.4-18.3], while the placebo group had a PFS of 10.9 months (9.0-13.2; P = 0.2). The median OS was similar in the two groups (31.0 months with nicotinamide vs. 29.4 months with placebo; P = 0.2). Notably, subgroup analyses revealed a significant reduction in mortality risk for females (P = 0.01) and never-smokers (P = 0.03) treated with nicotinamide. CONCLUSIONS: The addition of nicotinamide with EGFR-TKIs demonstrated potential improvements in PFS and OS, with notable survival benefits for female patients and those who had never smoked (ClinicalTrials.gov Identifier: NCT02416739).

Runx3 Restoration Regresses K-Ras-Activated Mouse Lung Cancers and Inhibits Recurrence.

Oncogenic K-RAS mutations occur in approximately 25% of human lung cancers and are most frequently found in codon 12 (G12C, G12V, and G12D). Mutated K-RAS inhibitors have shown beneficial results in many patients; however, the inhibitors specifically target K-RASG12C and acquired resistance is a common occurrence. Therefore, new treatments targeting all kinds of oncogenic K-RAS mutations with a durable response are needed. RUNX3 acts as a pioneer factor of the restriction (R)-point, which is critical for the life and death of cells. RUNX3 is inactivated in most K-RAS-activated mouse and human lung cancers. Deletion of mouse lung Runx3 induces adenomas (ADs) and facilitates the development of K-Ras-activated adenocarcinomas (ADCs). In this study, conditional restoration of Runx3 in an established K-Ras-activated mouse lung cancer model regressed both ADs and ADCs and suppressed cancer recurrence, markedly increasing mouse survival. Runx3 restoration suppressed K-Ras-activated lung cancer mainly through Arf-p53 pathway-mediated apoptosis and partly through p53-independent inhibition of proliferation. This study provides in vivo evidence supporting RUNX3 as a therapeutic tool for the treatment of K-RAS-activated lung cancers with a durable response.

The TGFß→TAK1→LATS→YAP1 Pathway Regulates the Spatiotemporal Dynamics of YAP1.

The Hippo kinase cascade functions as a central hub that relays input from the "outside world" of the cell and translates it into specific cellular responses by regulating the activity of Yes-associated protein 1 (YAP1). How Hippo translates input from the extracellular signals into specific intracellular responses remains unclear. Here, we show that transforming growth factor ß (TGFß)-activated TAK1 activates LATS1/2, which then phosphorylates YAP1. Phosphorylated YAP1 (p-YAP1) associates with RUNX3, but not with TEAD4, to form a TGFß-stimulated restriction (R)-point-associated complex which activates target chromatin loci in the nucleus. Soon after, p-YAP1 is exported to the cytoplasm. Attenuation of TGFß signaling results in re-localization of unphosphorylated YAP1 to the nucleus, where it forms a YAP1/TEAD4/SMAD3/AP1/p300 complex. The TGFß-stimulated spatiotemporal dynamics of YAP1 are abrogated in many cancer cells. These results identify a new pathway that integrates TGFß signals and the Hippo pathway (TGFß→TAK1→LATS1/2→YAP1 cascade) with a novel dynamic nuclear role for p-YAP1.

Role of RUNX3 in Restriction Point Regulation.

A cell cycle is a series of events that takes place in a cell as it grows and divides. At the G1 phase of cell cycle, cells monitor their cumulative exposure to specific signals and make the critical decision to pass through the restriction (R)-point. The R-point decision-making machinery is fundamental to normal differentiation, apoptosis, and G1-S transition. Deregulation of this machinery is markedly associated with tumorigenesis. Therefore, identification of the molecular mechanisms that govern the R-point decision is one of the fundamental issues in tumor biology. RUNX3 is one of the genes frequently inactivated in tumors by epigenetic alterations. In particular, RUNX3 is downregulated in most K-RAS-activated human and mouse lung adenocarcinomas (ADCs). Targeted inactivation of Runx3 in the mouse lung induces adenomas (ADs), and markedly shortens the latency of ADC formation induced by oncogenic K-Ras. RUNX3 participates in the transient formation of R-point-associated activator (RPA-RX3-AC) complexes, which measure the duration of RAS signals and thereby protect cells against oncogenic RAS. This review focuses on the molecular mechanism by which the R-point participates in oncogenic surveillance.

A Point Mutation R122C in RUNX3 Promotes the Expansion of Isthmus Stem Cells and Inhibits Their Differentiation in the Stomach.

BACKGROUND & AIMS: RUNX transcription factors play pivotal roles in embryonic development and neoplasia. We previously identified the single missense mutation R122C in RUNX3 from human gastric cancer. However, how RUNX3R122C mutation disrupts stem cell homeostasis and promotes gastric carcinogenesis remained unclear. METHODS: To understand the oncogenic nature of this mutation in vivo, we generated the RUNX3R122C knock-in mice. Stomach tissues were harvested, followed by histologic and immunofluorescence staining, organoid culture, flow cytometry to isolate gastric corpus isthmus and nonisthmus epithelial cells, and RNA extraction for transcriptomic analysis. RESULTS: The corpus tissue of RUNX3R122C/R122C homozygous mice showed a precancerous phenotype such as spasmolytic polypeptide-expressing metaplasia. We observed mucous neck cell hyperplasia; massive reduction of pit, parietal, and chief cell populations; as well as a dramatic increase in the number of rapidly proliferating isthmus stem/progenitor cells in the corpus of RUNX3R122C/R122C mice. Transcriptomic analyses of the isolated epithelial cells showed that the cell-cycle-related MYC target gene signature was enriched in the corpus epithelial cells of RUNX3R122C/R122C mice compared with the wild-type corpus. Mechanistically, RUNX3R122C mutant protein disrupted the regulation of the restriction point where cells decide to enter either a proliferative or quiescent state, thereby driving stem cell expansion and limiting the ability of cells to terminally differentiate. CONCLUSIONS: RUNX3R122C missense mutation is associated with the continuous cycling of isthmus stem/progenitor cells, maturation arrest, and development of a precancerous state. This work highlights the importance of RUNX3 in the prevention of metaplasia and gastric cancer.

Runx3 regulates iron metabolism via modulation of BMP signalling.

OBJECTIVES: Runx3, a member of the Runx family of transcription factors, has been studied as a tumour suppressor and key player of organ development. In a previous study, we reported differentiation failure and excessive angiogenesis in the liver of Runx3 knock-out (KO) mice. Here, we examined a function of the Runx3 in liver, especially in iron metabolism. METHODS: We performed histological and immunohistological analyses of the Runx3 KO mouse liver. RNA-sequencing analyses were performed on primary hepatocytes isolated from Runx3 conditional KO (cKO) mice. The effect of Runx3 knock-down (KD) was also investigated using siRNA-mediated KD in functional human hepatocytes and human hepatocellular carcinoma cells. RESULT: We observed an iron-overloaded liver with decreased expression of hepcidin in Runx3 KO mice. Expression of BMP6, a regulator of hepcidin transcription, and activity of the BMP pathway were decreased in the liver tissue of Runx3 KO mice. Transcriptome analysis on primary hepatocytes isolated from Runx3 cKO mice also revealed that iron-induced increase in BMP6 was mediated by Runx3. Similar results were observed in Runx3 knock-down experiments using HepaRG cells and HepG2 cells. Finally, we showed that Runx3 enhanced the activity of the BMP6 promoter by responding to iron stimuli in the hepatocytes. CONCLUSION: In conclusion, we suggest that Runx3 plays important roles in iron metabolism of the liver through regulation of BMP signalling.

RUNX3 methylation drives hypoxia-induced cell proliferation and antiapoptosis in early tumorigenesis.

Inactivation of tumor suppressor Runt-related transcription factor 3 (RUNX3) plays an important role during early tumorigenesis. However, posttranslational modifications (PTM)-based mechanism for the inactivation of RUNX3 under hypoxia is still not fully understood. Here, we demonstrate a mechanism that G9a, lysine-specific methyltransferase (KMT), modulates RUNX3 through PTM under hypoxia. Hypoxia significantly increased G9a protein level and G9a interacted with RUNX3 Runt domain, which led to increased methylation of RUNX3 at K129 and K171. This methylation inactivated transactivation activity of RUNX3 by reducing interactions with CBFß and p300 cofactors, as well as reducing acetylation of RUNX3 by p300, which is involved in nucleocytoplasmic transport by importin-α1. G9a-mediated methylation of RUNX3 under hypoxia promotes cancer cell proliferation by increasing cell cycle or cell division, while suppresses immune response and apoptosis, thereby promoting tumor growth during early tumorigenesis. Our results demonstrate the molecular mechanism of RUNX3 inactivation by G9a-mediated methylation for cell proliferation and antiapoptosis under hypoxia, which can be a therapeutic or preventive target to control tumor growth during early tumorigenesis.

K-Ras-Activated Cells Can Develop into Lung Tumors When Runx3-Mediated Tumor Suppressor Pathways Are Abrogated.

K-RAS is frequently mutated in human lung adenocarcinomas (ADCs), and the p53 pathway plays a central role in cellular defense against oncogenic K-RAS mutation. However, in mouse lung cancer models, oncogenic K-RAS mutation alone can induce ADCs without p53 mutation, and loss of p53 does not have a significant impact on early K-RAS-induced lung tumorigenesis. These results raise the question of how K-RAS-activated cells evade oncogene surveillance mechanisms and develop into lung ADCs. RUNX3 plays a key role at the restriction (R)-point, which governs multiple tumor suppressor pathways including the p14ARF-p53 pathway. In this study, we found that K-RAS activation in a very limited number of cells, alone or in combination with p53 inactivation, failed to induce any pathologic lesions for up to 1 year. By contrast, when Runx3 was inactivated and K-RAS was activated by the same targeting method, lung ADCs and other tumors were rapidly induced. In a urethane-induced mouse lung tumor model that recapitulates the features of K-RAS-driven human lung tumors, Runx3 was inactivated in both adenomas (ADs) and ADCs, whereas K-RAS was activated only in ADCs. Together, these results demonstrate that the R-point-associated oncogene surveillance mechanism is abrogated by Runx3 inactivation in AD cells and these cells cannot defend against K-RAS activation, resulting in the transition from AD to ADC. Therefore, K-RAS-activated lung epithelial cells do not evade oncogene surveillance mechanisms; instead, they are selected if they occur in AD cells in which Runx3 has been inactivated.

Role of RUNX Family Members in G1 Restriction-Point Regulation.

When cells are stimulated by growth factors, they make a critical choice in early G1 phase: proceed forward to S phase, remain in G1, or revert to G0 phase. Once the critical decision is made, cells execute a fixed program independently of extracellular signals. The specific stage at which the critical decision is made is called the restriction point or R-point. The existence of the R-point raises a major question: what is the nature of the molecular machinery that decides whether or not a cell in G1 will continue to advance through the cell cycle or exit from the cell cycle? The R-point program is perturbed in nearly all cancer cells. Therefore, exploring the nature of the R-point decision-making machinery will provide insight into how cells consult extracellular signals and intracellular status to make an appropriate R-point decision, as well into the development of cancers. Recent studies have shown that expression of a number of immediate early genes is associated with the R-point decision, and that the decision-making program constitutes an oncogene surveillance mechanism. In this review, we briefly summarize recent findings regarding the mechanisms underlying the context-dependent R-point decision.

Lung Cancer Staging and Associated Genetic and Epigenetic Events.

The first step in treating lung cancer is to establish the stage of the disease, which in turn determines the treatment options and prognosis of the patient. Many factors are involved in lung cancer staging, but all involve anatomical information. However, new approaches, mainly those based on the molecular biology of cancer, have recently changed the paradigm for lung cancer treatment and have not yet been incorporated into staging. In a group of patients of the same stage who receive the same treatment, some may experience unexpected recurrence or metastasis, largely because current staging methods do not reflect the findings of molecular biological studies. In this review, we provide a brief summary of the latest research on lung cancer staging and the molecular events associated with carcinogenesis. We hope that this paper will serve as a bridge between clinicians and basic researchers and aid in our understanding of lung cancer.

Reciprocal regulation of YAP/TAZ by the Hippo pathway and the Small GTPase pathway.

Yes-associated protein 1 (YAP) and transcriptional co-activator with PDZ-binding motif (TAZ) (YAP/TAZ) are transcriptional coactivators that regulate genes involved in proliferation and transformation by interacting with DNA-binding transcription factors. Remarkably, YAP/TAZ are essential for cancer initiation or growth of most solid tumors. Their activation induces cancer stem cell attributes, proliferation, and metastasis. The oncogenic activity of YAP/TAZ is inhibited by the Hippo cascade, an evolutionarily conserved pathway that is governed by two kinases, mammalian Ste20-like kinases 1/2 (MST1/2) and Large tumor suppressor kinase 1/2 (LATS1/2), corresponding to Drosophila's Hippo (Hpo) and Warts (Wts), respectively. One of the most influential aspects of YAP/TAZ biology is that these factors are transducers of cell structural features, including polarity, shape, and cytoskeletal organization. In turn, these features are intimately related to the cell's ability to attach to other cells and to the surrounding extracellular matrix (ECM), and are also influenced by the cell's microenvironment. Thus, YAP/TAZ respond to changes that occur at the level of whole tissues. Notably, small GTPases act as master organizers of the actin cytoskeleton. Recent studies provided convincing genetic evidence that small GTPase signaling pathways activate YAP/TAZ, while the Hippo pathway inhibits them. Biochemical studies showed that small GTPases facilitate the YAP-Tea domain transcription factor (TEAD) interaction by inhibiting YAP phosphorylation in response to serum stimulation, while the Hippo pathway facilitates the YAP-RUNX3 interaction by increasing YAP phosphorylation. Therefore, small GTPase pathways activate YAP/TAZ by switching its DNA-binding transcription factors. In this review, we summarize the relationship between the Hippo pathway and small GTPase pathways in the regulation of YAP/TAZ.

Involvement of RUNX and BRD Family Members in Restriction Point.

A tumor is an abnormal mass of tissue that arises when cells divide more than they should or do not die when they should. The cellular decision regarding whether to undergo division or death is made at the restriction (R)-point. Consistent with this, an increasingly large body of evidence indicates that deregulation of the R-point decision-making machinery accompanies the formation of most tumors. Although the R-point decision is literally a matter of life and death for the cell, and thus critical for the health of the organism, it remains unclear how a cell chooses its own fate. Recent work demonstrated that the R-point constitutes a novel oncogene surveillance mechanism operated by R-point-associated complexes of which RUNX3 and BRD2 are the core factors (Rpa-RX3 complexes). Here, we show that not only RUNX3 and BRD2, but also other members of the RUNX and BRD families (RUNX1, RUNX2, BRD3, and BRD4), are involved in R-point regulation.

Runx3 inhibits endothelial progenitor cell differentiation and function via suppression of HIF-1α activity.

Endothelial progenitor cells (EPCs) are bone marrow (BM)­derived progenitor cells that can differentiate into mature endothelial cells, contributing to vasculogenesis in the blood vessel formation process. Runt­related transcription factor 3 (RUNX3) belongs to the Runt domain family and is required for the differentiation of specific immune cells and neurons. The tumor suppressive role of RUNX3, via the induction of apoptosis and cell cycle arrest in a variety of cancers, and its deletion or frequent silencing by epigenetic mechanisms have been studied extensively; however, its role in the differentiation of EPCs is yet to be investigated. Therefore, in the present study, adult BM­derived hematopoietic stem cells (HSCs) were isolated from Runx3 heterozygous (Rx3+/­) or wild­type (WT) mice. The differentiation of EPCs from the BM­derived HSCs of Rx3+/­ mice was found to be significantly increased compared with those of the WT mice, as determined by the number of small or large colony­forming units. The migration and tube formation abilities of Rx3+/­ EPCs were also observed to be significantly increased compared with those of WT EPCs. Furthermore, the number of circulating EPCs, defined as CD34+/vascular endothelial growth factor receptor 2 (VEGFR2)+ cells, was also significantly increased in Rx3+/­ mice. Hypoxia­inducible factor (HIF)­1α was upregulated in Rx3+/­ EPCs compared with WT EPCs, even under normoxic conditions. Furthermore, in a hindlimb ischemic mouse models, the recovery of blood flow was observed to be highly stimulated in Rx3+/­ mice compared with WT mice. Also, in a Lewis lung carcinoma cell allograft model, the tumor size in Rx3+/­ mice was significantly larger than that in WT mice, and the EPC cell population (CD34+/VEGFR2+ cells) recruited to the tumor was greater in the Rx3+/­ mice compared with the WT mice. In conclusion, the present study revealed that Runx3 inhibits vasculogenesis via the inhibition of EPC differentiation and functions via the suppression of HIF­1α activity.

RUNX3 regulates cell cycle-dependent chromatin dynamics by functioning as a pioneer factor of the restriction-point.

The cellular decision regarding whether to undergo proliferation or death is made at the restriction (R)-point, which is disrupted in nearly all tumors. The identity of the molecular mechanisms that govern the R-point decision is one of the fundamental issues in cell biology. We found that early after mitogenic stimulation, RUNX3 binds to its target loci, where it opens chromatin structure by sequential recruitment of Trithorax group proteins and cell-cycle regulators to drive cells to the R-point. Soon after, RUNX3 closes these loci by recruiting Polycomb repressor complexes, causing the cell to pass through the R-point toward S phase. If the RAS signal is constitutively activated, RUNX3 inhibits cell cycle progression by maintaining R-point-associated genes in an open structure. Our results identify RUNX3 as a pioneer factor for the R-point and reveal the molecular mechanisms by which appropriate chromatin modifiers are selectively recruited to target loci for appropriate R-point decisions.

Functional relationship between p53 and RUNX proteins.

RUNX genes belong to a three-membered family of transcription factors, which are well established as master regulators of development. Of them, aberrations in RUNX3 expression are frequently observed in human malignancies primarily due to epigenetic silencing, which is often overlooked. At the G1 phase of the cell cycle, RUNX3 regulates the restriction (R)-point, a mechanism that decides cell cycle entry. Deregulation at the R-point or loss of RUNX3 results in premature entry into S phase, leading to a proliferative advantage. Inactivation of Runx1 and Runx2 induce immortalization of mouse embryo fibroblast. As a consequence, RUNX loss induces pre-cancerous lesions independent of oncogene activation. p53 is the most extensively studied tumour suppressor. p53 plays an important role to prevent tumour progression but not tumour initiation. Therefore, upon oncogene activation, early inactivation of RUNX genes and subsequent mutation of p53 appear to result in tumour initiation and progression. Recently, transcription-independent DNA repairing roles of RUNX3 and p53 are emerging. Being evolutionarily old genes, it appears that the primordial function of p53 is to protect genome integrity, a function that likely extends to the RUNX gene as well. In this review, we examine the mechanism and sequence of actions of these tumour suppressors in detail.

DNA binding partners of YAP/TAZ.

Hippo signaling plays critical roles in regulation of tissue homeostasis, organ size, and tumorigenesis by inhibiting YES-associated protein (YAP) and PDZ-binding protein TAZ through MST1/2 and LATS1/2 pathway. It is also engaged in cross-talk with various other signaling pathways, including WNT, BMPs, Notch, GPCRs, and Hedgehog to further modulate activities of YAP/TAZ. Because YAP and TAZ are transcriptional coactivators that lack DNA-binding activity, both proteins must interact with DNA-binding transcription factors to regulate target gene's expression. To activate target genes involved in cell proliferation, TEAD family members are major DNA-binding partners of YAP/TAZ. Accordingly, YAP/TAZ were originally classified as oncogenes. However, YAP might also play tumor-suppressing role. For example, YAP can bind to DNA-binding tumor suppressors including RUNXs and p73. Thus, YAP might act either as an oncogene or tumor suppressor depending on its binding partners. Here, we summarize roles of YAP depending on its DNA-binding partners and discuss context-dependent functions of YAP/TAZ. [BMB Reports 2018; 51(3): 126-133].

RUNX3 and p53: How Two Tumor Suppressors Cooperate Against Oncogenic Ras?

RUNX family members play pivotal roles in both normal development and neoplasia. In particular, RUNX1 and RUNX2 are essential for determination of the hematopoietic and osteogenic lineages, respectively. RUNX3 is involved in lineage determination of various types of epithelial cells. Analysis of mouse models and human cancer specimens revealed that RUNX3 acts as a tumor suppressor via multiple mechanisms. p53-related pathways play central roles in tumor suppression through the DNA damage response and oncogene surveillance, and RUNX3 is involved in both processes. In response to DNA damage, RUNX3 facilitates p53 phosphorylation by the ATM/ATR pathway and p53 acetylation by p300. When oncogenes are activated, RUNX3 induces ARF, thereby stabilizing p53. Here, we summarize the molecular mechanisms underlying the p53-mediated tumor-suppressor activity of RUNX3.

Nigral dopaminergic PAK4 prevents neurodegeneration in rat models of Parkinson's disease.

Parkinson's disease (PD) is characterized by progressive loss of dopaminergic (DA) neurons in the substantia nigra. No neuroprotective treatments have successfully prevented the progression of this disease. We report that p21-activated kinase 4 (PAK4) is a key survival factor for DA neurons. We observed PAK4 immunoreactivity in rat and human DA neurons in brain tissue, but not in microglia or astrocytes. PAK4 activity was markedly decreased in postmortem brain tissue from PD patients and in rodent models of PD. Expression of constitutively active PAK4S445N/S474E (caPAK4) protected DA neurons in both the 6-hydroxydopamine and α-synuclein rat models of PD and preserved motor function. This neuroprotective effect of caPAK4 was mediated by phosphorylation of CRTC1 [CREB (adenosine 3',5'-monophosphate response element-binding protein)-regulated transcription coactivator] at S215. The nonphosphorylated form of CRTC1S215A compromised the ability of caPAK4 to induce the expression of the CREB target proteins Bcl-2, BDNF, and PGC-1α. Our results support a neuroprotective role for the PAK4-CRTC1S215-CREB signaling pathway and suggest that this pathway may be a useful therapeutic target in PD.

Core Binding Factor ß Plays a Critical Role During Chondrocyte Differentiation.

Core binding factor ß (Cbfß) is a partner protein of Runx family transcription factors with minimally characterized function in cartilage. Here we address the role of Cbfß in cartilage by generating chondrocyte-specific Cbfß-deficient mice (Cbfb(Δch/Δch) ) from Cbfb-floxed mice crossed with mice expressing Cre from the Col2a1 promoter. Cbfb(Δch/Δch) mice died soon after birth and exhibited delayed endochondral bone formation, shorter appendicular skeleton length with increased proliferative chondrocytes, and nearly absent hypertrophic chondrocyte zones. Immunohistochemical and quantitative real-time PCR analyses showed that the number and size of proliferative chondrocytes increased and the expression of chondrocyte maturation markers at the growth plates, including Runx2, osterix, and osteopontin, significantly diminished in Cbfb(Δch/Δch) mice compared to wild type mice. With regard to signaling pathways, both PTHrP-Ihh and BMP signaling were compromised in Cbfb(Δch/Δch) mice. Mechanistically, Cbfß deficiency in chondrocytes caused a decrease of protein levels of Runx transcription factors by accelerating polyubiquitination-mediated proteosomal degradation in vitro. Indeed, Runx2 and Runx3, but not Runx1, decreased in Cbfb(Δch/Δch) mice. Collectively, these findings indicate that Cbfß plays a critical role for chondrocyte differentiation through stabilizing Runx2 and Runx3 proteins in cartilage.