A Runx1-enhancer Element eR1 Identified Lineage Restricted Mammary Luminal Stem Cells.

Mammary gland homeostasis is maintained by adult tissue stem-progenitor cells residing within the luminal and basal epithelia. Dysregulation of mammary stem cells is a key mechanism for cancer development. However, stem cell characterization is challenging because reporter models using cell-specific promoters do not fully recapitulate the mammary stem cell populations. We previously found that a 270-basepair Runx1 enhancer element, named eR1, marked stem cells in the blood and stomach. Here, we identified eR1 activity in a rare subpopulation of the ERα-negative luminal epithelium in mouse mammary glands. Lineage-tracing using an eR1-CreERT2 mouse model revealed that eR1+ luminal cells generated the entire luminal lineage and milk-secreting alveoli-eR1 therefore specifically marks lineage-restricted luminal stem cells. eR1-targeted-conditional knockout of Runx1 led to the expansion of luminal epithelial cells, accompanied by elevated ERα expression. Our findings demonstrate a definitive role for Runx1 in the regulation of the eR1-positive luminal stem cell proliferation during mammary homeostasis. Our findings identify a mechanistic link for Runx1 in stem cell proliferation and its dysregulation in breast cancer. Runx1 inactivation is therefore likely to be an early hit in the cell-of-origin of ERα+ luminal type breast cancer.

Induction of Gastric Cancer by Successive Oncogenic Activation in the Corpus.

BACKGROUND & AIMS: Metaplasia and dysplasia in the corpus are reportedly derived from de-differentiation of chief cells. However, the cellular origin of metaplasia and cancer remained uncertain. Therefore, we investigated whether pepsinogen C (PGC) transcript-expressing cells represent the cellular origin of metaplasia and cancer using a novel Pgc-specific CreERT2 recombinase mouse model. METHODS: We generated a Pgc-mCherry-IRES-CreERT2 (Pgc-CreERT2) knock-in mouse model. Pgc-CreERT2/+ and Rosa-EYFP mice were crossed to generate Pgc-CreERT2/Rosa-EYFP (Pgc-CreERT2/YFP) mice. Gastric tissues were collected, followed by lineage-tracing experiments and histologic and immunofluorescence staining. We further established Pgc-CreERT2;KrasG12D/+ mice and investigated whether PGC transcript-expressing cells are responsible for the precancerous state in gastric glands. To investigate cancer development from PGC transcript-expressing cells with activated Kras, inactivated Apc, and Trp53 signaling pathways, we crossed Pgc-CreERT2/+ mice with conditional KrasG12D, Apcflox, Trp53flox mice. RESULTS: Expectedly, mCherry mainly labeled chief cells in the Pgc-CreERT2 mice. However, mCherry was also detected throughout the neck cell and isthmal stem/progenitor regions, albeit at lower levels. In the Pgc-CreERT2;KrasG12D/+ mice, PGC transcript-expressing cells with KrasG12D/+ mutation presented pseudopyloric metaplasia. The early induction of proliferation at the isthmus may reflect the ability of isthmal progenitors to react rapidly to Pgc-driven KrasG12D/+ oncogenic mutation. Furthermore, Pgc-CreERT2;KrasG12D/+;Apcflox/flox mice presented intramucosal dysplasia/carcinoma and Pgc-CreERT2;KrasG12D/+;Apcflox/flox;Trp53flox/flox mice presented invasive and metastatic gastric carcinoma. CONCLUSIONS: The Pgc-CreERT2 knock-in mouse is an invaluable tool to study the effects of successive oncogenic activation in the mouse corpus. Time-course observations can be made regarding the responses of isthmal and chief cells to oncogenic insults. We can observe stomach-specific tumorigenesis from the beginning to metastatic development.

Iqgap3-Ras axis drives stem cell proliferation in the stomach corpus during homoeostasis and repair.

OBJECTIVE: Tissue stem cells are central regulators of organ homoeostasis. We looked for a protein that is exclusively expressed and functionally involved in stem cell activity in rapidly proliferating isthmus stem cells in the stomach corpus. DESIGN: We uncovered the specific expression of Iqgap3 in proliferating isthmus stem cells through immunofluorescence and in situ hybridisation. We performed lineage tracing and transcriptomic analysis of Iqgap3 +isthmus stem cells with the Iqgap3-2A-tdTomato mouse model. Depletion of Iqgap3 revealed its functional importance in maintenance and proliferation of stem cells. We further studied Iqgap3 expression and the associated gene expression changes during tissue repair after tamoxifen-induced damage. Immunohistochemistry revealed elevated expression of Iqgap3 in proliferating regions of gastric tumours from patient samples. RESULTS: Iqgap3 is a highly specific marker of proliferating isthmus stem cells during homoeostasis. Iqgap3+isthmus stem cells give rise to major cell types of the corpus unit. Iqgap3 expression is essential for the maintenance of stem potential. The Ras pathway is a critical partner of Iqgap3 in promoting strong proliferation in isthmus stem cells. The robust induction of Iqgap3 expression following tissue damage indicates an active role for Iqgap3 in tissue regeneration. CONCLUSION: IQGAP3 is a major regulator of stomach epithelial tissue homoeostasis and repair. The upregulation of IQGAP3 in gastric cancer suggests that IQGAP3 plays an important role in cancer cell proliferation.

Aurora kinase-induced phosphorylation excludes transcription factor RUNX from the chromatin to facilitate proper mitotic progression.

The Runt-related transcription factors (RUNX) are master regulators of development and major players in tumorigenesis. Interestingly, unlike most transcription factors, RUNX proteins are detected on the mitotic chromatin and apparatus, suggesting that they are functionally active in mitosis. Here, we identify key sites of RUNX phosphorylation in mitosis. We show that the phosphorylation of threonine 173 (T173) residue within the Runt domain of RUNX3 disrupts RUNX DNA binding activity during mitotic entry to facilitate the recruitment of RUNX proteins to mitotic structures. Moreover, knockdown of RUNX3 delays mitotic entry. RUNX3 phosphorylation is therefore a regulatory mechanism for mitotic entry. Cancer-associated mutations of RUNX3 T173 and its equivalent in RUNX1 further corroborate the role of RUNX phosphorylation in regulating proper mitotic progression and genomic integrity.

Roles of RUNX in Solid Tumors.

All RUNX genes have been implicated in the development of solid tumors, but the role each RUNX gene plays in the different tumor types is complicated by multiple interactions with major signaling pathways and tumor heterogeneity. Moreover, for a given tissue type, the specific role of each RUNX protein is distinct at different stages of differentiation. A regulatory function for RUNX in tissue stem cells points sharply to a causal effect in tumorigenesis. Understanding how RUNX dysregulation in cancer impinges on normal biological processes is important for identifying the molecular mechanisms that lead to malignancy. It will also indicate whether restoration of proper RUNX function to redirect cell fate is a feasible treatment for cancer. With the recent advances in RUNX research, it is time to revisit the many mechanisms/pathways that RUNX engage to regulate cell fate and decide whether cells proliferate, differentiate or die.

Aberrant Upregulation of RUNX3 Activates Developmental Genes to Drive Metastasis in Gastric Cancer.

Gastric cancer metastasis is a major cause of mortality worldwide. Inhibition of RUNX3 in gastric cancer cell lines reduced migration, invasion, and anchorage-independent growth in vitro. Following splenic inoculation, CRISPR-mediated RUNX3-knockout HGC-27 cells show suppression of xenograft growth and liver metastasis. We interrogated the potential of RUNX3 as a metastasis driver in gastric cancer by profiling its target genes. Transcriptomic analysis revealed strong involvement of RUNX3 in the regulation of multiple developmental pathways, consistent with the notion that Runt domain transcription factor (RUNX) family genes are master regulators of development. RUNX3 promoted "cell migration" and "extracellular matrix" programs, which are necessary for metastasis. Of note, we found pro-metastatic genes WNT5A, CD44, and VIM among the top differentially expressed genes in RUNX3 knockout versus control cells. Chromatin immunoprecipitation sequencing and HiChIP analyses revealed that RUNX3 bound to the enhancers and promoters of these genes, suggesting that they are under direct transcriptional control by RUNX3. We show that RUNX3 promoted metastasis in part through its upregulation of WNT5A to promote migration, invasion, and anchorage-independent growth in various malignancies. Our study therefore reveals the RUNX3-WNT5A axis as a key targetable mechanism for gastric cancer metastasis. SIGNIFICANCE: Subversion of RUNX3 developmental gene targets to metastasis program indicates the oncogenic nature of inappropriate RUNX3 regulation in gastric cancer.

RUNX family: Regulation and diversification of roles through interacting proteins.

The Runt-related transcription factors (RUNX) belong to an ancient family of metazoan genes involved in developmental processes. Through multiple protein-interacting partners, RUNX proteins have been implicated in diverse signaling pathways and cellular processes. The frequent inactivation of RUNX genes in cancer indicates crucial roles for RUNX in tumor suppression. This review discusses the abilities of RUNX proteins, in particular RUNX3, to integrate oncogenic signals or environmental cues and to initiate appropriate tumor suppressive responses.

Protocol for identification and validation of IGF2BP1 target genes in pluripotent human embryonic carcinoma cells.

We present a detailed protocol to identify and validate IGF2BP1 target genes in pluripotent human embryonic carcinoma cells (NTERA-2). We first identify the target genes through RNA-immunoprecipitation (RIP) sequencing. We then validate the identified targets through the use of RIP-qPCR assays, determine the m6A status of target genes by m6A-IP, and perform functional validation by quantifying changes in mRNA or protein expression levels upon knockdown of IGF2BP1 or methyltransferases in NTERA-2. For complete details on the use and execution of this protocol, please refer to Myint et al. (2022).1.

RUNX3 in Stem Cell and Cancer Biology.

The runt-related transcription factors (RUNX) play prominent roles in cell cycle progression, differentiation, apoptosis, immunity and epithelial-mesenchymal transition. There are three members in the mammalian RUNX family, each with distinct tissue expression profiles. RUNX genes play unique and redundant roles during development and adult tissue homeostasis. The ability of RUNX proteins to influence signaling pathways, such as Wnt, TGFß and Hippo-YAP, suggests that they integrate signals from the environment to dictate cell fate decisions. All RUNX genes hold master regulator roles, albeit in different tissues, and all have been implicated in cancer. Paradoxically, RUNX genes exert tumor suppressive and oncogenic functions, depending on tumor type and stage. Unlike RUNX1 and 2, the role of RUNX3 in stem cells is poorly understood. A recent study using cancer-derived RUNX3 mutation R122C revealed a gatekeeper role for RUNX3 in gastric epithelial stem cell homeostasis. The corpora of RUNX3R122C/R122C mice showed a dramatic increase in proliferating stem cells as well as inhibition of differentiation. Tellingly, RUNX3R122C/R122C mice also exhibited a precancerous phenotype. This review focuses on the impact of RUNX3 dysregulation on (1) stem cell fate and (2) the molecular mechanisms underpinning early carcinogenesis.

RUNX3 inactivates oncogenic MYC through disruption of MYC/MAX complex and subsequent recruitment of GSK3ß-FBXW7 cascade.

MYC is one of the most commonly dysregulated proto-oncogenes in cancer. MYC promotes cancer initiation and maintenance by regulating multiple biological processes, such as proliferation and stem cell function. Here, we show that developmental regulator RUNX3 targets MYC protein for rapid degradation through the glycogen synthase kinase-3 beta-F-box/WD repeat-containing protein 7 (GSK3ß-FBXW7) proteolytic pathway. The evolutionarily conserved Runt domain of RUNX3 interacts directly with the basic helix-loop-helix leucine zipper of MYC, resulting in the disruption of MYC/MAX and MYC/MIZ-1 interactions, enhanced GSK3ß-mediated phosphorylation of MYC protein at threonine-58 and its subsequent degradation via the ubiquitin-proteasomal pathway. We therefore uncover a previously unknown mode of MYC destabilization by RUNX3 and provide an explanation as to why RUNX3 inhibits early-stage cancer development in gastrointestinal and lung mouse cancer models.

Identifying Adult Stomach Tissue Stem/Progenitor Cells Using the Iqgap3-2A-CreERT2 Mouse.

Identification of unique gene markers of normal and cancer stem cells is an effective strategy to study cells of origin and understand tumor behavior. Lineage tracing experiments using the Cre recombinase driven by a stem cell-specific promoter in the CreERT2 reporter mouse model enables identification of adult stem cells and delineation of stem cell activities in vivo. In our recent research on the mouse stomach, Iqgap3 was identified as a homeostatic stem cell marker located in the isthmus of the stomach epithelium. Lineage tracing with the Iqgap3-2A-CreERT2;Rosa26-LSL-tdTomato mouse model demonstrated stem cell activity in Iqgap3-expressing cells. Using the Iqgap3-2A-CreERT2 mouse model to target oncogenic KrasG12D expression to Iqgap3-expressing cells, we observed the rapid development of precancerous metaplasia in the stomach and proposed that aberrant Iqgap3-expressing cells may be critical determinants of early carcinogenesis. In this chapter, we detail a lineage tracing protocol to assess stem cell activity in the murine stomach. We also describe the procedure of inducing KrasG12D expression in Iqgap3-expressing homeostatic stem cells to explore their role as cells of origin and to trace the early cellular changes that precede neoplastic transformation.

Loss of Runx3 is a key event in inducing precancerous state of the stomach.

BACKGROUND & AIMS: RUNX3 is a tumor suppressor originally identified in gastric cancer. The mutation R122C in RUNX3 promotes gastric carcinogenesis by unclear mechanisms. We investigated how Runx3-deficiency contributes to distinct changes in the gastric epithelium that precede neoplasia. METHODS: Runx3-deficient (Runx3(-/-)) and wild-type BALB/c adult mice were subjected to histological analyses. Gastric cancer formation after administration of N-methyl-N-nitrosourea was evaluated. Runx3(+/+) and Runx3(-/-) gastric epithelial cell lines were used to investigate the molecular basis underlying Runx3 function. RESULTS: The gastric epithelia in Runx3(-)/(-) adult mice was hyperplastic, with loss of chief cells and development of mucin 6- and trefoil factor-2-expressing metaplasia. The gastric epithelium of Runx3(-)/(-) mice had an intestinal phenotype that expressed Cdx2. After addition of N-methyl-N-nitrosourea, Runx3- mice, unlike wild-type mice, consistently developed adenocarcinomas, indicating that Runx3-deficiency leads to premalignant changes in the gastric epithelia. RUNX3, but not the RUNX3 mutant R122C, repressed Cdx2 expression by attenuation of oncogenic beta(symbol)-catenin and Tcfs. CONCLUSIONS: Runx3-deficiency leads to a precancerous state in the gastric epithelia of mice, characterized by loss of chief cells but not parietal cells; inflammation did not appear to be involved.

Oncofetal protein IGF2BP1 regulates IQGAP3 expression to maintain stem cell potential in cancer.

We reported earlier that IQGAP3 is an important stem cell factor in rapidly proliferating isthmus stem cells in the stomach and that IQGAP3 expression is robustly induced in terminally differentiated chief cells and de-differentiated cells following tissue damage. The elevated IQGAP3 expression in cancer and its association with metastasis suggest a fundamental role for IQGAP3 in proliferating cancer stem cells. What causes IQGAP3 upregulation in cancer is unclear. Here, we show that IGF2BP1 and IQGAP3 expression levels are highest in the blastocyst, with both decreasing during adulthood. This suggests that IQGAP3, like IGF2BP1, is an early developmental gene that is aberrantly upregulated upon re-expression of IGF2BP1 during carcinogenesis. IGF2BP1 binds and stabilizes m6A-modified IQGAP3 transcripts. Downstream targets of IGF2BP1, namely SRF and FOXM1, also upregulate IQGAP3 expression. These multiple layers of IQGAP3 regulation, which may safeguard against inappropriate stem cell proliferation, present additional drug targets to inhibit IQGAP3-driven malignant growth.

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 functions as an oncogene in ovarian cancer.

OBJECTIVE: The Runt domain transcription factor, RUNX3, has been shown to be a tumor suppressor in a variety of cancers including gastric, colon and breast cancer. Interestingly, an oncogenic role for RUNX3 has also been suggested in basal cell carcinoma and head and neck cancer. Here, we explore the role of RUNX3 in ovarian cancer. METHODS: Expression of RUNX3 mRNA and protein was evaluated in human ovarian cancer cell lines. In addition, subcellular localization of RUNX3 was also examined in cell lines and ovarian cancer tissues. Effect of exogenous RUNX3 expression and knockdown on cell proliferation was investigated by proliferation assays and a soft agar assay. RESULTS: Expression of RUNX3 was detected in the nucleus of ovarian cancer cell lines and ovarian cancer tissues and was found to play a growth stimulatory role. RUNX3 knockdown resulted in a decrease in cell proliferation in liquid media as well as in soft agar. Despite the fact that exogenous expression of RUNX3 strongly inhibits cell growth in many cell types, RUNX3 promoted cell growth in ovarian cancer cell lines not expressing RUNX3. CONCLUSION: RUNX3 is frequently expressed in the nuclei of ovarian cancer cell lines and plays an oncogenic role in ovarian cancer.

The Multiple Interactions of RUNX with the Hippo-YAP Pathway.

The Hippo-YAP signaling pathway serves roles in cell proliferation, stem cell renewal/maintenance, differentiation and apoptosis. Many of its functions are central to early development, adult tissue repair/regeneration and not surprisingly, tumorigenesis and metastasis. The Hippo pathway represses the activity of YAP and paralog TAZ by modulating cell proliferation and promoting differentiation to maintain tissue homeostasis and proper organ size. Similarly, master regulators of development RUNX transcription factors have been shown to play critical roles in proliferation, differentiation, apoptosis and cell fate determination. In this review, we discuss the multiple interactions of RUNX with the Hippo-YAP pathway, their shared collaborators in Wnt, TGFß, MYC and RB pathways, and their overlapping functions in development and tumorigenesis.

The RUNX1 Enhancer Element eR1: A Versatile Marker for Adult Stem Cells.

The identification of adult stem cells is challenging because of the heterogeneity and plasticity of stem cells in different organs. Within the same tissue, stem cells may be highly proliferative, or maintained in a quiescent state and only to be activated after tissue damage. Although various stem cell markers have been successfully identified, there is no universal stem cell marker, which is exclusively expressed in all stem cells. Here, we discuss the roles of master developmental regulator RUNX1 in stem cells and the development of a 270 base pair fragment of the Runx1 enhancer (eR1) for use as stem cell marker. Using eR1 to identify stem cells offers a distinct advantage over gene promoters, which might not be expressed exclusively in stem cells. Moreover, RUNX1 has been strongly implicated in various cancer types, such as leukemia, breast, esophageal, prostate, oral, skin, and ovarian cancers?it has been suggested that RUNX1 dysfunction promotes stem cell dysfunction and proliferation. As tissue stem cells are potential candidates for cancer cells-of-origin and cancer stem cells, we will also discuss the use of eR1 to target oncogenic gene manipulations in stem cells and to track subsequent neoplastic changes.

RUNX Poly(ADP-Ribosyl)ation and BLM Interaction Facilitate the Fanconi Anemia Pathway of DNA Repair.

The Fanconi anemia (FA) pathway is a pivotal genome maintenance network that orchestrates the repair of DNA interstrand crosslinks (ICLs). The tumor suppressors RUNX1 and RUNX3 were shown to regulate the FA pathway independent of their canonical transcription activities, by controlling the DNA damage-dependent chromatin association of FANCD2. Here, in further biochemical characterization, we demonstrate that RUNX3 is modified by PARP-dependent poly(ADP-ribosyl)ation (PARylation), which in turn allows RUNX binding to DNA repair structures lacking transcription-related RUNX consensus motifs. SILAC-based mass spectrometric analysis revealed significant association of RUNX3 with core DNA repair complexes, including PARP1, even in unstressed cells. After DNA damage, the increased interaction between RUNX3 and BLM facilitates efficient FANCD2 chromatin localization. RUNX-Walker motif mutations from breast cancers are impaired for DNA damage-inducible PARylation, unveiling a potential mechanism for FA pathway inactivation in cancers. Our results reinforce the emerging paradigm that RUNX proteins are tumor suppressors with genome gatekeeper function.

TGFß Promotes Genomic Instability after Loss of RUNX3.

Studies of genomic instability have historically focused on intrinsic mechanisms rather than extrinsic mechanisms based in the tumor microenvironment (TME). TGFß is the most abundantly secreted cytokine in the TME, where it imparts various aggressive characteristics including invasive migration, drug resistance, and epithelial-to-mesenchymal transition (EMT). Here we show that TGFß also promotes genomic instability in the form of DNA double strand breaks (DSB) in cancer cells that lack the tumor suppressor gene RUNX3 Loss of RUNX3 resulted in transcriptional downregulation of the redox regulator heme oxygenase-1 (HO-1 or HMOX1). Consequently, elevated oxidative DNA damage disrupted genomic integrity and triggered cellular senescence, which was accompanied by tumor-promoting inflammatory cytokine expression and acquisition of the senescence-associated secretory phenotype (SASP). Recapitulating the above findings, tumors harboring a TGFß gene expression signature and RUNX3 loss exhibited higher levels of genomic instability. In summary, RUNX3 creates an effective barrier against further TGFß-dependent tumor progression by preventing genomic instability. These data suggest a novel cooperation between cancer cell-extrinsic TGFß signaling and cancer cell-intrinsic RUNX3 inactivation as aggravating factors for genomic instability.Significance: RUNX3 inactivation in cancer removes an antioxidant barrier against DNA double strand breaks induced by TGFß expressed in the tumor microenvironment. Cancer Res; 78(1); 88-102. ©2017 AACR.

Molecular mechanism and therapeutic implications of selinexor (KPT-330) in liposarcoma.

Exportin-1 mediates nuclear export of multiple tumor suppressor and growth regulatory proteins. Aberrant expression of exportin-1 is noted in human malignancies, resulting in cytoplasmic mislocalization of its target proteins. We investigated the efficacy of selinexor against liposarcoma cells both in vitro and in vivo. Exportin-1 was highly expressed in liposarcoma samples and cell lines as determined by immunohistochemistry, western blot, and immunofluorescence assay. Knockdown of endogenous exportin-1 inhibited proliferation of liposarcoma cells. Selinexor also significantly decreased cell proliferation as well as induced cell cycle arrest and apoptosis of liposarcoma cells. The drug also significantly decreased tumor volumes and weights of liposarcoma xenografts. Importantly, selinexor inhibited insulin-like growth factor 1 (IGF1) activation of IGF-1R/AKT pathway through upregulation of insulin-like growth factor binding protein 5 (IGFBP5). Further, overexpression and knockdown experiments showed that IGFBP5 acts as a tumor suppressor and its expression was restored upon selinexor treatment of liposarcoma cells. Selinexor decreased aurora kinase A and B levels in these cells and inhibitors of these kinases suppressed the growth of the liposarcoma cells. Overall, our study showed that selinexor treatment restored tumor suppressive function of IGFBP5 and inhibited aurora kinase A and B in liposarcoma cells supporting the usefulness of selinexor as a potential therapeutic strategy for the treatment of this cancer.