Store-operated calcium (Ca2+) entry (SOCE) represents a major route of Ca2+ permeation across the plasma membrane (PM) in nonexcitable cells, which plays an indispensable role in maintaining intracellular Ca2+ homeostasis. This process is orchestrated through the dynamic coupling between the endoplasmic reticulum (ER)-localized Ca2+ sensor stromal interaction molecule 1 (STIM1) and the PM-resident ORAI1 channel. Upon depletion of ER Ca2+ stores, STIM1 undergoes conformational rearrangements and oligomerization, leading to the translocation of activated STIM1 toward the PM. This movement is facilitated by the physical interactions between positively charged cytosolic domains within STIM1 and negatively charged phospholipids embedded in the PM, ultimately enabling its binding to and activation of the PM-embedded ORAI1 channel. In this mini-review, we provide an overview of STIM1-mediated Ca2+ signaling at ER-PM contact sites, highlighting the regulatory roles of phospholipids in the inner leaflet and sphingolipids in the outer leaflet of the PM. We also discuss the development of molecular tools that enable real-time visualization and manipulation of membrane contact sites (MCSs) at ER-PM junctions. Finally, we highlight recent progress in developing targeted therapies for human diseases linked to STIM1 mutations and dysregulated Ca2+ signaling at ER-PM MCSs.

Abstract RAS mutations are found in 30% of human cancers and are characterized by hotspot mutations at codons G12, G13, and Q61. This results in constitutively activated RAS proteins. Despite recent advances in drug design and the emergence of covalent inhibitors, effectively targeting mutant RAS remains challenging. Our group previously identified the tumor suppressor protein DIRAS3, which directly binds Ras and inhibits its function by disrupting KRAS dimers/nanoclusters and blocking effector activation. DIRAS3 expression reduced cancer cell growth in pancreatic and ovarian cancers. To determine whether DIRAS3 plays a definitive role in inhibiting mutant KRAS, we comprehensively characterized DIRAS3's RAS-inhibitory effects using RASless mouse embryo fibroblast (MEF) cells as a model system lacking endogenous RAS isoforms. We ectopically expressed various KRAS mutations in these cells and then examined DIRAS3's effect on each KRAS mutant clone. We analyzed downstream RAS signaling, colony formation, cell viability, and toxicity. Our results showed that DIRAS3 significantly inhibited RAS-dependent colony formation in KRAS mutant (G12C, G12D, G12V, G13D, G12R, Q61R, Q61L) but not wild-type KRAS clones. However, when we added epidermal growth factor (EGF) to the medium, DIRAS3 expression inhibited wild-type KRAS activity, decreasing colony formation. This suggests that DIRAS3 selectively affects the active, GTP-bound form of KRAS and downstream signaling. Additionally, DIRAS3 expression decreased Erk1/2 phosphorylation in MEF cells with mutant KRAS, but not in the cells with a BRAFV600E mutation. This indicates that DIRAS3 specifically inhibits the KRAS-mediated MEK/ERK signaling, cell proliferation, and cytotoxicity. Moreover, to determine the critical region for DIRAS3's RAS-inhibitory function, we compared wild-type DIRAS3 and deletions (ΔNTE, ΔCTE, ΔNCTE). Our results showed both termini are required to effectively suppress clonogenic growth, emphasizing the requirement for DIRAS3 to anchor to the plasma membrane and interact with the RAS dimer interface to inhibit RAS-dependent tumor growth. In conclusion, our findings underscore the potent and specific inhibitory effects of DIRAS3 on RAS-driven oncogenesis, positioning it as a promising pan-RAS inhibitor for RAS-mutant cancers. Citation Format: Gamze Bildik, Junchen Liu, Weiqun Mao, Hailing Yang, John F. Hancock, Robert C. Bast, Zhen Lu. DIRAS3 inhibits oncogenic RAS signaling and RAS-dependent cell growth driven by prevalent KRAS hot spot mutations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1215.

KRAS is frequently mutated in cancer, contributing to 20% of all human cancer especially pancreatic, colorectal and lung cancer. Signaling of the constitutively active KRAS oncogenic mutants is mostly compartmentalized to proteolipid nanoclusters on the plasma membrane (PM). Signaling nanoclusters of many KRAS mutants selectively enrich phosphatidylserine (PS) lipids with unsaturated sn-2 acyl chains, but not the fully saturated PS species. Thus, remodeling PS acyl chains may suppress KRAS oncogenesis. Lysophosphatidylcholine acyltransferases (LPCATs) remodel sn-2 acyl chains of phospholipids, with LPCAT1 preferentially generating the fully saturated lipids. Here, we show that stable expression of LPCAT1 depletes major PS species with unsaturated sn-2 chains while decreasing minor phosphatidylcholine (PC) species with the corresponding acyl chains. LPCAT1 expression more effectively disrupts the nanoclustering of oncogenic GFP-KRAS G12V , which is restored by acute addback of exogenous unsaturated PS. LPCAT1 expression compromises signaling and oncogenic activities of the KRAS-dependent pancreatic tumor lines. LPCAT1 expression sensitizes human pancreatic tumor MiaPaCa-2 cells to KRAS G12C specific inhibitor, Sotorasib. Statistical analyses of patient data further reveal that pancreatic cancer patients with KRAS mutations express less LPCAT1. Higher LPCAT1 expression also improves survival probability of pancreatic and lung adenocarcinoma patients with KRAS mutations. Thus, PS acyl chain remodeling selectively suppresses KRAS oncogenesis.

RAP1 proteins belong to the RAS family of small GTPases that operate as molecular switches by cycling between GDP-bound inactive and GTP-bound active states. The C-terminal anchors of RAP1 proteins are known to direct membrane localization, but how these anchors organize RAP1 on the plasma membrane (PM) has not been investigated. Using high-resolution imaging, we show that RAP1A and RAP1B form spatially segregated nanoclusters on the inner leaflet of the PM, with further lateral segregation between GDP-bound and GTP-bound proteins. The C-terminal polybasic anchors of RAP1A and RAP1B differ in their amino acid sequences and exhibit different lipid binding specificities, which can be modified by single-point mutations in the respective polybasic domains (PBD). Molecular dynamics simulations reveal that single PBD mutations substantially reduce the interactions of the membrane anchors with the PM lipid phosphatidylserine. In summary, we show that aggregate lipid binding specificity encoded within the C-terminal anchor determines PM association and nanoclustering of RAP1A and RAP1B. Taken together with previous observations on RAC1 and KRAS, the study reveals that the PBD sequences of small GTPase membrane anchors can encode distinct lipid binding specificities that govern PM interactions.

Oncogenic KRAS expression generates a metabolic dependency on aerobic glycolysis, known as the Warburg effect. We report an effect of increased glycolytic flux that feeds into glycosphingolipid biosynthesis and is directly linked to KRAS oncogenic function. High resolution imaging and genetic approaches show that a defined subset of outer leaflet glycosphingolipids, including GM3 and SM4, is required to maintain KRAS plasma membrane localization, with GM3 engaging in cross-bilayer coupling to maintain inner leaflet phosphatidylserine content. Thus, glycolysis is critical for KRAS plasma membrane localization and nanoscale spatial organization. Reciprocally oncogenic KRAS selectively upregulates cellular content of these same glycosphingolipids, whose depletion in turn abrogates KRAS oncogenesis in pancreatic cancer models. Our findings expand the role of the Warburg effect beyond ATP generation and biomass building to high-level regulation of KRAS function. The positive feedforward loop between oncogenic KRAS signaling and glycosphingolipid synthesis represents a vulnerability with therapeutic potential.

This file containing all the supplemental data supporting the statements indicated in the manuscripts.

RAS genes are frequently mutated in cancer. The primary signaling compartment of wild-type and constitutively active oncogenic mutant RAS proteins is the inner leaflet of the plasma membrane (PM). Thus, a better understanding of the unique environment of the PM inner leaflet is important to shed further light on RAS function. Over the past few decades, an integrated approach of superresolution imaging, molecular dynamic simulations, and biophysical assays has yielded new insights into the capacity of RAS proteins to sort lipids with specific headgroups and acyl chains, to assemble signaling nanoclusters on the inner PM. RAS proteins also sense and respond to changes in components of the outer PM leaflet, including glycophosphatidylinositol-anchored proteins, sphingophospholipids, glycosphingolipids, and galectins, as well as cholesterol that translocates between the two leaflets. Such communication between the inner and outer leaflets of the PM, called interleaflet coupling, allows RAS to potentially integrate extracellular mechanical and electrostatic information with intracellular biochemical signaling events, and reciprocally allows mutant RAS-transformed tumor cells to modify tumor microenvironments. Here, we review RAS-lipid interactions and speculate on potential mechanisms that allow communication between the opposing leaflets of the PM.

Significance Exquisite lipid-binding specificity for phosphatidylserine (PtdSer) is hardwired into the structure of the KRAS C-terminal plasma membrane (PM) anchor. This renders KRAS–PM localization and hence biological function potentially vulnerable to perturbations of PM PtdSer content. Here, we show that all components of the recently described lipid transport machinery that maintain PM PtdSer content are indeed required to support KRAS oncogenic function. In this context, we demonstrate that the enzyme, PI4KIIIα, in particular has merit as a druggable target for inhibiting KRAS -dependent tumors. More generally, we provide insight into how PM phospholipids can regulate oncogene signaling and how PM lipid composition may be successfully targeted to exploit tumor vulnerabilities.

K-ras mutations are major genetic events that drive cancer development associated with aggressive malignant phenotypes, while expression of the immune checkpoint molecule PD-L1 plays a key role in cancer evasion of the immune surveillance that also profoundly affects the patient outcome. However, the relationship between K-ras oncogenic signal and PD-L1 expressions as an important area that requires further investigation. Using both in vitro and in vivo experimental models of K-ras-driven cancer, we found that oncogenic K-ras significantly enhanced PD-L1 expression through a redox-mediated mechanism. Activation of K-ras(G12V) promoted ROS generation and induced FGFR1 expression, leading to a significant upregulation of PD-L1. We further showed that exogenous ROS such as hydrogen peroxide alone was sufficient to activate FGFR1 and induce PD-L1, while antioxidants could largely abrogate PD-L1 expression in K-ras mutant cells, indicating a critical role of redox regulation. Importantly, genetic knockout of FGFR1 led to a decrease in PD-L1 expression, and impaired tumor growth in vivo due to a significant increase of T cell infiltration in the tumor tissues and thus enhanced T-cell-mediated tumor suppression. Our study has identified a novel mechanism by which K-ras promotes PD-L1 expression, and suggests that modulation of ROS or inhibition of the FGFR1 pathway could be a novel strategy to abrogate PD-L1-mediated immunosuppression and thus potentially improve the efficacy of immunotherapy in K-ras-driven cancers.

Prior studies demonstrated that the irreversible ERBB1/2/4 inhibitor neratinib caused plasma membrane-associated mutant K-RAS to localize in intracellular vesicles, concomitant with its degradation. Herein, we discovered that neratinib interacted with the chemically distinct irreversible ERBB1/2/4 inhibitor afatinib to reduce expression of ERBB1, ERBB2, K-RAS and N-RAS; this was associated with greater-than-additive cell killing of pancreatic tumor cells. Knock down of Beclin1, ATG16L1, Rubicon or cathepsin B significantly lowered the ability of neratinib to reduce ERBB1 and K-RAS expression, and to cause tumor cell death. Knock down of ATM-AMPK suppressed vesicle formation and knock down of cathepsin B-AIF significantly reduced neratinib lethality. PKG phosphorylates K-RAS and HMG CoA reductase inhibitors reduce K-RAS farnesylation both of which remove K-RAS from the plasma membrane, abolishing its activity. Neratinib interacted with the PKG activator sildenafil and the HMG CoA reductase inhibitor atorvastatin to further reduce K-RAS expression, and to further enhance cell killing. Neratinib is also a Ste20 kinase family inhibitor and in carcinoma cells, and hematopoietic cancer cells lacking ERBB1/2/4, it reduced K-RAS expression and the phosphorylation of MST1/3/4/Ezrin by ~ 30%. Neratinib increased LATS1 phosphorylation as well as that of YAP and TAZ also by ~ 30%, caused the majority of YAP to translocate into the cytosol and reduced YAP/TAZ protein levels. Neratinib lethality was enhanced by knock down of YAP. Neratinib, in a Rubicon-dependent fashion, reduced PAK1 phosphorylation and that of its substrate Merlin. Our data demonstrate that neratinib coordinately suppresses both mutant K-RAS and YAP function to kill pancreatic tumor cells.

Abstract The acquisition of ectopic fibroblast growthfactor receptor 1 (FGFR1) expression is well documented in prostate cancer progression. How it contributes to prostate cancer progression is not fully understood, although it is known to confer a growth advantage and promote cell survival. Here, we report that FGFR1 tyrosine kinase reprograms the energy metabolism of prostate cancer cells by regulating the expression of lactate dehydrogenase (LDH) isozymes. FGFR1 increased LDHA stability through tyrosine phosphorylation and reduced LDHB expression by promoting its promoter methylation, thereby shifting cell metabolism from oxidative phosphorylation to aerobic glycolysis. LDHA depletion compromised, whereas LDHB depletion enhanced the tumorigenicity of prostate cancer cells. Furthermore, FGFR1 overexpression and aberrant LDH isozyme expression were associated with short overall survival and biochemical recurrence times in patients with prostate cancer. Our results indicate that ectopic FGFR1 expression reprograms the energy metabolism of prostate cancer cells, representing a hallmark change in prostate cancer progression. Significance: FGF signaling drives the Warburg effect through differential regulation of LDHA and LDHB, thereby promoting the progression of prostate cancer. Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/78/16/4459/F1.large.jpg. Cancer Res; 78(16); 4459–70. ©2018 AACR.

Initiation of expression of fibroblast growth factor receptor 1 (FGFR1) concurrent with loss of FGFR2 expression is a well-documented event in the progression of prostate cancer (PCa). Although it is known that some FGFR isoforms confer advantages in cell proliferation and survival, the mechanism by which the subversion of different FGFR isoforms contributes to PCa progression is incompletely understood. Here, we report that fibroblast growth factor (FGF) promotes NF-κB signaling in PCa cells and that this increase is associated with FGFR1 expression. Disruption of FGFR1 kinase activity abrogated both FGF activity and NF-κB signaling in PCa cells. Of note, the three common signaling pathways downstream of FGFR1 kinase, extracellular signal-regulated kinase 1/2 (ERK1/2), phosphoinositide 3-kinase (PI3K/AKT), and phosphoinositide phospholipase Cγ (PLCγ), were not required for FGF-mediated NF-κB signaling. Instead, transforming growth factor β-activating kinase 1 (TAK1), a central regulator of the NF-κB pathway, was required for FGFR1 to stimulate NF-κB signaling. Moreover, we found that FGFR1 promotes NF-κB signaling in PCa cells by reducing TAK1 degradation and thereby supporting sustained NF-κB activation. Consistently, Fgfr1 ablation in the transgenic adenocarcinoma of the mouse prostate (TRAMP) model reduced inflammation in the tumor microenvironment. In contrast, activation of the FGFR1 kinase in the juxtaposition of chemical-induced dimerization (CID) and kinase 1 (JOCK1) mouse model increased inflammation. As inflammation plays an important role in PCa initiation and progression, these findings suggest that ectopically expressed FGFR1 promotes PCa progression, at least in part, by increasing inflammation in the tumor microenvironment.

Fibroblast growth factor (FGF)/FGF receptor (FGFR) signaling facilitates tumor initiation and progression. Although currently approved inhibitors of FGFR kinase have shown therapeutic benefit in clinical trials, overexpression or mutations of FGFRs eventually confer drug resistance and thereby abrogate the desired activity of kinase inhibitors in many cancer types. In this study, we report that loss of myristoylation of fibroblast growth factor receptor substrate 2 (FRS2α), a scaffold protein essential for FGFR signaling, inhibits FGF/FGFR-mediated oncogenic signaling and FGF10-induced tumorigenesis. Moreover, a previously synthesized myristoyl-CoA analog, B13, which targets the activity of N-myristoyltransferases, suppressed FRS2α myristoylation and decreased the phosphorylation with mild alteration of FRS2α localization at the cell membrane. B13 inhibited oncogenic signaling induced by WT FGFRs or their drug-resistant mutants (FGFRsDRM). B13 alone or in combination with an FGFR inhibitor suppressed FGF-induced WT FGFR- or FGFRDRM-initiated phosphoinositide 3-kinase (PI3K) activity or MAPK signaling, inducing cell cycle arrest and thereby inhibiting cell proliferation and migration in several cancer cell types. Finally, B13 significantly inhibited the growth of xenograft tumors without pathological toxicity to the liver, kidney, or lung in vivo In summary, our study suggests a possible therapeutic approach for inhibiting FGF/FGFR-mediated cancer progression and drug-resistant FGF/FGFR mutants.

BUB1 mitotic checkpoint serine/threonine kinase B (BUB1B) is a member of the spindle assembly checkpoint protein family, which has been proven to be associated with many kinds of cancers. The aim of this study was to investigate whether BUB1B was correlated with progression and prognosis in patients with prostate cancer (PCa) and how BUB1B regulated the proliferation, migration, and invasion of PCa cell lines. Compared to benign prostate cells and tissues, both messenger RNA and protein expressions of BUB1B were statistically increased in PCa cell lines and tumor tissues. In vitro studies revealed that BUB1B overexpression enhanced the proliferation, migration, and invasion ability of PCa cell lines, whereas depletion of BUB1B did not affect the cell functions. Microarray analysis showed the positive staining of BUB1B was upregulated in the higher Gleason score group, which also correlated with advanced clinicopathological stage, higher serum prostate-specific antigen, metastasis, overall survival, and prostate-specific antigen failure. Furthermore, the survival analysis indicated that high expression of BUB1B was an independent predictor for shorter biochemical recurrence-free survival, which had no effect on overall survival. BUB1B plays an important role in tumor growth and progression, which can lead to its use as a potential biomarker for the diagnosis and prognosis of PCa.

Metastasis of tumors requires angiogenesis, which is comprised of multiple biological processes that are regulated by angiogenic factors. The fibroblast growth factor (FGF) is a potent angiogenic factor and aberrant FGF signaling is a common property of tumors. Yet, how the aberration in cancer cells contributes to angiogenesis in the tumor is not well understood. Most studies of its angiogenic signaling mechanisms have been in endothelial cells. FGF receptor substrate 2α (FRS2α) is an FGF receptor-associated protein required for activation of downstream signaling molecules that include those in the mitogen-activated protein and AKT kinase pathways. Herein, we demonstrated that overactivation and hyperactivity of FRS2α, as well as overexpression of cJUN and HIF1α, were positively correlated with vessel density and progression of human prostate cancer (PCa) toward malignancy. We also demonstrate that FGF upregulated the production of vascular endothelial growth factor A mainly by increasing expression of cJUN and HIF1α. This then promoted recruitment of endothelial cells and vessel formation for the tumor. Tumor angiogenesis in mouse PCa tissues was compromised by tissue-specific ablation of Frs2α in prostate epithelial cells. Depletion of Frs2α expression in human PCa cells and in a preclinical xenograft model, MDA PCa 118b, also significantly suppressed tumor angiogenesis accompanied with decreased tumor growth in the bone. The results underscore the angiogenic role of FRS2α-mediated signaling in tumor epithelial cells in angiogenesis. They provide a rationale for treating PCa with inhibitors of FGF signaling. They also demonstrate the potential of overexpressed FRS2α as a biomarker for PCa diagnosis, prognosis and response to therapies.

Background: FGF signaling controls development and regeneration of the prostate. However, its role in prostate stem cells is not clear. Results: FGFR2 in basal prostate stem cells (P-bSCs) controls their self-renewal and differentiation. FGFR2 deficiency in P-bSCs impaired postnatal development of the prostate. Conclusion: FGFR2 signaling maintains P-bSC stemness. Significance: The findings suggest a novel avenue to manipulate P-bSC self-renewal and dormancy. Prostate stem cells (P-SCs) are capable of giving rise to all three lineages of prostate epithelial cells, which include basal, luminal, and neuroendocrine cells. Two types of P-SCs have been identified in both human and mouse adult prostates based on prostasphere or organoid cultures, cell lineage tracing, renal capsule implantation, and expression of luminal- and basal-specific proteins. The sphere-forming P-SCs are from the basal cell compartment that express P63, and are therefore designated as basal P-SCs (P-bSCs). Luminal P-SCs (P-lSCs) express luminal cytokeratins and Nkx3.1. Herein, we report that the type 2 FGF receptor (FGFR2) signaling axis is crucial for preserving stemness and preventing differentiation of P-bSCs. FGFR2 signaling mediated by FGFR substrate 2α (FRS2α) is indispensable for formation and maintenance of prostaspheres derived from P63+ P-bSCs. Ablation of Fgfr2 in P63+ cells in vitro causes the disintegration of prostaspheres. Ablation of Fgfr2 in vivo reduces the number of P63-expressing basal cells and enriches luminal cells. This suggests a basal stem cell-to-luminal cell differentiation. In addition, ablation of Fgfr2 in P63+ cells causes defective postnatal development of the prostate. Therefore, the data indicate that FGFR2 signaling is critical for preserving stemness and preventing differentiation of P-bSCs.

Prostate stem cells (P-SCs) are capable of giving rise to all three lineages of prostate epithelial cells, including basal, luminal, and neuroendocrine cells. Multiple methods have been used to identify P-SCs in adult prostates. These include in vivo renal capsule implantation of a single epithelial cell with urogenital mesenchymal cells, in vitro prostasphere and organoid cultures, and lineage tracing with castration-resistant Nkx3.1 expression (CARN), in conjunction with expression of cell type-specific markers. Both organoid culture and CARN tracing show the existence of P-SCs in the luminal compartment. Although prostasphere cells predominantly express basal cell-specific cytokeratin and P63, the lineage of prostasphere-forming cells in the P-SC hierarchy remains to be determined. Using lineage tracing with P63(CreERT2), we show here that the sphere-forming P-SCs are P63-expressing cells and reside in the basal compartment. Therefore we designate them as basal P-SCs (P-bSCs). P-bSCs are capable of differentiating into AR(+) and CK18(+) organoid cells, but organoid cells cannot form spheres. We also report that prostaspheres contain quiescent stem cells. Therefore, the results show that P-bSCs represent stem cells that are early in the hierarchy of overall prostate tissue stem cells. Understanding the contribution of the two types of P-SCs to prostate development and prostate cancer stem cells and how to manipulate them may open new avenues for control of prostate cancer progression and relapse.

Bone metastasis is the major cause of morbidity and mortality of prostate cancer (PCa).Fibroblast growth factor 9 (FGF9) has been reported to promote PCa bone metastasis.However, the mechanism by which overexpression of FGF9 promotes PCa progression and metastasis is still unknown.Herein, we report that transgenic mice forced to express FGF9 in prostate epithelial cells (F9TG) developed high grade prostatic intraepithelial neoplasia (PIN) in an expression leveland time-dependent manner.Moreover, FGF9/TRAMP bigenic mice (F9TRAMP) grew advanced PCa earlier and had higher frequencies of metastasis than TRAMP littermates.We observed tumor microenvironmental changes including hypercellularity and hyperproliferation in the stromal compartment of F9TG and F9TRAMP mice.Expression of TGFβ1, a key signaling molecule overexpressed in reactive stroma, was increased in F9TG and F9TRAMP prostates.Both in vivo and in vitro data indicated that FGF9 promoted TGFβ1 expression via increasing cJun-mediated signaling.Moreover, in silico analyses showed that the expression level of FGF9 was positively associated with expression of TGFβ1 and its downstream signaling molecules in human prostate cancers.Collectively, our data demonstrated that overexpressing FGF9 in PCa cells augmented the formation of reactive stroma and promoted PCa initiation and progression.

A constant supply of epithelial cells from dental epithelial stem cell (DESC) niches in the cervical loop (CL) enables mouse incisors to grow continuously throughout life. Elucidation of the cellular and molecular mechanisms underlying this unlimited growth potential is of broad interest for tooth regenerative therapies. Fibroblast growth factor (FGF) signaling is essential for the development of mouse incisors and for maintenance of the CL during prenatal development. However, how FGF signaling in DESCs controls the self-renewal and differentiation of the cells is not well understood. Herein, we report that FGF signaling is essential for self-renewal and the prevention of cell differentiation of DESCs in the CL as well as in DESC spheres. Inhibiting the FGF signaling pathway decreased proliferation and increased apoptosis of the cells in DESC spheres. Suppressing FGFR or its downstream signal transduction pathways diminished Lgr5-expressing cells in the CL and promoted cell differentiation both in DESC spheres and the CL. Furthermore, disruption of the FGF pathway abrogated Wnt signaling to promote Lgr5 expression in DESCs both in vitro and in vivo. This study sheds new light on understanding the mechanism by which the homeostasis, expansion, and differentiation of DESCs are regulated.

Understanding the cellular and molecular mechanisms underlying the self-renewal and differentiation of dental epithelial stem cells (DESCs) that support the unlimited growth potential of mouse incisors is critical for developing novel tooth regenerative therapies and unraveling the pathogenesis of odontogenic tumors. However, analysis of DESC properties and regulation has been limited by the lack of an in vitro assay system and well-documented DESC markers. Here, we describe an in vitro sphere culture system to isolate the DESCs from postnatal mouse incisor cervical loops (CLs) where the DESCs are thought to reside. The dissociated cells from CLs were able to expand and form spheres for multiple generations in the culture system. Lineage tracing indicated that DESC within the spheres were epithelial in origin as evident by lineage tracing. Upon stimulation, the sphere cells differentiated into cytokeratin 14- and amelogenin-expressing and mineral material-producing cells. Compared to the CL tissue, sphere cells expressed high levels of expression of Sca-1, CD49f (also designated as integrin α6), and CD44. Fluorescence-activated cell sorting (FACS) analyses of mouse incisor CL cells further showed that the CD49f(Bright) population was enriched in sphere-forming cells. In addition, the CD49f(Bright) population includes both slow-cycling and Lgr5(+) DESCs. The in vitro sphere culture system and identification of CD49f(Bright) as a DESC marker provide a novel platform for enriching DESCs, interrogating how maintenance, cell fate determination, and differentiation of DESCs are regulated, and developing tooth regenerative therapies.