Disruption of stem cell niche-confined R-spondin 3 expression leads to impaired hematopoiesis.

Self-renewal and differentiation of stem and progenitor cells are tightly regulated to ensure tissue homeostasis. This regulation is enabled both remotely by systemic circulating cues, such as cytokines and hormones, and locally by various niche-confined factors. R-spondin 3 (RSPO3) is one of the most potent enhancers of Wnt signaling, and its expression is usually restricted to the stem cell niche where it provides localized enhancement of Wnt signaling to regulate stem cell expansion and differentiation. Disruption of this niche-confined expression can disturb proper tissue organization and lead to cancers. Here, we investigate the consequences of disrupting the niche-restricted expression of RSPO3 in various tissues, including the hematopoietic system. We show that normal Rspo3 expression is confined to the perivascular niche in the bone marrow. Induction of increased systemic levels of circulating RSPO3 outside of the niche results in prominent loss of early B-cell progenitors and anemia but surprisingly has no effect on hematopoietic stem cells. Using molecular, pharmacologic, and genetic approaches, we show that these RSPO3-induced hematopoietic phenotypes are Wnt and RSPO3 dependent and mediated through noncanonical Wnt signaling. Our study highlights a distinct role for a Wnt/RSPO3 signaling axis in the regulation of hematopoiesis, as well as possible challenges related to therapeutic use of RSPOs for regenerative medicine.

Collagen-rich airway smooth muscle cells are a metastatic niche for tumor colonization in the lung.

Metastases account for the majority of cancer deaths. While certain steps of the metastatic cascade are well characterized, identification of targets to block this process remains a challenge. Host factors determining metastatic colonization to secondary organs are particularly important for exploration, as those might be shared among different cancer types. Here, we showed that bladder tumor cells expressing the collagen receptor, CD167a, responded to collagen I stimulation at the primary tumor to promote local invasion and utilized the same receptor to preferentially colonize at airway smooth muscle cells (ASMCs)-a rich source of collagen III in lung. Morphologically, COL3-CD167a-driven metastatic foci are uniquely distinct from typical lung alveolar metastatic lesions and exhibited activation of the CD167a-HSP90-Stat3 axis. Importantly, metastatic lung colonization could be abrogated using an investigational drug that attenuates Stat3 activity, implicating this seed-and-soil interaction as a therapeutic target for eliminating lung metastasis.

Stem cell plasticity enables hair regeneration following Lgr5+ cell loss.

Under injury conditions, dedicated stem cell populations govern tissue regeneration. However, the molecular mechanisms that induce stem cell regeneration and enable plasticity are poorly understood. Here, we investigate stem cell recovery in the context of the hair follicle to understand how two molecularly distinct stem cell populations are integrated. Utilizing diphtheria-toxin-mediated cell ablation of Lgr5+ (leucine-rich repeat-containing G-protein-coupled receptor 5) stem cells, we show that killing of Lgr5+ cells in mice abrogates hair regeneration but this is reversible. During recovery, CD34+ (CD34 antigen) stem cells activate inflammatory response programs and start dividing. Pharmacological attenuation of inflammation inhibits CD34+ cell proliferation. Subsequently, the Wnt pathway controls the recovery of Lgr5+ cells and inhibition of Wnt signalling prevents Lgr5+ cell and hair germ recovery. Thus, our study uncovers a compensatory relationship between two stem cell populations and the underlying molecular mechanisms that enable hair follicle regeneration.

A distinct role for Lgr5+ stem cells in primary and metastatic colon cancer.

Cancer stem cells (CSCs) have been hypothesized to represent the driving force behind tumour progression and metastasis, making them attractive cancer targets. However, conclusive experimental evidence for their functional relevance is still lacking for most malignancies. Here we show that the leucine-rich repeat-containing G-protein-coupled receptor 5 (Lgr5) identifies intestinal CSCs in mouse tumours engineered to recapitulate the clinical progression of human colorectal cancer. We demonstrate that selective Lgr5+ cell ablation restricts primary tumour growth, but does not result in tumour regression. Instead, tumours are maintained by proliferative Lgr5- cells that continuously attempt to replenish the Lgr5+ CSC pool, leading to rapid re-initiation of tumour growth upon treatment cessation. Notably, CSCs are critical for the formation and maintenance of liver metastasis derived from colorectal cancers. Together, our data highlight distinct CSC dependencies for primary versus metastasic tumour growth, and suggest that targeting CSCs may represent a therapeutic opportunity for managing metastatic disease.

Cellular Hierarchy as a Determinant of Tumor Sensitivity to Chemotherapy.

Chemotherapy has been shown to enrich cancer stem cells in tumors. Recently, we demonstrated that administration of chemotherapy to human bladder cancer xenografts could trigger a wound-healing response that mobilizes quiescent tumor stem cells into active proliferation. This phenomenon leads to a loss of sensitivity to chemotherapy partly due to an increase in the number of tumor stem cells, which typically respond to chemotherapy-induced cell death less than more differentiated cells. Different bladder cancer xenografts, however, demonstrate differential sensitivities to chemotherapy, the basis of which is not understood. Using mathematical models, we show that characteristics of the tumor cell hierarchy can be crucial for determining the sensitivity of tumors to drug therapy, under the assumption that stem cell enrichment is the primary basis for drug resistance. Intriguingly, our model predicts a weaker response to therapy if there is negative feedback from differentiated tumor cells that inhibits the rate of tumor stem cell division. If this negative feedback is less pronounced, the treatment response is predicted to be enhanced. The reason is that negative feedback on the rate of tumor cell division promotes a permanent rise of the tumor stem cell population over time, both in the absence of treatment and even more so during drug therapy. Model application to data from chemotherapy-treated patient-derived xenografts indicates support for model predictions. These findings call for further research into feedback mechanisms that might remain active in cancers and potentially highlight the presence of feedback as an indication to combine chemotherapy with approaches that limit the process of tumor stem cell enrichment. Cancer Res; 77(9); 2231-41. ©2017 AACR.

Positive association of collagen type I with non-muscle invasive bladder cancer progression.

PURPOSE: Non-muscle invasive bladder cancers (NMIBC) are generally curable, while ~15% progresses into muscle-invasive cancer with poor prognosis. While efforts have been made to identify genetic alternations associated with progression, the extracellular matrix (ECM) microenvironment remains largely unexplored. Type I collagen is a major component of the bladder ECM, and can be altered during cancer progression. We set out to explore the association of type I collagen with NMIBC progression. EXPERIMENTAL DESIGN: The associations of COL1A1 and COL1A2 mRNA levels with progression were evaluated in a multi-center cohort of 189 patients with NMIBCs. Type I collagen protein expression and structure were evaluated in an independent single-center cohort of 80 patients with NMIBCs. Immunohistochemical analysis was performed and state-of-the-art multi-photon microscopy was used to evaluate collagen structure via second harmonic generation imaging. Progression to muscle invasion was the primary outcome. Kaplan-Meier method, Cox regression, and Wilcoxon rank-sum were used for statistical analysis. RESULTS: There is a significant association of high COL1A1 and COL1A2 mRNA expression in patients with poor progression-free survival (P=0.0037 and P=0.011, respectively) and overall survival (P=0.024 and P=0.012, respectively). Additionally, immunohistochemistry analysis of type I collagen protein deposition revealed a significant association with progression (P=0.0145); Second-harmonic generation imaging revealed a significant lower collagen fiber curvature ratio in patients with invasive progression (P = 0.0018). CONCLUSIONS: Alterations in the ECM microenvironment, particularly type I collagen, likely contributes to bladder cancer progression. These findings will open avenues to future functional studies to investigate ECM-tumor interaction as a potential therapeutic intervention to treat NMIBCs.

Blocking PGE2-induced tumour repopulation abrogates bladder cancer chemoresistance.

Cytotoxic chemotherapy is effective in debulking tumour masses initially; however, in some patients tumours become progressively unresponsive after multiple treatment cycles. Previous studies have demonstrated that cancer stem cells (CSCs) are selectively enriched after chemotherapy through enhanced survival. Here we reveal a new mechanism by which bladder CSCs actively contribute to therapeutic resistance via an unexpected proliferative response to repopulate residual tumours between chemotherapy cycles, using human bladder cancer xenografts. Further analyses demonstrate the recruitment of a quiescent label-retaining pool of CSCs into cell division in response to chemotherapy-induced damages, similar to mobilization of normal stem cells during wound repair. While chemotherapy effectively induces apoptosis, associated prostaglandin E2 (PGE2) release paradoxically promotes neighbouring CSC repopulation. This repopulation can be abrogated by a PGE2-neutralizing antibody and celecoxib drug-mediated blockade of PGE2 signalling. In vivo administration of the cyclooxygenase-2 (COX2) inhibitor celecoxib effectively abolishes a PGE2- and COX2-mediated wound response gene signature, and attenuates progressive manifestation of chemoresistance in xenograft tumours, including primary xenografts derived from a patient who was resistant to chemotherapy. Collectively, these findings uncover a new underlying mechanism that models the progressive development of clinical chemoresistance, and implicate an adjunctive therapy to enhance chemotherapeutic response of bladder urothelial carcinomas by abrogating early tumour repopulation.

CD49d is the strongest flow cytometry-based predictor of overall survival in chronic lymphocytic leukemia.

PURPOSE: Although CD49d is an unfavorable prognostic marker in chronic lymphocytic leukemia (CLL), definitive validation evidence is lacking. A worldwide multicenter analysis was performed using published and unpublished CLL series to evaluate the impact of CD49d as an overall (OS) and treatment-free survival (TFS) predictor. PATIENTS AND METHODS: A training/validation strategy was chosen to find the optimal CD49d cutoff. The hazard ratio (HR) for death and treatment imposed by CD49d was estimated by pooled analysis of 2,972 CLLs; Cox analysis stratified by center and stage was used to adjust for confounding variables. The importance of CD49d over other flow cytometry-based prognosticators (eg, CD38, ZAP-70) was ranked by recursive partitioning. RESULTS: Patients with ≥ 30% of neoplastic cells expressing CD49d were considered CD49d+. Decrease in OS at 5 and 10 years among CD49d+ patients was 7% and 23% (decrease in TFS, 26% and 25%, respectively). Pooled HR of CD49d for OS was 2.5 (2.3 for TFS) in univariate analysis. This HR remained significant and of similar magnitude (HR, 2.0) in a Cox model adjusted for clinical and biologic prognosticators. Hierarchic trees including all patients or restricted to those with early-stage disease or those age ≤ 65 years always selected CD49d as the most important flow cytometry-based biomarker, with negligible additional prognostic information added by CD38 or ZAP-70. Consistently, by bivariate analysis, CD49d reliably identified patient subsets with poorer outcome independent of CD38 and ZAP-70. CONCLUSION: In this analysis of approximately 3,000 patients, CD49d emerged as the strongest flow cytometry-based predictor of OS and TFS in CLL.

Normal and neoplastic urothelial stem cells: getting to the root of the problem.

Most epithelial tissues contain self-renewing stem cells that mature into downstream progenies with increasingly limited differentiation potential. It is not surprising that cancers arising from such hierarchically organized epithelial tissues retain features of cellular differentiation. Accumulating evidence suggests that the urothelium of the urinary bladder is a hierarchically organized tissue, containing tissue-specific stem cells that are important for both normal homeostasis and injury response. The phenotypic and functional properties of cancer stem cells (CSCs; also known as tumour-initiating cells) from bladder cancer tissue have been studied in detail. Urothelial CSCs are not isolated by a 'one-marker-fits-all' approach; instead, various cell surface marker combinations (possibly reflecting the cell-of-origin) are used to isolate CSCs from distinct differentiation subtypes of urothelial carcinomas. Additional CSC markers, including cytokeratin 14 (CK14), aldehyde dehydrogenase 1 family, member A1 (ALDH1A1), and tumour protein 63 (p63), have revealed prognostic value for urothelial carcinomas. Signalling pathways involved in normal stem cell self-renewal and differentiation are implicated in the malignant transformation of different subsets of urothelial carcinomas. Early expansion of primitive CK14+ cells--driven by genetic pathways such as STAT3--can lead to the development of carcinoma in situ, and CSC-enriched urothelial carcinomas are associated with poor clinical outcomes. Given that bladder CSCs are the proposed root of malignancy and drivers of cancer initiation and progression for urothelial carcinomas, these cells are ideal targets for anticancer therapies.

Three differentiation states risk-stratify bladder cancer into distinct subtypes.

Current clinical judgment in bladder cancer (BC) relies primarily on pathological stage and grade. We investigated whether a molecular classification of tumor cell differentiation, based on a developmental biology approach, can provide additional prognostic information. Exploiting large preexisting gene-expression databases, we developed a biologically supervised computational model to predict markers that correspond with BC differentiation. To provide mechanistic insight, we assessed relative tumorigenicity and differentiation potential via xenotransplantation. We then correlated the prognostic utility of the identified markers to outcomes within gene expression and formalin-fixed paraffin-embedded (FFPE) tissue datasets. Our data indicate that BC can be subclassified into three subtypes, on the basis of their differentiation states: basal, intermediate, and differentiated, where only the most primitive tumor cell subpopulation within each subtype is capable of generating xenograft tumors and recapitulating downstream populations. We found that keratin 14 (KRT14) marks the most primitive differentiation state that precedes KRT5 and KRT20 expression. Furthermore, KRT14 expression is consistently associated with worse prognosis in both univariate and multivariate analyses. We identify here three distinct BC subtypes on the basis of their differentiation states, each harboring a unique tumor-initiating population.

Diverse marrow stromal cells protect CLL cells from spontaneous and drug-induced apoptosis: development of a reliable and reproducible system to assess stromal cell adhesion-mediated drug resistance.

Marrow stromal cells (MSCs) provide important survival and drug resistance signals to chronic lymphocytic leukemia (CLL) cells, but current models to analyze CLL-MSC interactions are heterogeneous. Therefore, we tested different human and murine MSC lines and primary human MSCs for their ability to protect CLL cells from spontaneous and drug-induced apoptosis. Our results show that both human and murine MSCs are equally effective in protecting CLL cells from fludarabine-induced apoptosis. This protective effect was sustained over a wide range of CLL-MSC ratios (5:1 to 100:1), and the levels of protection were reproducible in 4 different laboratories. Human and murine MSCs also protected CLL cells from dexamethasone- and cyclophosphamide-induced apoptosis. This protection required cell-cell contact and was virtually absent when CLL cells were separated from the MSCs by micropore filters. Furthermore, MSCs maintained Mcl-1 and protected CLL cells from spontaneous and fludarabine-induced Mcl-1 and PARP cleavage. Collectively, these studies define common denominators for CLL cocultures with MSCs. They also provide a reliable, validated tool for future investigations into the mechanism of MSC-CLL cross talk and for drug testing in a more relevant fashion than the commonly used suspension cultures.

B-cell antigen receptor signaling enhances chronic lymphocytic leukemia cell migration and survival: specific targeting with a novel spleen tyrosine kinase inhibitor, R406.

Antigenic stimulation through the B-cell antigen receptor (BCR) is considered to promote the expansion of chronic lymphocytic leukemia (CLL) B cells. The spleen tyrosine kinase (Syk), a key component of BCR signaling, can be blocked by R406, a small-molecule Syk inhibitor, that displayed activity in CLL patients in a first clinical trial. In this study, we investigated the effects of BCR stimulation and R406 on CLL cell survival and migration. The prosurvival effects promoted by anti-IgM stimulation and nurselike cells were abrogated by R406. BCR triggering up-regulated adhesion molecules, and increased CLL cell migration toward the chemokines CXCL12 and CXCL13. BCR activation also enhanced CLL cell migration beneath marrow stromal cells. These responses were blocked by R406, which furthermore abrogated BCR-dependent secretion of T-cell chemokines (CCL3 and CCL4) by CLL cells. Finally, R406 inhibited constitutive and BCR-induced activation of Syk, extracellular signal-regulated kinases, and AKT, and blocked BCR-induced calcium mobilization. These findings suggest that BCR activation favors CLL cell homing, retention, and survival in tissue microenvironments. R406 effectively blocks these BCR-dependent responses in CLL cells, providing an explanation for the activity of R406 in patients with CLL.

Isoform-selective phosphoinositide 3'-kinase inhibitors inhibit CXCR4 signaling and overcome stromal cell-mediated drug resistance in chronic lymphocytic leukemia: a novel therapeutic approach.

Phosphoinositide 3-kinases (PI3Ks) are among the most frequently activated signaling pathways in cancer. In chronic lymphocytic leukemia (CLL), signals from the microenvironment are critical for expansion of the malignant B cells, and cause constitutive activation of PI3Ks. CXCR4 is a key receptor for CLL cell migration and adhesion to marrow stromal cells (MSCs). Because of the importance of CXCR4 and PI3Ks for CLL-microenvironment cross-talk, we investigated the activity of novel, isoform-selective PI3K inhibitors that target different isoforms of the p110-kDa subunit. Inhibition with p110alpha inhibitors (PIK-90 and PI-103) resulted in a significant reduction of chemotaxis and actin polymerization to CXCL12 and reduced migration beneath MSC (pseudoemperipolesis). Western blot and reverse phase protein array analyses consistently demonstrated that PIK-90 and PI-103 inhibited phosphorylation of Akt and S6, whereas p110delta or p110beta/p110delta inhibitors were less effective. In suspension and MSC cocultures, PI-103 and PIK-90 were potent inducers of CLL cell apoptosis. Moreover, these p110alpha inhibitors enhanced the cytotoxicity of fludarabine and reversed the protective effect of MSC on fludarabine-induced apoptosis. Collectively, our data demonstrate that p110alpha inhibitors antagonize stromal cell-derived migration, survival, and drug-resistance signals and therefore provide a rational to explore the therapeutic activity of these promising agents in CLL.

Mantle cell lymphoma cells express high levels of CXCR4, CXCR5, and VLA-4 (CD49d): importance for interactions with the stromal microenvironment and specific targeting.

Mantle cell lymphoma (MCL) is characterized by an early, widespread dissemination and residual disease after conventional treatment, but the mechanisms responsible for lymphoma cell motility and drug resistance are largely unknown. There is growing evidence suggesting that chemokine receptors and adhesion molecules are critical for malignant B-cell trafficking and homing to supportive tissue microenvironments, where they receive survival and drug resistance signals. Therefore, we examined chemokine receptor and adhesion molecule expression and function in MCL cells and their importance for migration and adhesion to marrow stromal cells (MSCs). We found that MCL cells display high levels of functional CXCR4 and CXCR5 chemokine receptors and VLA-4 adhesion molecules. We also report that MCL cells adhere and spontaneously migrate beneath MSCs in a CXCR4- and VLA-4-dependent fashion (pseudoemperipolesis). Moreover, we demonstrate that MSCs confer drug resistance to MCL cells, particularly to MCL cells that migrate beneath MSC. To target MCL-MSC interactions, we tested Plerixafor, a CXCR4 antagonist, and natalizumab, a VLA-4 antibody. Both agents blocked functional responses to the respective ligands and inhibited adhesive interactions between MCL cells and MSCs. These findings provide a rationale to further investigate the therapeutic potential of these drugs in MCL.