Hedgehog Signaling in Normal and Malignant Hematopoiesis.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3381-3381 ◽  
Author(s):  
Akil A. Merchant ◽  
Giselle A. Joseph ◽  
Evan Jones ◽  
Tara Lin ◽  
B. Doug Smith ◽  
...  

Abstract The Hedgehog (Hh) signaling pathway is critical for normal development and dictates the self-renewal, proliferation and differentiation of normal stem cells and progenitors. Aberrant reactivation of Hh signaling has been described in a wide variety of human cancers and its role in normal stem cells suggest that pathway dysregulation contributes to oncogenesis and influences the cell fate decisions in cancer stem cells (CSC). Like their normal counterparts, CSC appear to undergo self-renewal as well as give rise to differentiated progeny, and these properties implicate that CSC are responsible for continual tumor cell production that underlies the initiation, maintenance and progression of clinical disease. Myeloid leukemias have long served as the model system for human CSC, but the cellular processes responsible for regulating these rare biologically distinct cell populations have remained unclear. We hypothesized that Hh pathway activation contributes to the pathogenesis of acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS) and studied Hh signaling in these settings. Using both RT-PCR for pathway components and a Gli1 reporter assay, we have found that Hh signaling is active in several human AML derived cell lines (Kasumi-1, KG1, KG1a) and primary AML and MDS samples. Approximately 80% (19/24) of primary AML samples tested express the downstream effectors GLI1 or GLI2 indicative of active Hh signaling. Furthermore, inhibition of Hh signaling with the naturally derived SMOOTHENED antagonist cyclopamine reduces the clonogenic growth of KG1 cells implicating the pathway in self-renewal. In contrast, cyclopamine failed to affect colony growth in the HL-60 cell line that lacks expression of Hh pathway signaling components, confirming that the effect of Hh inhibition is specific. In addition, the ectopic expression of Gli1 in KG1 cells partially rescued the effect of cyclopamine on colony formation further demonstrating the specific nature of this compound. We also studied normal CD34+ bone marrow cells and found that they expressed components of Hh pathway by RT-PCR. However, in contrast to KG1 cells, cyclopamine had little effect on the recovery of either normal hematopoietic progenitors or stem cells in an in vitro long-term culture assay. Therefore, it appears that Hh inhibition may preferentially inhibit myeloid leukemias. We further studied the role of Hh pathway activation on normal hematopoiesis and developed a transgenic mouse model in which SMOOTHENED is conditionally over-expressed in the myeloid lineage via Cre recombinase activity regulated by the Lysozyme promoter. Analysis of these mice demonstrated only subtle changes in peripheral blood counts, but further analysis of cells expressing the transgene revealed a significant reduction in the number of mature myeloid cells. This was confirmed by analyzing blood cells for the granulocyte marker Gr1 and pan-myeloid marker Mac1, both of which were significantly reduced in the SMOOTHENED over-expressing cells. These defects are reminiscent of MDS and further suggest that the Hh signaling pathway plays a role in normal hematopoiesis. Therefore, aberrant Hh pathway activation is a feature of myeloid leukemias and inhibitors such as cyclopamine may have a therapeutic role in the treatment of AML and MDS.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3582-3582
Author(s):  
Qiuju Wang ◽  
Craig Peacock ◽  
Kortney Hensley ◽  
Sarah Brennan ◽  
Akil Merchant ◽  
...  

Abstract Aberrant self-renewal is a hallmark of cancer and is central to the initiation, maintenance and relapse of clinical disease. The cellular processes responsible for self-renewal have not been delineated in most human cancers, but it is likely that conserved pathways required for the regulation of normal stem cells are involved. Notably several highly conserved signaling pathways that regulate stem cell fate decisions during embryonic development, such as Notch, Hedgehog (Hh) and Wingless (Wnt), are inappropriately activated in a wide variety of human cancers. We recently demonstrated that the Hh signaling pathway is required for the maintenance of cancer stem cells in the plasma cell malignancy, multiple myeloma. Since myeloma stem cells phenotypically resemble normal B cells, we hypothesized that aberrant Hh signaling is a feature of other B cell malignancies. We studied established human cell lines derived from patients with classical Hodgkin lymphoma (L428, KM-H2), diffuse large B cell NHL (HT, Pfeiffer, RL, and Hs 602), and mantle cell NHL (Granta 519, Jeko-1, Rec-1) and found that expression of the Hh signaling pathway components PATCHED (PTCH), SMOOTHENED (SMO), and GLI1, 2 or 3 by RT-PCR was markedly elevated compared to normal B cells in the majority of cell lines from each subtype of lymphoma. In order to examine the functional role of Hh signaling on human lymphomas, cells were treated with recombinant human sonic Hh ligand (SHh) or the naturally occurring inhibitor of SMO, cyclopamine, followed by evaluation of clonogenic growth in methylcellulose. Resulting colony formation was significantly increased in response to activating SHh ligand, whereas treatment with cyclopamine significantly inhibited clonogenic recovery. Similarly, the inhibition of pathway signaling by neutralizing anti-Hh antibodies limited colony formation suggesting that ligand binding by PTCH was required for pathway activation similar to other non-Gorlins tumors such as small cell lung cancer, pancreatic carcinoma, and metastatic prostate cancer that lack mutation of pathway components. Furthermore, we examined the activity of a novel semi-synthetic cyclopamine analogue, IPI-609, and found that it also limited clonogenic lymphoma growth. The effects of IPI-609 were highly specific as the clonogenic recovery of cell lines lacking expression of Hh pathway components was not affected by treatment. Our previous studies in multiple myeloma have suggested that cancer stem cells can be identified by their relatively higher activity of the intracellular enzyme retinaldehyde dehydrogenase (ALDH) similar to normal hematopoietic and neural stem cells. We found that high ALDH activity could also identify rare cell populations with greater clonogenic growth potential compared to ALDHlow/neg cells in the majority of lines and treatment with cyclopamine or IPI-609 significantly reduced the relative proportion of ALDHhigh cells. Therefore, the Hh signaling pathway may represent a novel therapeutic target in human lymphomas. Moreover, novel Hh inhibitors, such as IPI-609, may inhibit highly clonogenic lymphoma cancer stem cells responsible for disease relapse.


Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2578-2578
Author(s):  
Fabian Lang ◽  
Susanne Badura ◽  
Martin Ruthardt ◽  
Michael A. Rieger ◽  
Oliver G. Ottmann

Abstract Abstract 2578 The Hedgehog (Hh) pathway plays a functional role in embryonic development and promotes tumorigenesis in a diversity of cancers. Constitutive activation of Smo, an essential component of the Hh pathway, augments stem cell number and accelerates disease in BCR-ABL positive CML, whereas loss of Smo depletes CML stem cells by inhibition of self-renewal. Phase I clinical trials using Hh inhibitors have started in BCR-ABL pos ALL and CML. The role of Hh signalling on stem cell behaviour in BCR-ABL neg ALL has not yet been examined. The phenotype of leukemic stem cells (LSCs) and the target cells for transformation in ALL are controversial, but only a small subpopulation of cells seem to act as LSCs. These cells may be the most relevant targets for treatment regimens for compounds that interfere with self-renewal programs and that provide promising therapeutic options for improving treatment of adult ALL. Aims of the study are characterization of different genetically and phenotipically defined ALLs, using our twelve patient derived long term cultures (LTCs), according to their biologic behaviour including leukemia initiating capacity, assessment of the impact of Hh inhibition on proliferation, apoptosis and clonogenic capacity and LSC function and dissection of the role of different components of the Hh signalling pathway on cell fate decisions by means of single cell video microscopy. These studies are anticipated to yield information on the therapeutic potential of modulation of Hh signalling in both BCR-ABL pos and neg ALL and the potential value of combination therapy. As models of BCR-ABL pos and neg leukemias we used serum-, cytokine- and stroma-free long term cultures of primary ALL blasts. Clonogenic growth of ALL cells was assessed in semi solid methylcellulose based media. Cell subpopulations were isolated on the basis of CD20, CD34 and CD38 expression via FACS based sorting (BD FACS Aria). Cell proliferation was measured using XTT assays and Annexin V and 7 AAD FACS staining were used to quantitate apoptosis. Quantitative RT PCR of Hh signalling pathway components using predeveloped Taqman assays (Applied). Single cell video tracking to determine cell fate decisions was performed as previously described (Rieger et all, Science 2009), adapted to facilitate the analysis of ALL LTCs. Two novel Smo inhibitors being currently in clinical testing, LDE225 and BMS833923 were kindly provided by Novartis and Bristol Myers Squibb. Results: The expression pattern of surface markers varied profoundly between the different LTCs studied. In preliminary experiments designed to identify functionally distinct subpopulations of long term cultured ALL blasts, cells were isolated to greater than 90% purity based on CD20, CD34 and CD38 expression. With the exception of CD34 positive cells, the surface marker distribution rapidly reverted to an identical pattern as determined prior to culture in three cell lines studied. In two ALL LTCs, CD34 expression was associated with slower proliferation. All three cell lines displayed clonogenic capacity ranging from 0,25% to 8% and are able to engraft in NSG mice. Analysis of Hh Signalling in ALL LTCs by RT PCR demonstrated expression of Shh, Ptch, Smo, and the transcription factors Gli 1 + 3, indicating active Hh signalling in ALL. Interestingly the transcription factor Gli 2 was not expressed, the functional relevance of which remains as yet unclear. The Hh inhibitors LDE225 and BMS833923 (0,01μM to 5μM) showed no dose dependent effect on inhibition of proliferation or induction of apoptosis in ALL LTCs. In conclusion we found evidence of Hh activation in both BCR-ABL pos and neg LTC ALL cells. No impact of Smo inhibition on proliferation and apoptosis was observed in response to the Smo inhibitors LDE225 and BMS833923, consistent with the hypothesis that Hh signalling in these cells may affect primarily self-renewal mechanisms. Single cell imaging of ALL LTCs has been successfully established for up to nine days of culture and will be applied to the testing of Hh modulation on cell fate decisions. Disclosures: No relevant conflicts of interest to declare.


2020 ◽  
Vol 34 (4) ◽  
pp. 5128-5143
Author(s):  
Lilei Peng ◽  
Yang Ming ◽  
Ling Zhang ◽  
Jie Zhou ◽  
Wei Xiang ◽  
...  

2021 ◽  
Author(s):  
Hong-Chen Yan ◽  
Yu Sun ◽  
Ming-Yu Zhang ◽  
Shu-Er Zhang ◽  
Jia-Dong Sun ◽  
...  

Abstract Background Skin-derived stem cells (SDSCs) are a class of adult stem cells (ASCs) that have the ability to self-renew and differentiate. The regulation mechanisms involved in the differentiation of ASCs is a hot topic. Porcine models have close similarities to humans and porcine SDSCs (pSDSCs) offer an ideal in vitro model to investigate human ASCs. To date, studies concerning the role of yes-associated protein (YAP) in ASCs are limited, and the mechanism of its influence on self-renewal and differentiation of ASCs remain unclear. In this paper, we explore the link between the transcriptional regulator YAP and the fate of pSDSCs. Results We found that YAP promotes the pluripotent state of pSDSCs by maintaining the high expression of the pluripotency genes Sox2, Oct4. The overexpression of YAP prevented the differentiation of pSDSCs and the depletion of YAP by small interfering RNA (siRNAs) suppressed the self-renewal of pSDSCs. In addition, we found that YAP regulates the fate of pSDSCs through a mechanism related to the Wnt/β-catenin signaling pathway. When an activator of the Wnt/β-catenin signaling pathway, CHIR99021, was added to pSDSCs overexpressing YAP the ability of pSDSCs to differentiate was partially restored. Conversely, when XAV939 an inhibitor of Wnt/β-catenin signaling pathway, was added to YAP knockdown pSDSCs a higher self-renewal ability resulted. Conclusions our results suggested that, YAP and the Wnt/β-catenin signaling pathway interact to regulate the fate of pSDSCs.


2019 ◽  
Vol 51 (11) ◽  
pp. 1-20 ◽  
Author(s):  
Jun-Cheng Guo ◽  
Yi-Jun Yang ◽  
Jin-Fang Zheng ◽  
Jian-Quan Zhang ◽  
Min Guo ◽  
...  

AbstractHepatocellular carcinoma (HCC) is a major cause of cancer-related deaths, but its molecular mechanisms are not yet well characterized. Long noncoding RNAs (lncRNAs) play crucial roles in tumorigenesis, including that of HCC. However, the role of homeobox A11 antisense (HOXA11-AS) in determining HCC stem cell characteristics remains to be explained; hence, this study aimed to investigate the effects of HOXA11-AS on HCC stem cell characteristics. Initially, the expression patterns of HOXA11-AS and HOXA11 in HCC tissues, cells, and stem cells were determined. HCC stem cells, successfully sorted from Hep3B and Huh7 cells, were transfected with short hairpin or overexpression plasmids for HOXA11-AS or HOXA11 overexpression and depletion, with an aim to study the influences of these mediators on the self-renewal, proliferation, migration, and tumorigenicity of HCC stem cells in vivo. Additionally, the potential relationship and the regulatory mechanisms that link HOXA11-AS, HOXA11, and the Wnt signaling pathway were explored through treatment with Dickkopf-1 (a Wnt signaling pathway inhibitor). HCC stem cells showed high expression of HOXA11-AS and low expression of HOXA11. Both HOXA11-AS silencing and HOXA11 overexpression suppressed the self-renewal, proliferation, migration, and tumorigenicity of HCC stem cells in vivo, as evidenced by the decreased expression of cancer stem cell surface markers (CD133 and CD44) and stemness-related transcription factors (Nanog, Sox2, and Oct4). Moreover, silencing HOXA11-AS inactivated the Wnt signaling pathway by decreasing the methylation level of the HOXA11 promoter, thereby inhibiting HCC stem cell characteristics. Collectively, this study suggested that HOXA11-AS silencing exerts an antitumor effect, suppressing HCC development via Wnt signaling pathway inactivation by decreasing the methylation level of the HOXA11 promoter.


2019 ◽  
Vol 234 (12) ◽  
pp. 23461-23474 ◽  
Author(s):  
Jun‐Cheng Guo ◽  
Yi‐Jun Yang ◽  
Jian‐Quan Zhang ◽  
Min Guo ◽  
Li Xiang ◽  
...  

2010 ◽  
Vol 207 (3) ◽  
pp. 475-489 ◽  
Author(s):  
Yoon-Chi Han ◽  
Christopher Y. Park ◽  
Govind Bhagat ◽  
Jinping Zhang ◽  
Yulei Wang ◽  
...  

The function of microRNAs (miRNAs) in hematopoietic stem cells (HSCs), committed progenitors, and leukemia stem cells (LSCs) is poorly understood. We show that miR-29a is highly expressed in HSC and down-regulated in hematopoietic progenitors. Ectopic expression of miR-29a in mouse HSC/progenitors results in acquisition of self-renewal capacity by myeloid progenitors, biased myeloid differentiation, and the development of a myeloproliferative disorder that progresses to acute myeloid leukemia (AML). miR-29a promotes progenitor proliferation by expediting G1 to S/G2 cell cycle transitions. miR-29a is overexpressed in human AML and, like human LSC, miR-29a-expressing myeloid progenitors serially transplant AML. Our data indicate that miR-29a regulates early hematopoiesis and suggest that miR-29a initiates AML by converting myeloid progenitors into self-renewing LSC.


2017 ◽  
Vol 09 (06) ◽  
Author(s):  
Tetsuzo Tauchi ◽  
Seiichi Okabe ◽  
Seiichiro Katagiri ◽  
Yuko Tanaka ◽  
Kaoru Tohyama ◽  
...  

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