Cancer Research Aziza Shad Telomeres
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online
Cancer Research 68, 961, February 15, 2008. doi: 10.1158/0008-5472.CAN-07-5500
© 2008 American Association for Cancer Research
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Lindemann, R. K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Lindemann, R. K.
Related Collections
Right arrow Cellular Pathobiology
Right arrow Cellular Pathobiology: Proliferation, Senescence, and Death
Right arrow Therapeutics and Targets: Identification, Validation, and Markers

Reviews

Stroma-Initiated Hedgehog Signaling Takes Center Stage in B-Cell Lymphoma

Ralph K. Lindemann

National Center for Tumor Diseases, Department of Translational Oncology G100, Heidelberg, Germany

Requests for reprints: Ralph K. Lindemann, National Center for Tumor Diseases, Department of Translational Oncology G100, Im Neuenheimer Feld 581, D-69120 Heidelberg, Germany. Phone: 49-6221-421602; Fax: 49-6221-421611; E-mail: ralph.lindemann{at}nct-heidelberg.de.


   Abstract
Top
 Abstract
Background
 Stromal Hedgehog Regulates...
 Discussion
 References
 
Hedgehog-mediated signaling has been shown to promote growth and dissemination of solid cancers, most prominently basal cell carcinomas and medulloblastoma. Recent findings indicate that hedgehog signals are also important for tumor growth in hematologic malignancies. Hedgehog ligands secreted by stromal cells could elicit Patched/Smoothened-mediated antiapoptotic signaling in mouse B-cell lymphomas. Inhibition of hedgehog signaling induced apoptosis in lymphoma cells and prolonged survival of lymphoma-bearing mice. Depletion of tumor cells proceeded in the absence of p53 via the mitochondrial apoptotic pathway. These and other recently published data on hedgehog inhibition in cancer cells and their implications will be discussed. [Cancer Res 2008;68(4):961–4]


   Background
Top
 Abstract
 Background
Stromal Hedgehog Regulates...
 Discussion
 References
 
More than 25 years ago, Nüsslein-Volhard and Wieschhaus (1) identified the hedgehog gene as a crucial regulator of Drosophila embryonic patterning. Subsequent research has shown that hedgehog ligands bind to the Patched receptor, which blocks the inhibitory action of Patched on the transmembrane signaling molecule Smoothened. This leads to activation of (a) zinc-finger transcription factor(s) (Ci in Drosophila and GLI1-3 in mammals) with downstream regulation of target genes such as patched, ci, or gli, respectively, and certain cyclins (2, 3). In mammals, three hedgehog homologues have been described: Sonic hedgehog (Shh), Indian hedgehog (Ihh), and Desert hedgehog, whose binding to the Patched receptor (PTCH1 in humans and Ptch1 in mouse) results in derepression of Smoothened and activation of three different GLI transcription factors: GLI1, GLI2, and GLI3, the latter of which can be processed into a repressive factor (GLI3-R) in the absence of hedgehog signaling (2). Mutations in Shh in humans result in defective forebrain development (holoprosencephaly) and facial abnormalities, and disruption of the shh gene in mice results in cyclopia (2).

It is now clear that the hedgehog/PTCH1/Smoothened/GLI signal transduction pathway is a key regulator of tumorigenesis. In humans, this is best exemplified by Gorlin syndrome, which is caused by a mutation in the gene encoding for PTCH1, resulting in activation of Smoothened and GLI transcription factors. As a consequence, affected individuals develop basal cell carcinomas (BCC) and are predisposed to medulloblastoma and other cancers (4). Accordingly, sporadically arising BCCs, small-cell lung cancer, medulloblastoma, cancers of the digestive tract, and pancreatic cancer frequently show activation of hedgehog-dependent signaling pathways. These data are supported by experiments performed in mice or cultured tumor cells and by using the Smoothened inhibitor cyclopamine, a steroidal alkaloid from the corn lily (Veratrum californicum) and a widely used antagonist of hedgehog signaling (4). A number of mechanisms have been suggested by which hedgehog signaling could foster tumorigenesis: (a) promoting cellular proliferation by elevating cyclin and cyclin-dependent kinase levels; (b) disabling of the apoptotic apparatus by up-regulating Bcl-2 and Bcl-xL and, in late-stage tumor progression, (c) regulating epithelial-to-mesenchymal transition; and (d) enhancing vascular endothelial growth factor (VEGF) expression, both being integral for tumor cell metastasis and angiogenesis, respectively (4).

Upon deregulated expression of oncoproteins such as c-myc in healthy cells, activation of apoptotic pathways serves as a fail-safe mechanism to protect against malignant transformation (5). Consequently, selective pressure exists to inactivate components of the apoptotic machinery (e.g., through epigenetic silencing of caspase-encoding genes) or enhance antiapoptotic signaling (e.g., through overexpression of Bcl-2 or Bcl-xL) to bypass oncogene-induced cell death (5). The acquired ability to evade proapoptotic signals during tumorigenesis can induce drug resistance during anticancer treatment, effectively linking (epi-)genetic changes during tumorigenesis to treatment response (5). Interestingly, overexpression of the hedgehog effector GLI1 can induce expression of Bcl-2 (6, 7), and Shh overexpression results in up-regulation of Bcl-2 and Bcl-xL protein levels in murine pancreatic cancer (8), indicating that hedgehog-mediated signal transduction can lead to inactivation of apoptotic pathways.


   Stromal Hedgehog Regulates Growth of Murine and Human B-Cell Lymphomas
Top
 Abstract
 Background
 Stromal Hedgehog Regulates...
Discussion
 References
 
Although it has been shown that hedgehog ligands produced by follicular dendritic cells promote survival of germinal center B cells (9), activities of hedgehog signaling pathways have not been thoroughly examined in hematologic malignancies. Now, Dierks et al. (10) report that hedgehog ligands produced by stromal cells support proliferation and survival of B-cell lymphomas. Inhibition of hedgehog signaling impinged on tumor growth in lymphomas, most likely by inducing mitochondrial apoptosis (10). For their studies, they used the well-established Eµ-myc model of B-cell lymphoma, a mouse model of Burkitts lymphoma driven by transgenic B-cell–specific overexpression of c-myc (11). This model has been a valuable tool for genetically dissecting the apoptotic pathways underlying therapeutic efficacy of specific anticancer drugs (12, 13). Eµ-myc lymphomas that arise in transgenic mice can be transplanted into fully immunocompetent recipient mice where they grow in lymph nodes, spleen, and bone marrow. Consequently, growth characteristics and treatment response can be studied in tumors that grow at their "natural site" with an intact microenvironment.

Dierks et al. (10) isolated lymphoma cells from Eµ-myc transgenic mice and cultured them on bone marrow or spleen stromal cells. Removing the lymphoma cells from the stroma halted their proliferation, eventually leading to apoptosis. Among a number of soluble factors, only Ihh and Shh could sustain growth of lymphoma cells cultured without stroma, which was blocked by cotreatment with a hedgehog-neutralizing antibody or the Smoothened inhibitor cyclopamine. Furthermore, cyclopamine decreased the viability of ~75% of all tested lymphoma cell lines and of non–Hodgkins lymphoma and multiple myeloma samples from patients. As indicated by Annexin V positivity, cyclopamine-treated tumor cells had undergone apoptosis. Importantly, cyclopamine-induced death is markedly reduced in Eµ-myc lymphomas overexpressing the hedgehog-activated transcription factors GLI1 or Fused, showing that the effects of cyclopamine are directly connected to inhibition of hedgehog signaling. Immunohistochemistry showed that—in contrast to solid cancers—hedgehog ligands are produced only by host cells, whereas Smoothened and GLI1 expression could be detected in lymphoma cells. It is possible that these findings reflect hedgehog signaling in germinal centers where follicular dendritic cells provide hedgehog ligands as prosurvival signals for B cells (9).

Dierks et al. (10) then proceeded to genetically dissect the mechanism by which cyclopamine induced apoptosis in lymphoma cells. For this purpose, they harvested bone marrow cells from mice with defined genetic defects in apoptotic pathways and obtained lymphomas by transducing the bone marrow cells with a myc-expressing retrovirus. Stroma withdrawal from those lymphomas or cyclopamine treatment resulted in cell death only in cells deficient for Trp53 or the CDKN2A/ARF locus, respectively, whereas some protection from death was seen in cells deficient for Bax or Caspase-3. Only Bcl-2–overexpressing lymphomas proved completely resistant to stroma withdrawal or cyclopamine-induced apoptosis, indicating that mitochondrial apoptosis is the anticancer effector mechanism in Eµ-myc lymphomas upon inhibition of the hedgehog signaling pathway. Did the same genetic requirements exist for therapeutic efficacy of cyclopamine against Eµ-myc lymphomas? Cyclopamine treatment was shown to prolong the survival of lymphoma-bearing mice when treated for 3 weeks starting on day 2 after tumor injection. Using luciferase-expressing lymphomas, the authors performed bioluminescence imaging of established lymphomas (10 days after tumor injection) treated with cyclopamine and could show that overexpression of Bcl-2, but not genetic deletion of Trp53, prevented subsequent reduction in lymphoma mass. It remains to be shown that cyclopamine directly induced apoptosis in vivo e.g., using deoxynucleotidyl transferase-mediated dUTP-biotin end labeling (TUNEL)–based detection of apoptotic cells. Using further optimized cyclopamine regimen, it will now also be possible to assess whether inactivation of the mitochondrial apoptotic pathway indeed predicts chemoresistance to cyclopamine in long-term therapy assays.

Taken together, Dierks et al. (10) show that stroma-cell derived Hedgehog sustains growth of murine and human B-cell lymphomas, and that pharmacologic inhibition of the central hedgehog signaling mediator Smoothened has anticancer effects in vitro and in vivo by inducing mitochondria-regulated apoptosis (Fig. 1 ).


Figure 1
View larger version (58K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 1. Schematic representation of stroma-initiated hedgehog signaling in Eµ-myc lymphomas as described by Dierks et al. (10). A, stroma-derived hedgehog ligands bind to the Patched receptor on lymphoma cells, which relieves Ptch-1–mediated suppression of Smoothened. This results in activation of GLI transcription factors and increased transcription of hedgehog signaling target genes, such as ptch1, gli, and bcl-2, subsequently enhancing lymphoma cell survival. B, in the absence of hedgehog ligands, Smoothened is repressed by Ptch1 and expression of prosurvival genes such as bcl-2 is low, tipping the death/life balance toward apoptotic cell death. C, inhibition of Smoothened by cyclopamine mimics absence of hedgehog ligands and leads to down-regulation of Bcl-2 levels and subsequent apoptotic cell death, allowing for depletion of Eµ-myc lymphomas in vivo.

 

   Discussion
Top
 Abstract
 Background
 Stromal Hedgehog Regulates...
 Discussion
References
 
Hedgehog-mediated signal transduction has been shown to induce prosurvival signaling by up-regulation of the antiapoptotic Bcl-2 family members Bcl-2 and Bcl-xL (6, 7). Most likely, this is one of the mechanisms by which hedgehog signaling enhances tumor development. According to Dierks et al. (10), cyclopamine treatment of murine B-cell lymphomas down-regulates bcl-2 mRNA and Bcl-2 protein levels, in parallel to down-regulation of Gli1 levels. Gli1/2 are direct regulators of bcl-2 gene transcription, so a probable mechanism of cyclopamine-induced cell death is lowering the cellular death threshold by depleting antiapoptotic proteins. Yet, additional prodeath pathways might be initiated in lymphomas upon hedgehog inhibition, such as up-regulation of components of the extrinsic apoptotic pathway. In UV-induced mouse BCCs, cyclopamine has been shown to be a potent regulator of Fas, one of the prodeath cell surface receptors of the extrinsic apoptotic machinery, and Fas ligand neutralization markedly inhibited cyclopamine-induced apoptosis in cultured BCC cells (14). It is also not known whether cyclopamine-mediated cell death requires the activity of one or more of the proapoptotic BH3-only proteins, which are part of the anticancer mechanism of diverse chemotherapeutic drugs such as DNA alkylating agents, microtubule-targeting agents, and histone deacetylase (HDAC) inhibitors (12, 15). In this regard, the genetic requirements for the antitumor effects of cyclopamine are remarkably similar to results from a therapeutic candidate screen we recently performed to identify apoptotic pathways underlying the therapeutic efficacy of the HDAC inhibitor Vorinostat (12). Vorinostat induced in vivo apoptosis in Eµ-myc lymphomas deficient for p53, and activity of the mitochondrial, but not the extrinsic apoptotic pathway was correlated with therapeutic outcome (12). Could inhibitors of hedgehog signaling and HDAC activity share anticancer effector pathways? HDAC1 is required for hedgehog-mediated Isl1 expression during motoneuron development in zebrafish (16), and exciting results presented at a recent workshop on hedgehog signaling in cancer indicate that the acetylation of GLI1—possibly by the acetyltransferase enzymes p300/PCAF—can negatively regulate its activity (8). Of note, the HDAC inhibitor Trichostatin A down-regulated GLI1 transcriptional activity and the expression of hedgehog target genes (8). Thus, it is tempting to speculate that anticancer effects of HDAC inhibitors, such as apoptotic and therapeutic efficacy against Trp53-deficient tumors, in part, mimic the action of inhibitors of hedgehog signaling.

The direct positive control of cell survival by hedgehog signal transduction could also link this pathway to resistance of tumors in the course of chemotherapy. Indeed, >80% of postchemotherapy esophageal tumor biopsies showed activation of hedgehog signaling in the remaining tumor mass as judged by cytoplasmic Shh and nuclear Gli1 expression (17). In the SEG-1 xenograft model of esophageal adenocarcinoma, activation of hedgehog signaling preceded regrowth of tumors subsequent to therapy (17). A study from the same group showed that inhibition of hedgehog signaling sensitizes cultured human cancer cells to a variety of chemotherapeutics, in part by down-regulating drug-efflux pumps (17). In addition, hedgehog signaling has the potential to perturb successful long-term eradication of tumor cells by yet another mechanism: In various types of cancers such as gliomas, mammary tumors and multiple myeloma, hedgehog signaling seems to be part of the molecular program that maintains the tumor stem cell compartment (18, 19). Taken together, there is an emerging theme that hedgehog signaling can contribute to multifactorial drug resistance in cancer, once again reiterating the need for development of clinically applicable hedgehog pathway inhibitors.

There are a number of possible mechanism by which hedgehog signaling can be activated during tumorigenesis, but they can broadly be divided in two categories (4): (a) tumor cell–intrinsic mechanisms, such as inactivating mutations of PTCH1 or activating mutations of Smoothened, or Gli proteins, respectively (alternatively, hedgehog signaling can be elicited in an autocrine fashion by tumor cell–derived hedgehog ligands) and (b) nontumor cell–intrinsic mechanisms, such as production of hedgehog ligands by tumor cells, which then bind to PTCH1 on stromal cells, resulting in the production of tumor-promoting factors including VEGF and insulin-like growth factor. In the case of Eµ-myc lymphomas, the stroma seems to supply hedgehog ligands, which then induce downstream signaling in lymphoma cells (10). Whether the lymphoma cells subsequently produce factors that, in turn, stimulate the stromal expression and secretion of hedgehog ligands as part of a positive regulatory loop is unclear. Dierks et al. (10) show that stroma derived from both healthy and Eµ-myc transgenic mice can support the growth of lymphomas to the same extent, implying that presence of lymphoma cells is not a prerequisite for stromal hedgehog production. If stroma-produced hedgehog ligands severely affect growth of lymphoma and possibly other cancer cells and their response to treatment, should not we be able to access previously unrecognized, clinically relevant information by studying tumor cells in a context that can, at least in part, mimic their natural environment? In fact, when compared with freshly isolated cells, serially passaged murine neuroblastoma cells down-regulated components of the hedgehog signaling machinery, resulting in markedly decreased responsiveness to hedgehog antagonists upon retransplantation into mice (20). It is likely that similar mechanisms operate for other signaling pathways. For the benefit of researchers, clinicians and, eventually, patients, tumor cells in the laboratory should get the microenvironment that they deserve.


   Acknowledgments
 

Received 9/18/07. Revised 10/30/07. Accepted 10/30/07.


   References
Top
 Abstract
 Background
 Stromal Hedgehog Regulates...
 Discussion
 References
 

  1. Nusslein-Volhard C, Wieschaus E. Mutations affecting segment number and polarity in Drosophila. Nature 1980;287:795–801.[CrossRef][Medline]
  2. Kalderon D. The mechanism of hedgehog signal transduction. Biochem Soc Trans 2005;33:1509–12.[CrossRef][Medline]
  3. Lum L, Beachy PA. The Hedgehog response network: sensors, switches, and routers. Science 2004;304:1755–9.[Abstract/Free Full Text]
  4. Rubin LL, de Sauvage FJ. Targeting the Hedgehog pathway in cancer. Nat Rev 2006;5:1026–33.
  5. Johnstone RW, Ruefli AA, Lowe SW. Apoptosis: a link between cancer genetics and chemotherapy. Cell 2002;108:153–64.[CrossRef][Medline]
  6. Bar EE, Chaudhry A, Farah MH, Eberhart CG. Hedgehog signaling promotes medulloblastoma survival via Bc/II. Am J Pathol 2007;170:347–55.[Abstract/Free Full Text]
  7. Bigelow RL, Chari NS, Unden AB, et al. Transcriptional regulation of bcl-2 mediated by the sonic hedgehog signaling pathway through gli-1. J Biol Chem 2004;279:1197–205.[Abstract/Free Full Text]
  8. Morton JP, Mongeau ME, Klimstra DS, et al. Sonic hedgehog acts at multiple stages during pancreatic tumorigenesis. Proc Natl Acad Sci U S A 2007;104:5103–8.[Abstract/Free Full Text]
  9. Sacedon R, Diez B, Nunez V, et al. Sonic hedgehog is produced by follicular dendritic cells and protects germinal center B cells from apoptosis. J Immunol 2005;174:1456–61.[Abstract/Free Full Text]
  10. Dierks C, Grbic J, Zirlik K, et al. Essential role of stromally induced hedgehog signaling in B-cell malignancies. Nat Med 2007;13:944–51.[CrossRef][Medline]
  11. Adams JM, Harris AW, Pinkert CA, et al. The c-myc oncogene driven by immunoglobulin enhancers induces lymphoid malignancy in transgenic mice. Nature 1985;318:533–8.[CrossRef][Medline]
  12. Lindemann RK, Newbold A, Whitecross KF, et al. Analysis of the apoptotic and therapeutic activities of histone deacetylase inhibitors by using a mouse model of B cell lymphoma. Proc Natl Acad Sci U S A 2007;104:8071–6.[Abstract/Free Full Text]
  13. Schmitt CA, Rosenthal CT, Lowe SW. Genetic analysis of chemoresistance in primary murine lymphomas. Nat Med 2000;6:1029–35.[CrossRef][Medline]
  14. Athar M, Li C, Tang X, et al. Inhibition of smoothened signaling prevents ultraviolet B-induced basal cell carcinomas through regulation of Fas expression and apoptosis. Cancer Res 2004;64:7545–52.[Abstract/Free Full Text]
  15. Willis SN, Adams JM. Life in the balance: how BH3-only proteins induce apoptosis. Curr Opin Cell Biol 2005;17:617–25.[CrossRef][Medline]
  16. Cunliffe VT. Histone deacetylase 1 is required to repress Notch target gene expression during zebrafish neurogenesis and to maintain the production of motoneurones in response to hedgehog signalling. Development 2004;131:2983–95.[Abstract/Free Full Text]
  17. Chen YJ, Sims-Mourtada J, Izzo J, Chao KS. Targeting the hedgehog pathway to mitigate treatment resistance. Cell Cycle 2007;6:1826–30.[Medline]
  18. Robbins DJ, Hebrok M. Hedgehogs: la dolce vita. Workshop on hedgehog-Gli signaling in cancer and stem cells. EMBO Rep 2007;8:451–5.[CrossRef][Medline]
  19. Peacock CD, Wang Q, Gesell GS, et al. Hedgehog signaling maintains a tumor stem cell compartment in multiple myeloma. Proc Natl Acad Sci U S A 2007;104:4048–53.[Abstract/Free Full Text]
  20. Sasai K, Romer JT, Lee Y, et al. Shh pathway activity is down-regulated in cultured medulloblastoma cells: implications for preclinical studies. Cancer Res 2006;66:4215–22.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 aacredbookHome page
J. Xie
Activation of Hedgehog Signaling in Human Cancer: Basic Mechanisms and Clinical Implications
Am. Assoc. Cancer Res. Educ. Book, April 18, 2009; 2009(1): 25 - 41.
[Full Text] [PDF]

Home page
 Mol Cancer ResHome page
G. V. Hegde, K. J. Peterson, K. Emanuel, A. K. Mittal, A. D. Joshi, J. D. Dickinson, G. J. Kollessery, R. G. Bociek, P. Bierman, J. M. Vose, et al.
Hedgehog-Induced Survival of B-Cell Chronic Lymphocytic Leukemia Cells in a Stromal Cell Microenvironment: A Potential New Therapeutic Target
Mol. Cancer Res., December 1, 2008; 6(12): 1928 - 1936.
[Abstract] [Full Text] [PDF]

Home page
 Molecular Cancer TherapeuticsHome page
G. V. Hegde, C. M. Munger, K. Emanuel, A. D. Joshi, T. C. Greiner, D. D. Weisenburger, J. M. Vose, and S. S. Joshi
Targeting of sonic hedgehog-GLI signaling: a potential strategy to improve therapy for mantle cell lymphoma
Mol. Cancer Ther., June 1, 2008; 7(6): 1450 - 1460.
[Abstract] [Full Text] [PDF]

Home page
 CRO ContentHome page
G. C. Prendergast
Murder or Suicide: Strategies to Selectively Eliminate Cancer Cells
Cancer Reviews Online Content, March 1, 2008; 2008(3): 5 - 6.
[Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Lindemann, R. K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Lindemann, R. K.
Related Collections
Right arrow Cellular Pathobiology
Right arrow Cellular Pathobiology: Proliferation, Senescence, and Death
Right arrow Therapeutics and Targets: Identification, Validation, and Markers

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online