Wendy Devereux

Hedgehog signaling maintains a tumor stem cell compartment in multiple myeloma

The cancer stem cell hypothesis suggests that malignant growth depends on a subset of tumor cells with stem cell-like properties of self-renewal. Because hedgehog (Hh) signaling regulates progenitor cell fate in normal development and homeostasis, aberrant pathway activation might be involved in the maintenance of such a population in cancer. Indeed, mutational activation of the Hh pathway is associated with medulloblastoma and basal cell carcinoma; pathway activity is also critical for growth of other tumors lacking such mutations, although the mechanism of pathway activation is poorly understood. Here we study the role and mechanism of Hh pathway activation in multiple myeloma (MM), a malignancy with a well defined stem cell compartment. In this model, rare malignant progenitors capable of clonal expansion resemble B cells, whereas the much larger tumor cell population manifests a differentiated plasma cell phenotype that pathologically defines the disease. We show that the subset of MM cells that manifests Hh pathway activity is markedly concentrated within the tumor stem cell compartment. The Hh ligand promotes expansion of MM stem cells without differentiation, whereas the Hh pathway blockade, while having little or no effect on malignant plasma cell growth, markedly inhibits clonal expansion accompanied by terminal differentiation of purified MM stem cells. These data reveal that Hh pathway activation is heterogeneous across the spectrum of MM tumor stem cells and their more differentiated progeny. The potential existence of similar relationships in other adult cancers may have important biologic and clinical implications for the study of aberrant Hh signaling.

A Primary Xenograft Model of Small-Cell Lung Cancer Reveals Irreversible Changes in Gene Expression Imposed by Culture In vitro

Traditional approaches to the preclinical investigation of cancer therapies rely on the use of established cell lines maintained in serum-based growth media. This is particularly true of small-cell lung cancer (SCLC), where surgically resected tissue is rarely available. Recent attention has focused on the need for better models that preserve the integrity of cancer stem cell populations, as well as three-dimensional tumor-stromal interactions. Here we describe a primary xenograft model of SCLC in which endobronchial tumor specimens obtained from chemo-naive patients are serially propagated in vivo in immunodeficient mice. In parallel, cell lines grown in conventional tissue culture conditions were derived from each xenograft line, passaged for 6 months, and then reimplanted to generate secondary xenografts. Using the Affymetrix platform, we analyzed gene expression in primary xenograft, xenograft-derived cell line, and secondary xenograft, and compared these data to similar analyses of unrelated primary SCLC samples and laboratory models. When compared with normal lung, primary tumors, xenografts, and cell lines displayed a gene expression signature specific for SCLC. Comparison of gene expression within the xenograft model identified a group of tumor-specific genes expressed in primary SCLC and xenografts that was lost during the transition to tissue culture and that was not regained when the tumors were reestablished as secondary xenografts. Such changes in gene expression may be a common feature of many cancer cell culture systems, with functional implications for the use of such models for preclinical drug development.

A crucial requirement for Hedgehog signaling in small cell lung cancer

Small-cell lung cancer (SCLC) is an aggressive neuroendocrine subtype of lung cancer for which there is no effective treatment. Using a mouse model in which deletion of Rb1 and Trp53 in the lung epithelium of adult mice induces SCLC, we found that the Hedgehog signaling pathway is activated in SCLC cells independently of the lung microenvironment. Constitutive activation of the Hedgehog signaling molecule Smoothened (Smo) promoted the clonogenicity of human SCLC in vitro and the initiation and progression of mouse SCLC in vivo. Reciprocally, deletion of Smo in Rb1 and Trp53-mutant lung epithelial cells strongly suppressed SCLC initiation and progression in mice. Furthermore, pharmacological blockade of Hedgehog signaling inhibited the growth of mouse and human SCLC, most notably following chemotherapy. These findings show a crucial cell-intrinsic role for Hedgehog signaling in the development and maintenance of SCLC and identify Hedgehog pathway inhibition as a therapeutic strategy to slow the progression of disease and delay cancer recurrence in individuals with SCLC.

Properties of purified recombinant human polyamine oxidase, PAOh1/SMO.

The discovery of an inducible oxidase whose apparent substrate preference is spermine indicates that polyamine catabolism is more complex than that originally proposed. To facilitate the study of this enzyme, the purification and characterization of the recombinant human PAOh1/SMO polyamine oxidase are reported. Purified PAOh1/SMO oxidizes both spermine (K(m)=1.6 microM) and N(1)-acetylspermine (K(m)=51 microM), but does not oxidize spermidine. The purified human enzyme also does not oxidize eight representative antitumor polyamine analogues; however, specific oligamine analogues were found to be potent inhibitors of the oxidation of spermine by PAOh1/SMO. The results of these studies are consistent with the hypothesis that PAOh1/SMO represents a new addition to the polyamine metabolic pathway that may represent a new target for antineoplastic drug development.

Cooperation between the Hic1 and Ptch1 tumor suppressors in medulloblastoma

Medulloblastoma is an embryonal tumor thought to arise from the granule cell precursors (GCPs) of the cerebellum. PATCHED (PTCH), an inhibitor of Hedgehog signaling, is the best-characterized tumor suppressor in medulloblastoma. However, <20% of medulloblastomas have mutations in PTCH. In the search for other tumor suppressors, interest has focused on the deletion events at the 17p13.3 locus, the most common genetic defect in medulloblastoma. This chromosomal region contains HYPERMETHYLATED IN CANCER 1 (HIC1), a transcriptional repressor that is a frequent target of epigenetic gene silencing in medulloblastoma. Here we use a mouse model of Ptch1 heterozygosity to reveal a critical tumor suppressor function for Hic1 in medulloblastoma. When compared with Ptch1 heterozygous mutants, compound Ptch1/Hic1 heterozygotes display a fourfold increased incidence of medulloblastoma. We show that Hic1 is a direct transcriptional repressor of Atonal Homolog 1 (Atoh1), a proneural transcription factor essential for cerebellar development, and show that ATOH1 expression is required for human medulloblastoma cell growth in vitro. Given that Atoh1 is also a putative target of Hh signaling, we conclude that the Hic1 and Ptch1 tumor suppressors cooperate to silence Atoh1 expression during a critical phase in GCP differentiation in which malignant transformation may lead to medulloblastoma.

Cloning and Characterization of Human Polyamine-modulated Factor-1, a Transcriptional Cofactor That Regulates the Transcription of the Spermidine/Spermine N 1-Acetyltransferase Gene

The increased transcription and ultimate superinduction of the spermidine/spermineN 1-acetyltransferase (SSAT) gene has been associated with the antineoplastic activity of several new antitumor polyamine analogues. In sensitive tumor cell types, the transcriptional induction appears to be regulated by the constitutive association of the transcription factor Nrf-2 with the recently discovered polyamine-responsive element. Using the yeast two-hybrid system, a new transcriptional cofactor, polyamine-modulated factor-1 (PMF-1), has been identified as a partner protein of Nrf-2 that, in combination with Nrf-2, regulates the polyamine analogue-induced transcription of SSAT. The human PMF-1 gene, located on chromosome 1 near the 1q12/1q21 border, yields an mRNA transcript of ∼1.2 kilobases that codes for a 165-amino acid protein with a predicted molecular mass of ∼20 kDa. The PMF-1 mRNA appears to increase in response to analogue exposure only in analogue-responsive cells. In addition to the transcriptional regulation of SSAT, PMF-1 or similar factors should be considered in the regulation of other polyamine-dependent genes.

Cloning and characterization of multiple human polyamine oxidase splice variants that code for isoenzymes with different biochemical characteristics

The recently cloned and characterized human polyamine oxidase (PAOh1) potentially represents a new class of catabolic enzymes in the mammalian polyamine metabolic pathway capable of the efficient oxidation of polyamines. Here the discovery of three additional human PAO splice variants is reported, and the data support the fact that the human PAO gene codes for at least four isoenzymes, each of which exhibit distinctive biochemical characteristics, suggesting the existence of additional levels of complexity in polyamine catabolism.

Induction of the PAOh1/SMO polyamine oxidase by polyamine analogues in human lung carcinoma cells

PurposeThe induction of polyamine catabolism has been directly associated with the cytotoxic response of various tumor types to the antitumor polyamine analogues. Initially, human polyamine catabolism was assumed to be under the control of a rate-limiting spermidine/spermine N1-acetyltransferase (SSAT) that provides substrate for an acetylpolyamine oxidase (PAO). We have recently cloned a new polyamine analogue-inducible human polyamine oxidase (PAOh1/SMO) that efficiently uses spermine as a substrate. The induction of PAOh1/SMO in response to multiple polyamine analogues was examined in representative lung tumor cell lines.MethodsRepresentatives of three different classes of antitumor polyamine analogues were examined for their ability to induce PAOh1/SMO.ResultsThe human adenocarcinoma line, NCI A549 was found to be the most responsive line with respect to induction of PAOh1/SMO in response to analogue exposure. Similar to previous observations with SSAT expression, PAOh1/SMO induction was found to occur primarily in non-small-cell lung cancers cell lines. Using a series of polyamine analogues, it was found that the most potent inducers of PAOh1/SMO possessed multiple three-carbon linkers between nitrogens, as typified by N1,N11-bis(ethyl)norspermine.ConclusionsSince PAOh1/SMO is an analogue-inducible enzyme that produces H2O2 as a metabolic product, it may play a significant role in determining the sensitivity of various human tumors to specific polyamine analogues.

The role of polyamine catabolism in anti-tumour drug response

Interest in polyamine catabolism has increased since it has been directly associated with the cytotoxic response of multiple tumour types to exposure to specific anti-tumour polyamine analogues. Human polyamine catabolism was considered to be a two-step pathway regulated by the rate-limiting enzyme spermidine/spermine N(1)-acetyltransferase (SSAT) that provides substrate for an acetylpolyamine oxidase (APAO). Further, the super-induction of SSAT by several anti-tumour polyamine analogues has been implicated in the cytotoxic response of specific solid-tumour phenotypes to these agents. This high induction of SSAT has been correlated with cellular response to the anti-tumour polyamine analogues in several systems and considerable progress has been made in understanding the molecular mechanisms that regulate the analogue-induced expression of SSAT. A polyamine response element has been identified and the transacting transcription factors that bind and stimulate transcription of SSAT have been cloned and characterized. The link between SSAT activity and cellular toxicity is thought to be based on the production of H(2)O(2) by the activity of the constitutive APAO that uses the SSAT-produced acetylated polyamines. The high induction of SSAT and the subsequent activity of APAO are linked to the cytotoxic response of some tumour cell types to specific polyamine analogues. However, we have recently cloned a variably spliced human polyamine oxidase (PAOh1) that is inducible by specific polyamine analogues, efficiently uses unacetylated spermine as a substrate, and also produces toxic H(2)O(2) as a product. The results of studies with PAOh1 suggest that it is an additional enzyme in polyamine catabolism that has the potential to significantly contribute to polyamine homoeostasis and drug response. Most importantly, PAOh1 is induced by specific polyamine analogues in a tumour-phenotype-specific manner in cell lines representative of the major forms of solid tumours, including lung, breast, colon and prostate. The sensitivity to these anti-tumour polyamine analogues can be significantly reduced if the tumour cells are co-treated with 250 microM of the polyamine oxidase inhibitor N (1), N (4)-bis(2,3-butadienyl)-1,4-butanediamine (MDL 72,527), suggesting that the H(2)O(2) produced by PAOh1 does in fact play a direct role in the observed cytotoxicity. These results strongly implicate PAOh1 as a new target that, in combination with SSAT, may be exploited for therapeutic advantage. The current understanding of the role and regulation of these two important polyamine catabolic enzymes are discussed.

Polyamine-modulated factor 1 binds to the human homologue of the 7a subunit of the Arabidopsis COP9 signalosome: Implications in gene expression

Polyamines have been identified to play a role in the transcription of various growth-related genes. The recently discovered polyamine responsive element and the associated trans-acting proteins involved in polyamine-regulated transcription have provided a model system for the study of the role of polyamines in transcription. Polyamine-modulated factor 1 (PMF-1) was identified as one of the transacting factors that binds to NF-E2 related factor-2 (Nrf-2) to regulate the transcription of spermidine/spermine N(1)-acetyltransferase (SSAT). The possibility that PMF-1 also binds to other proteins involved in transcriptional regulation cannot be ruled out. Using a yeast two-hybrid strategy, it was found that PMF-1 binds to a human homologue of the Arabidopsis COP9 signalosome subunit 7a (CSN 7) protein. In the present study, we describe human CSN 7, a 275-amino-acid- containing protein that may have a direct role in regulating gene expression. CSN 7 and PMF-1 bind to each other, as well as compete with each other for binding to Nrf-2. This competition for Nrf-2 binding and interaction with each other is implicated in the regulation of SSAT transcription. CSN 7 possesses a C-terminal coiled-coil domain similar to the domain that mediates the interaction between PMF-1 and Nrf-2, suggesting that coiled-coil domains also mediate the interaction between CSN 7 and PMF-1. Since CSN 7 does not contain a DNA-binding domain, its effects on transcription must occur in conjunction with binding to other proteins. The results presented here demonstrate that PMF-1 and Nrf-2 can act as protein partners of CSN 7.