Peter A. Steck

Identification of a candidate tumour suppressor gene, MMAC1, at chromosome 10q23.3 that is mutated in multiple advanced cancers

Deletions involving regions of chromosome 10 occur in the vast majority (>90%) of human glioblastoma multiformes. A region at chromosome 10q23–24 was implicated to contain a tumour suppressor gene and the identification of homozygous deletions in four glioma cell lines further refined the location. We have identified a gene, designated MMAC1, that spans these deletions and encodes a widely expressed 5.5-kb mRNA. The predicted MMAC1 protein contains sequence motifs with significant homology to the catalytic domain of protein phosphatases and to the cytoskeletal proteins, tensin and auxilin. MMAC1 coding-region mutations were observed in a number of glioma, prostate, kidney and breast carcinoma cell lines or tumour specimens. Our results identify a strong candidate tumour suppressor gene at chromosome 10q23.3, whose loss of function appears to be associated with the oncogenesis of multiple human cancers.

The PTEN/MMAC1/TEP tumor suppressor gene decreases cell growth and induces apoptosis and anoikis in breast cancer cells

The PTEN/MMAC1/TEP (PTEN) tumor suppressor gene at 10q23.3 is mutated in multiple types of sporadic tumors including breast cancers and also in the germline of patients with the Cowdens breast cancer predisposition syndrome. The PTEN gene encodes a multifunctional phosphatase capable of dephosphorylating the same sites in membrane phosphatidylinositols phosphorylated by phosphatidylinositol 3′-kinase (PI3K). We demonstrate herein that loss of PTEN function in breast cancer cells results in an increase in basal levels of phosphorylation of multiple components of the P13K signaling cascade as well as an increase in duration of ligand-induced signaling through the P13K cascade. These alterations are reversed by wild-type but not phosphatase inactive PTEN. In the presence of high concentrations of serum, enforced expression of PTEN induces a predominant G1 arrest consistent with the capacity of PTEN to evoke increases in the expression of the p27Kip1 cyclin dependent kinase inhibitor. In the presence of low concentrations of serum, enforced PTEN expression results in a marked increase in cellular apoptosis, a finding which is consistent with the capacity of PTEN to alter the phosphorylation, and presumably function, of the AKT, BAD, p70S6 kinase and GSK3α apoptosis regulators. Under anchorage-independent conditions, PTEN also induces anoikis, a form of apoptosis that occurs when cells are dissociated from the extracellular matrix, which is enhanced in conjunction with low serum culture conditions. Together, these data suggest that PTEN effects on the PI3K signaling cascade are influenced by the cell stimulatory context, and that depending on the exposure to growth factors and other exogenous stimuli such as integrin ligation, PTEN can induce cell cycle arrest, apoptosis or anoikis in breast cancer cells.

The autocrine loop of TGF-α/EGFR and brain tumors

Malignant human gliomas are the most common formsof primary tumors in the central nerve system.Due to their location and invasive nature, treatmentso far has been mainly palliative. Thus, understandingthe molecular detail of tumor transformation and progressionis crucial for developing effective therapeutic strategy forthis fetal tumor. Among the genetic alternations foundin these tumors, p53 inactivation and PDGF/PDGFR activationrepresent the early events, and the loss ofchromosome 10 and gene amplification and rearrangement ofEGFR represent the late events. Studies with bothglioma cell lines and primary tumor tissues havestrongly suggested that TGF-α and EGFR function asan important autocrine loop in supporting proliferation ofhuman glioma, especially in high grade glioma, sinceelevated TGF-α expression is also found in thesehigh grade tumors. Furthermore, down regulation of theexpression of TGF-α by antisense constructs has beenshown to inhibit several types of human tumorcell growth including glioma. Other means of therapeuticapproaches using this autocrine loop as a targetalso include the use of monoclonal antibodies andtheir cytotoxic conjugated. Considerable understanding of the EGFR-mediatedsignal transduction pathways has become available recently, whichincluding GRB2/mSOS1 mediated MAP kinase activation; JAK/STATs pathway;PLC-γ pathway. However, much work still needs tobe done before a specific component of thesepathways can be applied for effective control oftumor growth in the clinic.

MMAC1/PTEN inhibits cell growth and induces chemosensitivity to doxorubicin in human bladder cancer cells

The development and progression of bladder cancer is associated with multiple alterations in the genome, including loss of chromosome 10. Recently, MMAC1/PTEN, a phosphatidylinositol phosphatase, has been mapped to chromosome 10q23. We previously demonstrated that MMAC1/PTEN has tumor suppressive properties in glioblastoma and prostate cancer. To investigate the efficacy of gene therapy with MMAC1/PTEN, we examined whether the exogenous introduction of MMAC1/PTEN via an adenoviral vector (Ad-MMAC) can inhibit tumor growth and reverse drug resistance to doxorubicin in human bladder cancer cells. Human bladder cancer cell lines UM-UC-3 and T24 were infected with Ad-MMAC to induce exogenous expression of MMAC1/PTEN. The cells were then analysed for cell growth and expression of phosphorylated protein kinase B (Akt/PKB) and MMAC1/PTEN. UM-UC-6dox, a doxorubicin resistant subline, was infected with Ad-MMAC to evaluate its role in reversing drug resistance to doxorubicin. We found that MMAC1/PTEN suppressed tumor growth in UM-UC-3 and T24 cells with arrest in the G1 phase of the cell cycle. We also showed that gene therapy with MMAC1/PTEN abrogated phosphorylated Akt/PKB expression in UM-UC-3, T24 and UMUC-6dox cells, and restored doxorubicin sensitivity in UM-UC-6dox. These data demonstrate that MMAC1/PTEN can induce growth suppression and increase sensitivity to doxorubicin in bladder cancer cells and suggest that the MMAC1/PTEN gene and its pathways can be therapeutic targets for bladder cancer.

Deletion mapping of chromosome 4 in head and neck squamous cell carcinoma

Genomic deletions involving chromosome 4 have recently been implicated in several human cancers. To identify and characterize genetic events associated with the development of head and neck squamous cell carinoma (HNSCC), a fine mapping of allelic losses associated with chromosome 4 was performed on DNA isolated from 27 matched primary tumor specimens and normal tissues. Loss of heterozygosity (LOH) of at least one chromosome 4 polymorphic allele was seen in the majority of tumors (92%). Allelic deletions were confined to short arm loci in four tumors and to the long arm loci in 12 tumors, suggesting the presence of two regions of common deletion. One region of frequent deletion was centered at D4S405 on 4p and included the loci D4S1546 to D4S428 in ∼41% of the tumors. The common region of deletion on 4q was more complex and extended from D4S1571 to D4S1573. Frequent genetic alterations were observed within this region (4q25) and one marker, D4S407, exhibited a high frequency of LOH (>75%). These results indicate that alterations of chromosome 4 regions are associated with HNSCC tumorigenesis and further localizes the regions that may harbor tumor suppressor genes.

Role of plasminogen activator and of 92-KDa type IV collagenase in glioblastoma invasion using an in vitro matrigel model

SummaryThe invasive nature of human gliomas represents a major factor in preventing their total resection. The exact nature of the underlying mechanisms of tumor cell invasion are still unclear. In this study, we have quantitatively assayed a glioblastoma cell line for its ability to migrate through a polycarbonate filter coated with matrigel which contains a complex of multiple basement membrane components. At 48 h the glioblastoma cell line (U251) showed a rate of invasiveness of 42% and also dependent on the concentration of matrigel. The U251 cell line produced a urokinase type plasminogen activator and a 92-KDa type IV collagenase. Both enzymes were inhibited by the addition of uPA and 92-KDa type IV collagenase antibodies. Those same antibodies reduced the invasion rate of U251 cells from 42% to 12 and 21%, respectively. Similarly, the addition of ɛ-aminocaproic acid (a plasmin inhibitor) or tissue inhibitor of metalloprotease (TIMP2, a collagenase inhibitor) reduced the invasiveness of U251 cells from 42% to 14% and 10%, respectively. Additionally, the other two glioblastoma cell lines (LG11, UWR1) and astrocytes showed a rate of invasiveness at 41%, 61% and 12%, respectively. Finally, the addition of hyaluronic acid to the matrigel, a constituent of brain extracellular matrix, enhanced the rate of invasion. These findings provide evidence for the role of serine proteases and metalloproteases in facilitating the invasion of extracellular matrix components by glioblastoma cell line and suggest a therapeutic role for protease inhibitors in attempting to minimize the invasive propensity of gliomas.

Functional and molecular analyses of 10q deletions in human gliomas.

Extensive genomic deletions involving chromosome 10 are the most common genetic alteration in glioblastoma multiforme (GBM). To localize and examine the potential roles of two chromosome arm 10q tumor suppressor regions, we used two independent strategies: mapping of allelic deletions, and functional analysis of phenotypic suppression after transfer of chromosome 10 fragments. By allelic deletion analysis, the region of 10q surrounding the MMAC/PTEN locus was shown to be frequently lost in GBMs but maintained in most low‐grade astrocytic tumors. An additional region at 10q25 containing the DMBT1 locus was lost in all grades of gliomas examined. The potential biological significance of these two regions was further assessed by examining microcell hybrids that contained various fragments of 10q. Somatic cell hybrid clones that retained the MMAC/PTEN locus have a less transformed phenotype with clones exhibiting an inability to grow in soft agarose. However, presence or absence of DMBT1 did not correlate with any in vitro phenotype assessed in our model system. These results support a model of molecular progression in gliomas in which the frequent deletion of 10q25–26 is an early event and is followed by the deletion of the MMAC/PTEN during the progression to high‐grade GBMs. Genes Chromosomes Cancer 24:135–143, 1999.

Overexpression of elongation factor‐1γ protein in colorectal carcinoma

Elongation factor‐1 (EF‐1) is a cellular protein that plays a role in protein synthesis by mediating the transfer of aminoacyl‐tRNA to 80S ribosomes. It is comprised of four subunits: α, β, γ, and δ. EF‐1γ is a substrate for the maturation‐promoting factor, which determines entry into the M‐phase of the cell cycle in all eukaryotic cells. Previously, the authors showed that EF‐1γ RNA is overexpressed in a high proportion of colorectal carcinomas. At that time, there were no antibodies to EF‐1γ, so the EF‐1γ protein could not be examined. Because levels of RNA do not always parallel the levels of the protein it encodes, it was important to develop antibodies to EF‐1γ to examine its expression at the protein level in colorectal carcinoma.

Phenotypic analysis of human glioma cells expressing the MMAC1 tumor suppressor phosphatase.

MMAC1, also known as PTEN or TEP-1, was recently identified as a gene commonly mutated in a variety of human neoplasias. Sequence analysis revealed that MMAC1 harbored sequences similar to those found in several protein phosphatases. Subsequent studies demonstrated that MMAC1 possessed in vitro enzymatic activity similar to that exhibited by dual specificity phosphatases. To characterize the potential cellular functions of MMAC1, we expressed wild-type and several mutant variants of MMAC1 in the human glioma cell line, U373, that lacks endogenous expression. While expression of wild-type MMAC1 in these cells significantly reduced their growth rate and saturation density, expression of enzymatically inactive MMAC1 significantly enhanced growth in soft agar. Our observations indicate that while wild-type MMAC1 exhibits activities compatible with its proposed role as a tumor suppressor, cellular expression of MMAC1 containing mutations in the catalytic domain may yield protein products that enhance transformation characteristics.

Growth inhibitory effect of recombinant α and β interferon on human glioma cells

SummaryGrowth inhibitory activity of recombinant α and β interferon on two human glioma cell lines, EFC-2 and KE cells, was determined by two different growth assays. Recombinant β interferon showed β slight growth inhibitory effect on EFC-2 cells at day 3, and maximum inhibition was seen on day 6 with an ID50 of 50 U/ml. Recombinant α interferon showed no significant growth inhibition at any concentration. KE cells were resistant to both recombinant α and β interferon. The growth inhibitory activity of recombinant β interferon on EFC-2 cells was not blocked by recombinant α interferon, although recombinant α and β interferons shared same receptors on EFC-2 cells. Addition of DFMO (α-difluoromethylornithine) to interferon in the media showed additive effect rather than synergistic effect in growth inhibition of glioma cells. Out of 7 glioma cell lines tested, 4 showed heterogeneous sensitivity to recombinant β interferon, and all were resistant to recombinant α interferon. These results suggest β differential sensitivity of EFC-2 cells to recombinant β interferon, as well as a heterogeneous sensitivity to recombinant β interferon among different glioma cell lines.