Human Sporadic Tumors Research Articles

Alterations of the AKT Pathway in Sporadic Human Tumors, Inherited Susceptibility to Cancer, and Overgrowth Syndromes.

The AKT kinases are critical signaling molecules that regulate cellular physiology upon the activation of tyrosine kinase receptors and phosphatidylinositol 3-kinases (PI3K). AKT kinases govern many cellular processes considered hallmarks of cancer, including cell proliferation and survival, cell size, tumor invasion, metastasis, and angiogenesis. AKT signaling is regulated by multiple tumor suppressors and oncogenic proteins whose loss or activation, respectively, leads to dysregulation of this pathway, thereby contributing to oncogenesis. Herein, we review the enormous body of literature documenting how the AKT pathway becomes hyperactivated in sporadic human tumors and various hereditary cancer syndromes. We also discuss the role of activating mutations of AKT pathway genes in various chimeric overgrowth disorders, including Proteus syndrome, hypoglycemia with hypertrophy, CLOVES and SOLAMEN syndromes, and hemimegalencephaly.

PTEN as a Prognostic/Predictive Biomarker in Cancer: An Unfulfilled Promise?

Identifying putative biomarkers of clinical outcomes in cancer is crucial for successful enrichment, and for the selection of patients who are the most likely to benefit from a specific therapeutic approach. Indeed, current research in personalized cancer therapy focuses on the possibility of identifying biomarkers that predict prognosis, sensitivity or resistance to therapies. Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is a tumor suppressor gene that regulates several crucial cell functions such as proliferation, survival, genomic stability and cell motility through both enzymatic and non-enzymatic activities and phosphatidylinositol 3-kinase (PI3K)-dependent and -independent mechanisms. Despite its undisputed role as a tumor suppressor, assessment of PTEN status in sporadic human tumors has yet to provide clinically robust prognostic, predictive or therapeutic information. This is possibly due to the exceptionally complex regulation of PTEN function, which involves genetic, transcriptional, post-transcriptional and post-translational events. This review shows a brief summary of the regulation and function of PTEN and discusses its controversial aspects as a prognostic/predictive biomarker.

Abstract 1686: TSC2 enhances antitumor immunity and potentiates PD-1 and CTLA-4 blockade

Abstract Tuberous sclerosis complex (TSC) is an incurable multisystem genetic disease characterized by mTORC1-hyperactive hamartomatous tumors of brain, kidney, and lung. TSC is caused by mutations in the two TSC1 or TSC2 tumor-suppressor genes. mTORC1 inhibitors lead to partial antitumor responses in TSC, with tumor regrowth upon treatment cessation. Understanding the interaction between T cells and tumor cells has led to the development of successful checkpoint blockade immunotherapy targeting PD-1, programmed cell death-ligand 1 (PD-L1), and CTLA-4. These therapies have proven to be highly effective in treating several malignancies, including non-small cell lung cancer (NSCLC), renal cell carcinoma (RCC), melanoma, bladder cancer and Hodgkin's lymphoma. It is currently unknown whether benign tumors, such as those arising in TSC, will also respond to checkpoint blockade therapy. Using multiparametric flow cytometry and immunohistochemistry of human angiomyolipomas and pulmonary LAM, we discovered that PD-1 is highly expressed on tumor-infiltrating T cells. Using immunocompetent preclinical models of TSC, we found that dual blockade of PD-1 and CTLA-4 inhibited the growth of TSC2-deficient tumors by 77% (p < 0.0001). This was associated with increased tumor-infiltrating CD8+ and CD4+ T cells and decreased Tregs, G-MDSCs and regulatory CD11b+ DCs. Strikingly, we discovered that complete regression of established tumors can be achieved in 37% of the mice using anti-PD-1 monotherapy and in 62% of the mice using combined anti-PD-1 and anti-CTLA-4 therapy. We also assessed a treatment regimen of rapamycin followed by PD-1 blockade and found that this sequential treatment delays regrowth of TSC2-deficient tumors. Furthermore, TSC2 re-expression in TSC2-deficient tumors promoted antitumor responses via increasing T cells and decreasing CD11b+Ly6G+Ly6Cmed G-MDSCs and CD11b+ DCs infiltration into tumors. Importantly, we found that the potency of dual PD-1 and CTLA-4 blockade is significantly enhanced by TSC2 re-expression. Our data indicate that PD-1 is upregulated on T cells in TSC-associated tumors and that dual blockade of PD-1 and CTLA-4 pathways is effective in delaying tumor growth and improving long-term survival, with long-term complete tumor responses. Therefore, PD-1 and/or CTLA-4 blockade may represent a promising dual immunotherapy for patients with TSC-associated tumors and women with the sporadic form of LAM. Our data may have relevance for the many sporadic human tumors with mTORC1 hyperactivation (~50% of all human malignancies) and also for other hamartomatous tumor syndromes such as neurofibromatosis. Furthermore, the specific role of intratumoral TSC2 in antitumor T cell responses that we discovered may help to elucidate the response to immunotherapy in sporadic human malignancies with mutations in the TSC genes, which include a subset of bladder cancer and renal cell carcinoma. Citation Format: Heng-Jia Liu, Patrick Lizotte, Heng Du, Maria Speranza, Spencer Vaughan, Nicola Alesi, Kwok-Kin Wong, Gordon Freeman, Arlene Sharpe, Elizabeth Henske. TSC2 enhances antitumor immunity and potentiates PD-1 and CTLA-4 blockade [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1686.

Abstract P3-03-03: A mouse model of sporadic breast tumor with a conditional P53 mutation

Abstract Missense mutations in the tumor suppressor gene p53 are present in more than 50% of human tumors. In particular, the arginine-to-histidine mutation at codon 175 (p53R175H) has been found in more than 4% of human breast cancers. p53 mutations fall into two general categories: germline mutations that are associated with hereditary tumors, and somatic mutations that cause sporadic tumors. Mouse models for cancers induced by p53 germline mutations have been established and characterized. However, hitherto there are no accurate animal models for sporadic tumors induced by p53 somatic missense mutations, although this mechanism of inactivating p53 occurs in a large fraction of human cancers. We have generated a mouse allele, p53WM, that carries coding sequences for both wild type (WT) and R172H mutant p53 (corresponding to R175H in humans) at the p53 endogenous locus. The coding sequence for WT p53 is flanked by loxP sites and therefore can be deleted by Cre recombinase. In the absence of Cre, following radiation, the p53WM/+ mice have the similar ability as the p53 WT mice to activate p53, as demonstrated by transcriptional activation of p53 targets and induction of apoptosis. In addition, like WT mouse embryonic fibroblasts (MEFs), the p53WM/+ MEFs also underwent cell cycle arrest following radiation. After crossing p53WM/+ mice with Zp3-Cre mice which express the Cre recombinase in oocytes, the coding sequence for WT p53 was removed, and the mutant p53R172H allele was regenerated as determined by sequencing, western blot analysis and real time reverse transcription PCR. To investigate whether a somatic p53R172H mutation in mammary epithelium can induce breast tumors, adenoviruses expressing Cre (Ad-Cre) were injected into the mammary duct of p53WM/+ mice. Limiting the adenovirus dose allowed us to induce the p53R172H mutation in 1 of 20, 100 or 1000 cells, generating a mutant p53 surrounded by normal cells which more accurately mimics the clinical situation of sporadic tumors. Meanwhile, p53WM/+; K14-Cre and p53WM/+; WAP-Cre mice have been generated, in which p53R172H mutation are induced through Cre transgene specifically in the mammary epithelial cells. Lastly, to examine how somatic p53 mutation cooperates with an oncogene in mammary tumor induction, MMTV-neu; p53WM/+ mice were also subjected to the mammary intraductal Ad-Cre injection. We are currently monitoring mice and will further investigate the molecular mechanisms of tumorigenesis by studying the loss of heterozygosity, and the co-evolution of tumor epithelial cells and their microenvironment. This mouse model is more likely to share the underlying molecular pathology with human sporadic tumors. Therefore, it will be more predictive of human responses to drugs, and thus a more valuable tool in preclinical testing. Citation Format: Zhang Y, Xiong S, Pant V, El-Naggar A, Lozano G. A mouse model of sporadic breast tumor with a conditional P53 mutation. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P3-03-03.

Cowden Syndrome-Related Mutations in PTEN Associate with Enhanced Proteasome Activity

Germline mutations in PTEN have been described in a spectrum of syndromes that are collectively known as PTEN hamartoma tumor syndrome (PHTS). In addition to being mutated in the germline in PHTS, somatic loss-of-function PTEN mutations are seen in a wide range of sporadic human tumors. Here, we show evidence of upregulated proteasome activity in PHTS-derived lymphoblasts, Pten knock-in mice and cell lines expressing missense and nonsense PTEN mutations. Notably, elevated nuclear proteasome activity occurred in cells expressing the nuclear mislocalized PTEN-K62R mutant, whereas elevated cytosolic proteasome activity was observed in cells expressing the cytosolic-predominant mutant PTEN (M3M4 and C136R). Treatment with proteasome inhibitor MG-132 was able to restore both nonsense and missense mutant PTEN protein levels in vitro. PHTS patients with destabilizing PTEN mutations and proteasome hyperactivity are more susceptible to develop neurologic symptoms such as mental retardation and autism than mutation-positive patients with normal proteasome activity. A detailed molecular and functional analysis shows that PTEN mutants most likely cause proteasome hyperactivity via 2 different mechanisms, namely, induction of proteotoxic stress and loss of protein phosphatase activity. These results provide novel insights into the cellular functions of PTEN and reveal molecular mechanisms whereby PTEN mutations increase proteasome activity and lead to neurologic phenotypes.

Quantitative promoter hypermethylation analysis of RASSF1A in lung cancer: Comparison with methylation-specific PCR technique and clinical significance

Lung cancer is the major health problem and leading cause of cancer-related deaths worldwide owing to late diagnosis and poor prognosis. Aberrant promoter methylation is an important mechanism for silencing tumor-suppressor genes in cancer and a promising tool for the development of molecular biomarkers. Ras association domain family 1A (RASSF1A), a pivotal gatekeeper of cell cycle progression, is highly methylated in a wide range of human sporadic tumors, including lung cancer. However, no significant prognostic implications have been observed in most studies qualitatively analyzed by methylation-specific PCR (MSP). We found that the RASSF1A promoter was aberrantly methylated in 44.7 and 37.4% of the tumors by pyrosequencing (PS) and MSP methods, respectively. RASSF1A methylation evaluated by the two methods was more frequent in ever-smokers and tumors with TP53 mutation than in never-smokers and tumors without TP53 mutation, respectively. Univariate and multivariate analyses revealed that strong methylation was an unfavorable prognostic factor with stage I (adjusted HR, 2.25; 95% CI 1.03-4.90; P=0.003) and squamous cell carcinoma patients (adjusted HR=2.25, 95% CI 1.03-4.90, P=0.042). Taken together, these results suggested that quantitative PS could gain wider applications in clinical samples as a promising method for early detection screening and prognosis compared with MSP.

On hedgehogs and human cancer

Ariel Ruiz i Altaba and Frederic Varnat thank the editor for inviting a response to Dr. Curran's letter, which questions the specificity of cyclopamine and the xenografts used in our recent work on human sporadic colon carcinomas (CCs) (Varnat et al, 2009). To respond, in detail, we address five issues. First , a key issue is when and where is the pathway active in order to know if effects of HH‐GLI antagonists can be specific. We and others established that GLI1 transcription is, so far, the only reliable and general HEDGEHOG‐GLI (HH‐GLI) pathway activation marker ( e.g. Lee et al, 1997), and that it is expressed in many kinds of sporadic human tumours. Antisense ribonucleic acid (RNA) in situ hybridizations on fresh patient‐derived tissues have unambiguously shown the tumour cells themselves, not the surrounding stroma, express GLI1 and other HH‐GLI pathway components in basal cell carcinomas, melanomas, gliomas, medulloblastomas, prostate and colon cancers (Clement et al, 2007; Dahmane et al, 1997; Sanchez et al, 2004; Stecca et al, 2007; Varnat et al, 2009). Recent in situ hybridization images of pancreatic cancer are undecisive (see Lauth et al, 2010). In cases where antibodies to pathway components have been used, strong labelling is found in the tumour cells themselves ( e.g. Varnat et al, 2009; see also www.proteinatlas.org). So far there is no evidence of exclusive pathway activity in human stromal cells. In contrast, mice display paracrine Hh signalling in several organs but the use of human xenografts to determine mode of action appears inappropriate as these are chimeras. The epithelial …

Hippo Tumor Supressor Pathway: Novel Implications for the Treatment of Hepatocellular Carcinoma

Zhou D, Conrad C, Xia F, et al. (Department of Molecular Biology, Massachusetts General Hospital, Boston, Massachusetts). Mst1 and Mst2 maintain quiescence and suppress hepatocellular carcinoma development through inactivation of the Yap1 oncogene. Cancer Cell 2009;16:425–438.

P53: A Guide to Apoptosis

Approximately 50% of sporadic human tumors harbor somatic mutations in the p53 gene locus, while germ line mutations confer a high familial risk and are associated with Li-Fraumeni Syndrome patients. The p53 tumor suppressor protein is often referred to as the "guardian of the genome" since its response to DNA-damage or checkpoint failure gives rise to a series of anti-proliferative responses. One of the most important functions of p53 is its ability to induce apoptosis, while disruption of this route can promote tumor progression and chemo resistance. Besides its ability to promote apoptosis through transcription dependent mechanisms, p53 may also be able to activate apoptosis independent of transcriptional regulation. Therefore, to ensure normal cell growth, p53 levels and activity are tightly regulated. Upon diverse forms of cellular stress the steady state levels and transcriptional activity of p53 are considerably increased. The stabilization and activation of p53 are a result of hindered inhibition by its negative regulators, e.g. Mdmx (also known as Mdm4) and Mdm2, while on the other hand activators such as HIPK2 and DYRK2 enhance the p53 response. The continually increasing understanding of the mechanisms of regulation of p53 may provide the basis for new drug designs that could eventually lead to therapeutics to reactivate p53 in cancers.

Brain as a paradigm of organ growth: Hedgehog–Gli signaling in neural stem cells and brain tumors

The Hedgehog-Gli (Hh-Gli) signaling pathway is essential for numerous events during the development of many animal cell types and organs. In particular, it controls neural cell precursor proliferation in dorsal brain structures and regulates the number of neural stem cells in distinct embryonic, perinatal, and adult niches, such as the developing neocortex, the subventricular zone of the lateral ventricle of the forebrain, and the hippocampus. We have proposed that Hh-Gli signaling regulates dorsal brain growth during ontogeny and that its differential regulation underlays evolutionary change in the morphology (size and shape) of dorsal brain structures. It is also critically involved in sporadic brain tumorigenesis--as well as several other human cancer--suggesting that tumors derive from stem cells or progenitors maintaining an inappropriate active Hh-Gli pathway. Importantly, we and others have demonstrated that human sporadic tumors from the brain and other organs require sustained HH-GLI signaling for sustained growth and survival. Modulating HH-GLI signaling thus represents a novel rational avenue to treat, on one hand, brain degeneration and injury by inducing controlled HH-GLI-mediated regeneration and growth, and on the other hand, to combat cancer by blocking its abnormal activity in tumor cells.

Aberrations of the CHK2 gene are rare in pediatric solid tumors

In pediatric solid tumors, such as neuroblastoma (NB), it has been reported that the frequency of TP53 gene alterations is lower than that in adult tumors, suggesting that other tumor suppressor genes may play more important roles in the development of pediatric solid tumors. The CHK2 gene, whose product is a checkpoint kinase that plays a central role in DNA damage response and acts upstream of TP53, has been found to be mutated in a subset of Li-Fraumeni syndrome without mutations of TP53 and in some other sporadic human tumors, earmarking this serine/threonine kinase as a candidate tumor suppressor gene. Thus, we analyzed the CHK2 gene to address whether it is a candidate tumor suppressor gene for pediatric solid tumors. We screened for mutations of the CHK2 gene in 25 NB, 8 rhbdomyosarcoma, 12 Ewing sarcoma, and 26 other pediatric solid tumor cell lines as well as 77 fresh tumors including two cases of multiple cancers. Using polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) analysis and reverse transcriptase (RT)-PCR-SSCP followed by direct sequence analysis, we detected only one missense mutation (S505T) in one NB cell line and two silent mutations in one NB cell line and one NB fresh tumor, respectively. Through RT-PCR and subcloning analysis, we detected a similar expression of the CHK2 gene in all of the NB cell lines and fresh tumors; however, we identified at least three isoforms of the CHK2 gene, two of which have not been reported previously. These results suggest that aberrations of the CHK2 gene are rare in pediatric solid tumors.

Loss of the tumor suppressor gene PTEN marks the transition from intratubular germ cell neoplasias (ITGCN) to invasive germ cell tumors

PTEN/MMAC1/TEP1: (hereafter PTEN) is a tumor suppressor gene (located at 10q23) that is frequently mutated or deleted in sporadic human tumors. PTEN encodes a multifunctional phosphatase, which negatively regulates cell growth, migration and survival via the phosphatidylinositol 3'-kinase/AKT signalling pathway. Accordingly, Pten+/- mice develop various types of tumors including teratocarcinomas and teratomas. We have investigated PTEN expression in 60 bioptic specimens of germ cell tumors (32 seminomas, 22 embryonal carcinomas and six teratomas) and 22 intratubular germ cell neoplasias (ITGCN) adjacent to the tumors for PTEN protein and mRNA expression. In total, 10 testicular biopsies were used as controls. In the testis, PTEN was abundantly expressed in germ cells whereas it was virtually absent from 56% of seminomas as well as from 86% of embryonal carcinomas and virtually all teratomas. On the contrary, ITGCN intensely expressed PTEN, indicating that loss of PTEN expression is not an early event in testicular tumor development. The loss of PTEN expression occurs mainly at the RNA level as determined by in situ hybridization of cellular mRNA (17/22) but also it may involve some kind of post-transcriptional mechanisms in the remaining 25% of cases. Analysis of microsatellites D10S551, D10S541 and D10S1765 in GCTs (n=22) showed LOH at the PTEN locus at 10q23 in at least 36% of GCTs (three embryonal carcinoma, three seminoma, two teratoma); one seminoma and one embryonal (9%) carcinoma presented an inactivating mutation in the PTEN gene (2/22). Finally, we demonstrated that the phosphatidylinositol 3'-kinase/AKT pathway, which is regulated by the PTEN phosphatase, is crucial in regulating the proliferation of the NT2/D1 embryonal carcinoma cells, and that the cyclin-dependent kinase inhibitor p27(kip1) is a key downstream target of this pathway.

Characterization of novel non-clonal intrachromosomal rearrangements between the H4 and PTEN genes (H4/PTEN) in human thyroid cell lines and papillary thyroid cancer specimens

The two main forms of RET rearrangement in papillary thyroid carcinomas (PTC) arise from intrachromosomal inversions fusing the tyrosine kinase domain of RET with either the H4 (RET/PTC1) or the ELE1/RFG genes (RET/PTC3). PTEN codes for a dual-specificity phosphatase and maps to chromosome 10q22-23. Germline mutations confer susceptibility to Cowden syndrome whereas somatic mutations or deletions are common in several sporadic human tumors. Decreased PTEN expression has been implicated in thyroid cancer development. We report the characterization of a new chromosome 10 rearrangement involving H4 and PTEN. The initial H4 /PTEN rearrangement was discovered as a non-specific product of RT-PCR for RET/PTC1 in irradiated thyroid cell lines. Sequencing revealed a transcript consisting of exon 1 and 2 of H4 fused with exons 3–6 of PTEN. Nested RT-PCR with specific primers bracketing the breakpoints confirmed the H4 /PTEN rearrangements in irradiated KAT-1 and KAT-50 cells. Additional H4 /PTEN variants, generated by recombination of either exon 1 or exon 2 of H4 with exon 6 of PTEN, were found in non-irradiated KAK-1, KAT-50, ARO and NPA cells. Their origin through chromosomal recombination was confirmed by detection of the reciprocal PTEN /H4 product. H4 /PTEN recombination was not a clonal event in any of the cell lines, as Southern blots with appropriate probes failed to demonstrate aberrant bands, and multicolor FISH of KAK1 cells with BAC probes for H4 and PTEN did not show a signal overlap in all cells. Based on PCR of serially diluted samples, the minimal frequency of spontaneous recombination between these loci was estimated to be approximately 1/10 6 cells. H4/PTEN products were found by nested RT-PCR in 4/14 normal thyroid tissues (28%) and 14/18 PTC (78%) ( P < 0.01). H4 /PTEN is another example of recombination involving the H4 locus, and points to the high susceptibility of thyroid cells to intrachromosomal gene rearrangements. As this also represents a plausible mechanism for loss-of-function of PTEN, other thyroid neoplastic phenotypes and eventually other cancer types need to be screened for clonal H4 /PTEN rearrangements.

Application of inter-simple sequence repeat PCR to mouse models: assessment of genetic alterations in carcinogenesis.

Genomic instability is believed to play a significant role in cancer development by facilitating tumor progression and tumor heterogeneity. Inter-simple sequence repeat (inter-SSR) PCR has been proved to be a fast and reproducible technique for quantitation of genomic instability (amplifications, deletions, translocations, and insertions) in human sporadic tumors. However, the use of inter-SSR PCR in animal models of cancer has never been described. This new technique has been adapted in our laboratory for the analysis of spontaneous and induced mouse tumors. We established the best PCR conditions for each microsatellite-anchored primer and critically evaluated the reproducibility of the band patterns. We also studied the variation of the fingerprints between and within various inbred mouse strains, including wild-derived lines. Tumor-specific alterations were detected as gains, losses, or intensity changes in bands when compared with matched normal DNA. We quantitated the extent of alterations by dividing the number of altered bands in the tumor by the total number of bands in normal DNA (instability index). By means of inter-SSR PCR, we successfully analyzed genomic alterations in various mouse tumors, including spontaneous thymic lymphomas developed in Msh2 knockout mice as well as chemically induced squamous cell carcinomas and thymic lymphomas. Instability index values ranged between 0 and 9%, the highest levels observed in N-methyl-N-nitrosourea-induced thymic lymphomas generated in Trp53 (p53) nullizygote (-/-) mice. We report here, for the first time, the use of inter-SSR PCR to detect somatic mutations in mouse tumoral DNA, including laser-capture microdissected, methanol-fixed tissues. These PCR-based fingerprints provide a novel approach to assessing the number and onset of mutational events in mouse tumors and will help to understand better the mechanisms of carcinogenesis in mouse models.

Introduction of wild-type patched gene suppresses the oncogenic potential of human squamous cell carcinoma cell lines including A431.

Defects in a developmental signaling pathway involving the mammalian homologue of the Drosophila segment polarity gene, patched are associated with human tumors such as basal cell carcinoma, medulloblastoma and squamous cell carcinoma. Loss of heterozygosity (LOH) in some of these tumor cells suggests that patched functions as a tumor suppressor gene. To evaluate the biological significance of patched mutations in human sporadic tumor cells, we constructed a VSV-G pseudotyped retrovirus vector carrying the wild-type patched gene and transduced it into two human squamous cell carcinoma (SCC) cell lines, A431 and KA, that express only mutant patched mRNA. When SSC cells were transduced with Ptc virus, colony forming activity in soft agar was drastically reduced and these cells recovered anchorage independent growth when Sonic hedgehog (Shh), the ligand of Patched (Ptc), was added into the soft agar culture. Expression of exogenous patched, however, had no effect on anchorage independent growth of Ras-transformed NIH3T3 cells or SCC cell line, NA, which expresses wild-type patched mRNA. Cyclopamine, a specific inhibitor of the Shh/Ptc/Smo signaling pathway, efficiently suppressed anchorage independent growth of A431 and KA cells. These results indicate that loss of patched function plays a major role in the acquisition of oncogenic potential in these SCCs and further that Ptc virus would be an effective reagent for suppressing tumorigenicity of such SCCs.

Photodynamic therapy of DNA mismatch repair-deficient and -proficient tumour cells.

Loss of DNA mismatch repair is a common finding in hereditary nonpolyposis colon cancer as well as in many types of sporadic human tumours. DNA mismatch repair-deficient cells have been reported to be resistant to many chemotherapeutic agents and to radiotherapy, and to have the potential of rapidly acquiring additional mutations leading to tumour progression. Photodynamic therapy is a new treatment modality using light to activate a photosensitiser that preferentially localises in tumour cells. An oxygen dependent photochemical reaction ensues, resulting in selective tumour necrosis. The effect of loss of DNA mismatch repair activity on the sensitivity to photodynamic therapy was tested using pairs of cell lines proficient or deficient in mismatch repair due to loss of either MLH1 or MSH2 protein function. Cells were incubated with the photosensitiser 5,10,15,20-meta-tetra(hydroxyphenyl)chlorin and exposed to laser light at 652 nm with various optical doses ranging from 0–1 J cm−2. Cell survival was assessed using the clonogenic assay. Loss of MLH1 or MSH2 function was not associated with resistance to photodynamic therapy. MCF-7 cells repeatedly treated with photodynamic therapy expressed parental levels of MLH1, MSH2, MSH6, and PMS2. DNA mismatch repair-deficient and -proficient cells showed similar subcellular distributions of meta-tetra(hydroxyphenyl)chlorin as analysed by laser scanning and fluorescence microscopy. Therefore, repeated exposure of tumour cells to photodynamic therapy does not seem to result in loss of DNA mismatch repair, and loss of mismatch repair, in turn, does not seem to contribute to resistance to photodynamic therapy. Our results suggest recommending photodynamic therapy as a strategy for circumventing resistance due to loss of DNA mismatch repair.British Journal of Cancer (2002) 86, 1130–1135. DOI: 10.1038/sj/bjc/6600218 www.bjcancer.com© 2002 Cancer Research UK

Microsatellite instability in primary brain tumors

Microsatellite instability has been reported in hereditary colorectal cancer syndrome and in various kinds of human sporadic tumors. It has also been shown in brain tumors, although with conflicting results. In the present study, DNA samples obtained from 20 primary brain tumors (10 glioblastomas, three astrocytomas, five meningiomas, one ependymoma, one hemangiopericytoma) were analyzed to detect microsatellite instability. Nine microsatellites, mono, di-, tri- and tetranucleotide repeat markers, located on nine different chromosomes, were used. Four of the 20 neoplasias (20%) showed microsatellite alterations in tumor DNA with respect to normal DNA. Two glioblastomas and one atypical meningioma (15%) showed additional bands or bands with shift of electrophoretic mobility, whereas allelic loss was observed in two glioblastomas (10%). In one glioblastoma, one allelic loss and one extra allele were observed at two different loci. These data indicate that in primary brain tumors there is not a high genetic instability. Although we used markers with inherently high levels of instability, only sporadic microsatellite alterations were found. Consequently, alterations in the mechanisms of DNA mismatch-repair, the most important cause of replication errors in hereditary and sporadic colorectal cancers, do not seem to play a major role in the oncogenesis of primary brain tumors. [Neurol Res 2000; 22: 571-575]

Mutations of the human PTEN gene.

PTEN (phosphatase and tensin homolog deleted on chromosome ten), a recently discovered tumor suppressor gene, appears to negatively control the phosphoinositide 3-kinase signaling pathway for regulation of cell proliferation and cell survival by dephosphorylating the phosphatidylinositol 3,4,5-triphosphate. To date, 110 germline PTEN mutations have been reported in patients affected with two tumor predisposing syndromes, each having overlapping clinical features: Cowden disease and Bannayan-Riley-Ruvalcaba syndrome. These germline mutations are scattered along the length of the gene, with the exception of exon 9 (no mutation reported) and exon 1 (only two mutations reported). A mutational hot spot is found in exon 5, which encodes the phosphatase catalytic core motif, and recurrent mutations are also found at CpG dinucleotides suggesting deamination-induced mutations. PTEN has also been found to be defective in a large number of sporadic human tumors. In this article, 332 somatic point mutations of PTEN, occurring in primary tumors or metastasis, have been reviewed. Somatic PTEN mutations are more particularly involved in two types of human cancers: endometrial carcinomas and glioblastomas. In most cases, these somatic mutations result in protein inactivation and, as with germline mutations, recurrent somatic mutations are found in CpG dinucleotides. A mutagenesis by insertion-deletion in repetitive elements is however specifically observed in endometrial carcinomas.

Loss of DNA mismatch repair due to knockout of MSH2 or PMS2 results in resistance to cisplatin and carboplatin

Loss of DNA mismatch repair is a common finding in hereditary nonpolyposis colon cancer as well as in many types of sporadic human tumors. The effect of loss of DNA mismatch repair activity on sensitivity to cisplatin and carboplatin was tested using MSH2 and PMS2 knockout cell lines. The knockout dMsh2 embryonic stem cell line was 2.1-fold more resistant to cisplatin and 1.7-fold more resistant to carboplatin when compared to the isogenic wild-type wt-2 cell line. Likewise, the PMS2(-/-) mouse fibroblasts were 1.9-fold more resistant to cisplatin and 1.5-fold more resistant to carboplatin when compared to the isogenic PMS2(+/+) fibroblasts. These findings demonstrate that loss of mismatch repair due to knockout of either MSH2 or PMS2 results in low-level resistance to cisplatin and carboplatin, drugs that form the same types of adducts in DNA. These data validate results previously obtained using non-isogenic mismatch repair-proficient and -deficient cell lines, and indicate that simple recognition of the cisplatin adduct by the MSH2/MSH6 heterodimer is not sufficient for full detector function, but that PMS2 is also required for the pro-apoptotic signal to be generated from this detector.

Genetic changes induced by heterocyclic amines

Clarification of the mutational fingerprints of HCAs offers a promising approach in the investigation of the role of heterocyclic amines (HCAs) in human carcinogenesis. We analyzed mutations in the tumor related genes of tumors induced by HCAs, 2-amino-3,4-dimethylimidazo[4,5- f]quinoline (MeIQ), 2-amino-3-methylimidazo[4,5- f]quinoline (IQ), and 2-amino-1-methyl-6-phenylimidazo[4,5- b]pyridine (PhIP), which mainly yield DNA-adducts of C8-guanine. The G→T transversion at codon 13-2nd position in Ha- ras was predominantly observed in mouse forestomach and rat Zymbal gland tumors induced by MeIQ. In contrast, various types of mutation were detected in the ras family genes of rat Zymbal gland tumors induced by IQ; the presence of a methyl group at position 4 of imidazo[4,5- f]quinoline gave rise to a remarkable difference in the mutational fingerprint. Apc mutations were detected in PhIP- and IQ-induced rat colon tumors, with incidences of 50% (4/8) and 15% (2/13), respectively. All five mutations detected in the four PhIP-induced tumors consisted of a guanine deletion from the 5′-GGGA-3′ sequence, in contrast with T to C and C to T mutations in IQ-induced tumors. Four of these five mutations shared seven common nucleotides, -GTGGGAT- surrounding the guanine; indicating that PhIP leaves a characteristic mutational fingerprint in Apc. Colon tumors induced by PhIP were also found to have mutations in their microsatellite sequences, and similar results were detected in mammary gland tumors induced by PhIP, contrasting with no mutations in IQ-induced colon tumors and a very low frequency of mutations in 7,12-dimethylbenz[ a]anthracene (DMBA)-induced mammary tumors. Although the mechanisms involved in the induction of microsatellite mutations are not known yet, microsatellite mutations which can also be detected in sporadic human tumors, including colon and breast tumors, were indicated to be a characteristic of PhIP. Mammary tumors induced by PhIP showed loss of heterozygocity (LOH) at the distal part of chromosome 10, which shows synteny with the distal part of human chromosome 17, where LOH frequently occurs in human breast cancer. In conclusion, each heterocyclic amine leave a mutational fingerprint which is specific to each compound. Since the tumor-related genes involved in PhIP-induced tumors have characteristics in common with those in human cancers, further detailed analysis will provide us with useful information on mutational fingerprints, and on the possible contribution of PhIP to human colon cancer.