Fibroblast Growth Factor Receptors as Novel Therapeutic Targets in SNF5-Deleted Malignant Rhabdoid Tumors

Malignant rhabdoid tumors (MRTs) are aggressive pediatric cancers arising in brain, kidney and soft tissues, which are characterized by loss of the tumor suppressor SNF5/SMARCB1. MRTs are poorly responsive to chemotherapy and thus a high unmet clinical need exists for novel therapies for MRT patients. SNF5 is a core subunit of the SWI/SNF chromatin remodeling complex which affects gene expression by nucleosome remodeling. Here, we report that loss of SNF5 function correlates with increased expression of fibroblast growth factor receptors (FGFRs) in MRT cell lines and primary tumors and that re-expression of SNF5 in MRT cells causes a marked repression of FGFR expression. Conversely, siRNA-mediated impairment of SWI/SNF function leads to elevated levels of FGFR2 in human fibroblasts. In vivo, treatment with NVP-BGJ398, a selective FGFR inhibitor, blocks progression of a murine MRT model. Hence, we identify FGFR signaling as an aberrantly activated oncogenic pathway in MRTs and propose pharmacological inhibition of FGFRs as a potential novel clinical therapy for MRTs.     

Preclinical Evaluation of Engineered Oncolytic Herpes Simplex Virus for the Treatment of Pediatric Solid Tumors

Recently, investigators showed that mice with syngeneic murine gliomas that were treated with a neuroattenuated oncolytic herpes simplex virus-1 (oHSV), M002, had a significant increase in survival. M002 has deletions in both copies of the γ₁34.5 gene, enabling replication in tumor cells but precluding infection of normal cells. Previous studies have shown antitumor effects of other oHSV against a number of adult tumors including hepatocellular carcinoma and renal cell carcinoma. The purpose of the current study was to investigate the oncolytic potential of M002 against difficult to treat pediatric liver and kidney tumors. We showed that the oHSV, M002, infected, replicated, and decreased cell survival in hepatoblastoma, malignant rhabdoid kidney tumor, and renal sarcoma cell lines. In addition, we showed that in murine xenografts, treatment with M002 significantly increased survival and decreased tumor growth. Finally, these studies showed that the primary entry protein for oHSV, CD111 (nectin-1) was present in human hepatoblastoma and malignant rhabdoid kidney tumor specimens. We concluded that M002 effectively targeted these rare aggressive tumor types and that M002 may have potential for use in children with unresponsive or relapsed pediatric solid tumors.     

Malignant rhabdoid tumor of the kidney in a child: report of a case with recurrence in the contralateral kidney

BACKGROUND: Malignant rhabdoid tumor (MRT) of the kidney is a rare and aggressive neoplasm with a controversial histogenesis. Although their immunohistochemistry may be diverse, the rhabdoid phenotype and mutations of the INI1 gene are consistently exhibited by MRTs regardless of their location. CASE: MRT recurred in the contralateral kidney in a 12-month-old child within 6 months after the initial histologic diagnosis, nephrectomy and autologous stem cell transplant. The presence of widespread metastases at the time of the recurrence precluded any further chemical and surgical diagnostic or therapeutic intervention. CONCLUSION: To the best of our knowledge, only a few cases describing the cytologic diagnosis of MRT of the kidney in a child have been reported. This case illustrates the usefulness of cytologic diagnosis in an MRT recurrence.     

Differential expression of miRNAs in rhabdomyosarcoma and malignant rhabdoid tumor

Alveolar rhabdomyosarcoma (RMA) and malignant rhabdoid tumor (MRT) have a frequent metastatic spread and a poor prognosis. Aberrant miRNA expression is often found in metastatic tumors. The aim of this study was to identify specific miRNA expression patterns in these tumors. We analyzed the expression of miRNAs in RMA and MRT in tissue samples and in the rhabdomyosarcoma (RMS) cell lines (Rh30 and RD). Selected target miRNAs were modulated with mimic or inhibitor oligonucleotides. Functional analysis was monitored by flow cytometry and migration assays. A set of 107 differentially expressed miRNAs showed tissue-specific clustering of RMA and MRT. Comparison with the Sarcoma microRNA Expression Database revealed RMA- and MRT-specific miRNAs. Metastatic invasion associated miRNA miR-9^? was overexpressed in RMA. miR-200c—inhibiting migration—was lower expressed in RMA than in MRT. Transient transfection of RMS cells with a miR-200c mimic and miR-9^* inhibitor did neither increase the expression of the known target E-cadherin nor decrease migration. Expression of E-cadherin could be induced in RD cells using decitabine, but demethylation did not influence cell migration. Despite a comparable high rate of metastatic invasion pediatric RMA and MRT show a different pattern of miRNA expression possibly allowing risk stratification.     

Loss of the tumor suppressor Snf5 leads to aberrant activation of the Hedgehog-Gli pathway

Aberrant activation of the Hedgehog (Hh) pathway can drive tumorigenesis. To investigate the mechanism by which glioma-associated oncogene family zinc finger-1 (GLI1), a crucial effector of Hh signaling, regulates Hh pathway activation, we searched for GLI1-interacting proteins. We report that the chromatin remodeling protein SNF5 (encoded by SMARCB1, hereafter called SNF5), which is inactivated in human malignant rhabdoid tumors (MRTs), interacts with GLI1. We show that Snf5 localizes to Gli1-regulated promoters and that loss of Snf5 leads to activation of the Hh-Gli pathway. Conversely, re-expression of SNF5 in MRT cells represses GLI1. Consistent with this, we show the presence of a Hh-Gli-activated gene expression profile in primary MRTs and show that GLI1 drives the growth of SNF5-deficient MRT cells in vitro and in vivo. Therefore, our studies reveal that SNF5 is a key mediator of Hh signaling and that aberrant activation of GLI1 is a previously undescribed targetable mechanism contributing to the growth of MRT cells.     

FK506 confers chemosensitivity to anticancer drugs in glioblastoma multiforme cells by decreasing the expression of the multiple resistance-associated protein-1

Malignant rhabdoid tumor (MRT) is a rare and highly aggressive neoplasm of young children. MRT is characterized by inactivation of integrase interactor 1 (INI1). Cyclin-dependent kinase 4 (CDK4), which acts downstream of INI1, is required for the proliferation of MRT cells. Here we investigated the effects of PD 0332991 (PD), a potent inhibitor of CDK4, against five human MRT cell lines (MP-MRT-AN, KP-MRT-RY, G401, KP-MRT-NS, KP-MRT-YM). In all of the cell lines except KP-MRT-YM, PD inhibited cell proliferation >50%, (IC₅₀ values 0.01 to 0.6 μM) by WST-8 assay, and induced G1-phase cell cycle arrest, as shown by flow cytometry and BrdU incorporation assay. The sensitivity of the MRT cell lines to PD was inversely correlated with p16 expression (r = 0.951). KP-MRT-YM cells overexpress p16 and were resistant to the growth inhibitory effect of PD. Small interfering RNA against p16 significantly increased the sensitivity of KP-MRT-YM cells to PD (p < 0.05). These results suggest that p16 expression in MRT could be used to predict its sensitivity to PD. PD may be an attractive agent for patients with MRT whose tumors express low levels of p16.     

Genetics of Bladder Malignant Tumors in Childhood

Bladder masses are represented by either benign or malignant entities. Malignant bladder tumors are frequent causes of disease and death in western countries. However, in children they are less common. Additionally, different features are found in childhood, in which non epithelial tumors are more common than epithelial ones. Rhabdomyosarcoma is the most common pediatric bladder tumor, but many other types of lesions may be found, such as malignant rhabdoid tumor (MRT), inflammatory myofibroblastic tumor and neuroblastoma. Other rarer tumors described in literature include urothelial carcinoma and other epithelial neoplasms. Rhabdomyosarcoma is associated to a variety of genetic syndromes and many genes are involved in tumor development. PAX3-FKHR and PAX7-FKHR (P-F) fusion state has important implications in the pathogenesis and biology of RMS, and different genes alterations are involved in the pathogenesis of P-F negative and embryonal RMS, which are the subsets of tumors most frequently affecting the bladder. These genes include p53, MEF2, MYOG, Ptch1, Gli1, Gli3, Myf5, MyoD1, NF1, NRAS, KRAS, HRAS, FGFR4, PIK3CA, CTNNB1, FBXW7, IGF1R, PDGFRA, ERBB2/4, MET, BCOR. Malignant rhabdoid tumor (MRT) usually shows SMARCB1/INI1 alterations. Anaplastic lymphoma kinase (ALK) gene translocations are the most frequently associated alterations in inflammatory myofibroblastic tumor (IMT). Few genes alterations in urothelial neoplasms have been reported in the paediatric population, which are mainly related to deletion of p16/lnk4, overexpression of CK20 and overexpression of p53. Here, we reviewed available literature to identify genes associated to bladder malignancies in children and discussed their possible relationships with these tumors.     

Akt and XIAP regulate the sensitivity of human uterine cancer cells to cisplatin,doxorubicin and taxol

We have investigated the interrelationship between two anti-apoptotic factors, XIAP and Akt, and their role in chemoresistance of uterine cancer cells. We used one cervical cancer cell line (HeLa) and two endometrial cancer cell lines (KLE and Ishikawa) as a model. The three drugs decreased Akt and XIAP content and induced apoptosis in P-Akt-negative HeLa cells. In P-Akt1/3-positive Ishikawa cells apoptosis induction correlated with XIAP decrease. P-Akt1/2/3-positive KLE cells showed maximum chemoresistance as XIAP and Akt levels/phosphorylation remained stable in response to the three drugs, and only cisplatin could significantly induce apoptosis. We found that XIAP and Akt were functionally linked in uterine cancer cells, as downregulation of XIAP with RNAi decreased P-Akt levels, and inhibition of PI3-K/Akt activity using LY294002 decreased XIAP content. Overexpression of constitutively active Akt isoforms in HeLa cells induced isoform-specific sensitivity to doxorubicin and taxol but not cisplatin. XIAP RNAi increased the cell-specific sensitivity to cisplatin and doxorubicin but not taxol. Finally, we found P-Akt immunoreactivity in epithelial cells from multiple human endometrial carcinoma tumors, suggesting that Akt may also regulate chemosensitivity in uterine cancers in vivo. Altogether these results highlight an intertwined role for specific Akt isoforms and XIAP in chemoresistance of uterine cancer cells.     

The potential role of Akt phosphorylation in human cancers

Akt/protein kinase B (PKB) is a serine/threonine kinase which is implicated in mediating a variety of biological responses including cell growth, proliferation and survival. Akt is activated by phosphorylation on two critical residues, namely threonine 308 (Thr308) and serine 473 (Ser473). Several studies have found Akt2 to be amplified or overexpressed at the mRNA level in various tumor cell lines and in a number of human malignancies such as colon, pancreatic and breast cancers. Nevertheless, activation of Akt isoforms by phosphorylation appears to be more clinically significant than Akt2 amplification or overexpression. Many studies in the past 4-5 years have revealed a prognostic and/or predictive role of Akt phosphorylation in breast, prostate and non-small cell lung cancer. Several publications suggest a role of phosphorylated Akt also in endometrial, pancreatic, gastric, tongue and renal cancer. However, different types of assays were used in these studies. Before assessment of P-Akt can be incorporated into routine clinical practice, all aspects of the assay methodology will have to be standardized.     

Novel role for insulin as an autocrine growth factor for malignant brain tumour cells

AT/RTs (atypical teratoid/rhabdoid tumours) of the CNS (central nervous system) are childhood malignancies associated with poor survival rates due to resistance to conventional treatments such as chemotherapy. We characterized a panel of human AT/RT and MRT (malignant rhabdoid tumour) cell lines for expression of RTKs (receptor tyrosine kinases) and their involvement in tumour growth and survival. When compared with normal brain tissue, AT/RT cell lines overexpressed the IR (insulin receptor) and the IGFIR (insulin-like growth factor-I receptor). Moreover, insulin was secreted by AT/RT cells grown in serum-free medium. Insulin potently activated Akt (also called protein kinase B) in AT/RT cells, as compared with other growth factors, such as epidermal growth factor. Pharmacological inhibitors, neutralizing antibodies, or RNAi (RNA interference) targeting the IR impaired the growth of AT/RT cell lines and induced apoptosis. Inhibitors of the PI3K (phosphoinositide 3-kinase)/Akt pathway also impaired basal and insulin-stimulated AT/RT cell proliferation. Experiments using RNAi and isoform-specific pharmacological inhibitors established a key role for the class I(A) PI3K p110alpha isoform in AT/RT cell growth and insulin signalling. Taken together, our results reveal a novel role for autocrine signalling by insulin and the IR in growth and survival of malignant human CNS tumour cells via the PI3K/Akt pathway.     

INI1 expression induces cell cycle arrest and markers of senescence in malignant rhabdoid tumor cells

The INI1 gene, which encodes a functionally uncharacterized protein component of the hSWI/SNF chromatin remodeling complex, is often mutated or deleted in malignant rhabdoid tumor (MRT). Two isoforms of INI1, that differ by the variable inclusion of nine amino acids, potentially are produced by differential RNA splicing. To determine the effect of the two INI1 isoforms on cell growth, INI1-devoid (MRT) and INI1-expressing cell lines were transfected separately with mammalian expression vectors or transduced with adenoviruses. Transfection of the short form of INI1 into either INI1-deficient or expressing cell lines resulted in complete suppression of cell growth in colony formation assays. The longer splice variant induced moderate to severe growth suppression of MRT cells, but had a far milder effect on non-MRT cells. Transduction of MRT cells with adenoviruses expressing either isoform of INI1 led to a dramatic change in morphology, growth suppression, and cell cycle arrest. Furthermore, senescence-associated proteins were up-regulated after transduction, while levels of proteins implicated in cell cycle progression were down-regulated. Adenoviral delivery of INI1 into a non-MRT cell line, however, had no demonstrable effect on any of these parameters. These results support the genetic evidence that INI1 is a tumor suppressor gene gone awry in MRT cells, and also suggest that delivery of the INI1 gene to MRT cells by adenoviruses may lead to a more effective treatment of this highly aggressive malignancy.     

SWI/SNF mediates polycomb eviction and epigenetic reprogramming of the INK4b-ARF-INK4a locus

Stable silencing of the INK4b-ARF-INK4a tumor suppressor locus occurs in a variety of human cancers, including malignant rhabdoid tumors (MRTs). MRTs are extremely aggressive cancers caused by the loss of the hSNF5 subunit of the SWI/SNF chromatin-remodeling complex. We found previously that, in MRT cells, hSNF5 is required for p16(INK4a) induction, mitotic checkpoint activation, and cellular senescence. Here, we investigated how the balance between Polycomb group (PcG) silencing and SWI/SNF activation affects epigenetic control of the INK4b-ARF-INK4a locus in MRT cells. hSNF5 reexpression in MRT cells caused SWI/SNF recruitment and activation of p15(INK4b) and p16(INK4a), but not of p14(ARF). Gene activation by hSNF5 is strictly dependent on the SWI/SNF motor subunit BRG1. SWI/SNF mediates eviction of the PRC1 and PRC2 PcG silencers and extensive chromatin reprogramming. Concomitant with PcG complex removal, the mixed lineage leukemia 1 (MLL1) protein is recruited and active histone marks supplant repressive ones. Strikingly, loss of PcG complexes is accompanied by DNA methyltransferase DNMT3B dissociation and reduced DNA methylation. Thus, various chromatin states can be modulated by SWI/SNF action. Collectively, these findings emphasize the close interconnectivity and dynamics of diverse chromatin modifications in cancer and gene control.     

A masked NES in INI1/hSNF5 mediates hCRM1-dependent nuclear export: implications for tumorigenesis.

INI1 (integrase interactor 1)/hSNF5 is a component of the mammalian SWI/SNF complex and a tumor suppressor mutated in malignant rhabdoid tumors (MRT). We have identified a nuclear export signal (NES) in the highly conserved repeat 2 domain of INI1 that is unmasked upon deletion of a downstream sequence. Mutation of conserved hydrophobic residues within the NES, as well as leptomycin B treatment abrogated the nuclear export. Full-length INI1 specifically associated with hCRM1/exportin1 in vivo and in vitro. A mutant INI1 [INI1(1-319) delG950] found in MRT lacking the 66 C-terminal amino acids mislocalized to the cytoplasm. Full-length INI1 but not the INI1(1-319 delG950) mutant caused flat cell formation and cell cycle arrest in cell lines derived from MRT. Disruption of the NES in the delG950 mutant caused nuclear localization of the protein and restored its ability to cause cell cycle arrest. These observations demonstrate that INI1 has a masked NES that mediates regulated hCRM1/exportin1-dependent nuclear export and we propose that mutations that cause deregulated nuclear export of the protein could lead to tumorigenesis.     

Involvement of the chromatin-remodeling factor BRG1/SMARCA4 in human cancer

The SWI/SNF complex is a chromatin-remodeling complex that uses the energy of ATP hydrolysis to modify chromatin structure in order to regulate gene expression. The SWI/SNF complex is evolutionarily conserved in all eukaryotes and is comprised of a catalytic subunit, either of BRG1 (also known as SMARCA4) or of BRM (also known as SMARCA2), and a variety of associated proteins that can modulate the recruitment of the complex and its activity. Key observations link the SWI/SNF complex with cancer. First, two of its subunits (SNF5 and BRG1) bear cancer-inactivating mutations and thus are bona fide tumor suppressors. The SNF5 gene is biallelically inactivated in malignant rhabdoid tumors (MRTs) whereas BRG1 is mutated in cancer cell lines of several types, such as those of the breast, prostate, lung, pancreas and colon. Second, mice heterozygous for mutations at Snf5 and Brg1 are cancer-prone, and, third, BRG1 binds or is related to important tumor-suppressor proteins. The present review focuses on the biological function and genetics of BRG1, particularly with respect to its role as a tumor suppressor.     

Constitutional Mutations of the hSNF5/INI1 Gene Predispose to a Variety of Cancers

Biallelic, truncating mutations of the hSNF5/INI1 gene have recently been documented in malignant rhabdoid tumor (MRT), one of the most aggressive human cancers. This finding suggests that hSNF5/INI1 is a new tumor-suppressor gene for which germline mutations might predispose to cancer. We now report the presence of loss-of-function mutations of this gene in the constitutional DNA from affected members but not from healthy relatives in cancer-prone families. Furthermore, a constitutional mutation is documented in a patient with two successive primary cancers. In agreement with the two-hit model, the wild-type hSNF5/INI1 allele is deleted in the tumor DNA from mutation carriers. In all tested cases, DNA from parents demonstrated normal hSNF5/INI1 sequences, therefore indicating the de novo occurrence of the mutation, which was shown to involve the maternal allele in one case and the paternal allele in two other cases. These data indicate that constitutional mutation of the hSNF5/INI1 gene defines a new hereditary syndrome predisposing to renal or extrarenal MRT and to a variety of tumors of the CNS, including choroid plexus carcinoma, medulloblastoma, and central primitive neuroectodermal tumor. This condition, which we propose to term "rhabdoid predisposition syndrome," may account for previous observations of familial and multifocal cases of the aforementioned tumor types. It could also provide the molecular basis for cases of Li-Fraumeni syndrome without p53 germline mutations.     

Dormancy signatures and metastasis in estrogen receptor positive and negative breast cancer

Breast cancers can recur after removal of the primary tumor and treatment to eliminate remaining tumor cells. Recurrence may occur after long periods of time during which there are no clinical symptoms. Tumor cell dormancy may explain these prolonged periods of asymptomatic residual disease and treatment resistance. We generated a dormancy gene signature from published experimental models and applied it to both breast cancer cell line expression data as well as four published clinical studies of primary breast cancers. We found that estrogen receptor (ER) positive breast cell lines and primary tumors have significantly higher dormancy signature scores (P<0.0000001) than ER- cell lines and tumors. In addition, a stratified analysis combining all ER+ tumors in four studies indicated 2.1 times higher hazard of recurrence among patients whose tumors had low dormancy scores (LDS) compared to those whose tumors had high dormancy scores (HDS) (p<0.000005). The trend was shown in all four individual studies. Suppression of two dormancy genes, BHLHE41 and NR2F1, resulted in increased in vivo growth of ER positive MCF7 cells. The patient data analysis suggests that disseminated ER positive tumor cells carrying a dormancy signature are more likely to undergo prolonged dormancy before resuming metastatic growth. Furthermore, genes identified with this approach might provide insight into the mechanisms of dormancy onset and maintenance as well as dormancy models using human breast cancer cell lines.     

Circulating muscle-specific microRNA, miR-206, as a potential diagnostic marker for rhabdomyosarcoma

Presently there is no serum biomarker of rhabdomyosarcoma (RMS). Several studies have shown that profiles of microRNA (miRNA) expression differ among tumor types. Here we evaluated the feasibility of using muscle-specific miRNAs (miR-1, -133a, -133b and -206) as biomarkers of RMS. Expression of muscle-specific miRNAs, especially miR-206, was significantly higher in RMS cell lines than in other tumor cell lines, as well as in RMS tumor specimens. Further, serum levels of muscle-specific miRNAs were significantly higher in patients with RMS tumors than in patients with non-RMS tumors. Normalized serum miR-206 expression level could be used to differentiate between RMS and non-RMS tumors, with sensitivity of 1.0 and specificity of 0.913. These results raise the possibility of using circulating muscle-specific miRNAs, especially miR-206, as landmark biomarkers for RMS.     

Malignant rhabdoid tumor in the renal allograft of an adult transplant recipient: a unique case of a rare tumor

Background

Renal transplant recipients have increased risk for developing malignant diseases because of immunosuppression or donor-to-recipient transmission. Malignant rhabdoid tumor (MRT) is a rare, highly aggressive and lethal tumor primarily affecting the kidney of infants and young children. MRT has not been reported in the renal allograft of an adult recipient after kidney transplantation.

Case presentation

In this report, a 47-year-old woman who received a kidney transplantation from an infant donor and developed a mass in the transplanted kidney is presented. Pathological examinations revealed a malignant tumor with rhabdoid cells morphologically and the loss of INI1 expression immunohistochemically. The diagnosis of malignant rhabdoid tumor in the transplanted kidney was made. We confirmed that donor-to-recipient malignancy transmission was the cause of MRT in the transplanted kidney by fluorescence in situ hybridization (FISH) and short tandem repeat (STR) analysis.

Conclusion

To our knowledge, this is the first case of MRT in an adult renal allograft recipient. This report highlights the importance of the criteria for selection of donors to screen possible malignant tumors transmission.