Laurence Seabra

Sensitivity to cis-diamminedichloroplatinum in human cancer cells is related to expression of cyclin D1 but not c-raf-1 protein.

Although several oncogenes, including c‐myc,ras and c‐raf‐1, have been implicated in cellular resistance to ionising radiation, there is less information relating oncogene expression to cis‐diamminedochloroplatinum (CDDP) resistance. However, transfection of c‐myc or v‐H‐ras and activation of protein kinase C (PKC), which contributes to the RAF‐I, MAP kinase signal transduction pathway, can influence therapeutic response to CDDP. Activation of PKC increases CDDP sensitivity, whilst transfected c‐myc or v‐H‐ras induce CDDP resistance. We have previously reported that human in vitro cell lines show different patterns of sensitivity to CDDP and 4 MeV X‐irradiation. In these cells radiation sensitivity is related to high levels of expression of the c‐raf‐1 proto‐oncogene. We thus predicted that cells sensitive to CDDP might show a different relationship to c‐raf‐1 expression. In addition, because cyclin D1 expression can be upregulated by the myc or ras oncogenes, we also chose to study putative relationships between cyclin D1 protein levels and intrinsic cellular sensitivity to CDDP and γ‐irradiation. We report that in the 16 human cell lines which we have studied, high cyclin D1 expression is related to CDDP resistance but has no relationship with radiation responsiveness, whereas high c‐raf‐1 expression, although related to radiosensitivity has no relationship with CDDP responsiveness.

Folliculin interacts with p0071 (plakophilin-4) and deficiency is associated with disordered RhoA signalling, epithelial polarization and cytokinesis

Inherited mutations in the folliculin (FLCN) gene cause the Birt-Hogg-Dubé syndrome of familial hair follicle tumours (fibrofolliculomas), lung cysts and kidney tumours. Though folliculin has features of a tumour suppressor, the precise function of the FLCN gene product is not well characterized. We identified plakophilin-4 (p0071) as a potential novel folliculin interacting protein by yeast two-hybrid analysis. We confirmed the interaction of folliculin with p0071 by co-immunoprecipitation studies and, in view of previous studies linking p0071 to the regulation of rho-signalling, cytokinesis and intercellular junction formation, we investigated the effect of cell folliculin status on p0071-related functions. Folliculin and p0071 partially co-localized at cell junctions and in mitotic cells, at the midbody during cytokinesis. Previously, p0071 has been reported to regulate RhoA signalling during cytokinesis and we found that folliculin deficiency was associated with increased expression and activity of RhoA and evidence of disordered cytokinesis. Treatment of folliculin-deficient cells with a downstream inhibitor of RhoA signalling (the ROCK inhibitor Y-27632) reversed the increased cell migration phenotype observed in folliculin-deficient cells. Deficiency of folliculin and of p0071 resulted in tight junction defects and mislocalization of E-cadherin in mouse inner medullary collecting duct-3 renal tubular cells. These findings suggest that aspects of folliculin tumour suppressor function are linked to interaction with p0071 and the regulation of RhoA signalling.

Birt Hogg-Dubé syndrome-associated FLCN mutations disrupt protein stability.

Germline mutations in the FLCN gene cause Birt‐Hogg‐Dubé syndrome, familial spontaneous pneumothorax, or apparently nonsyndromic inherited RCC. The vast majority of reported FLCN mutations are predicted to result in a truncated/absent gene product and so infrequent missense and inframe‐deletion (IFD) FLCN mutations might indicate critical functional domains. To investigate this hypothesis we (1) undertook an in silico evolutionary analysis of the FLCN sequence and (2) investigated in vitro the functional effects of naturally occurring FLCN missense/IFD mutations. The folliculin protein sequence evolved more slowly and was under stronger purifying selection than the average gene, most notably at a region between codons 100 and 230. Pathogenic missense and IFD FLCN mutations that impaired folliculin tumor suppressor function significantly disrupted the stability of the FLCN gene product but two missense substitutions initially considered to be putative mutations did not impair protein stability, growth suppression activity, or intracellular localization of folliculin. These findings are consistent with the distribution of FLCN mutations throughout the coding sequence, and suggest that multiple protein domains contribute to folliculin stability and tumor suppressor activity. In vitro assessment of protein stability and tumor suppressor activity provides a practical strategy for assessing the pathogenicity of potential FLCN mutations. Hum Mutat 32:1–9, 2011.

Combined RAF1 protein expression and p53 mutational status provides a strong predictor of cellular radiosensitivity

The tumour suppressor gene, p53, and genes coding for positive signal transduction factors can influence transit through cell-cycle checkpoints and modulate radiosensitivity. Here we examine the effects of RAF1 protein on the rate of exit from a G2/M block induced by γ-irradiation in relation to intrinsic cellular radiosensitivity in human cell lines expressing wild-type p53 (wtp53) protein as compared to mutant p53 (mutp53) protein. Cell lines which expressed mutp53 protein were all relatively radioresistant and exhibited no relationship between RAF1 protein and cellular radiosensitivity. Cell lines expressing wtp53 protein, however, showed a strong relationship between RAF1 protein levels and the radiosensitivity parameter SF2. In addition, when post-irradiation perturbation of G2/M transit was compared using the parameter T50 (time after the peak of G2/M delay at which 50% of the cells had exited from a block induced by 2 Gy of irradiation), RAF1 was related to T50 in wtp53, but not mutp53, cell lines. Cell lines which expressed wtp53 protein and high levels of RAF1 had shorter T50s and were also more radiosensitive. These results suggest a cooperative role for wtp53 and RAF1 protein in determining cellular radiosensitivity in human cells, which involves control of the G2/M checkpoint.

Therapeutic Targeting the Loss of the Birt-Hogg-Dubé Suppressor Gene

Brit-Hogg-Dubé (BHD) syndrome, an autosomal dominant familial cancer, is associated with increased risk of kidney cancer. BHD syndrome is caused by loss-of-function mutations in the folliculin (FLCN) protein. To develop therapeutic approaches for renal cell carcinoma (RCC) in BHD syndrome, we adopted a strategy to identify tumor-selective growth inhibition in a RCC cell line with FLCN inactivation. The COMPARE algorithm was used to identify candidate anticancer drugs tested against the NCI-60 cell lines that showed preferential toxicity to low FLCN expressing cell lines. Fifteen compounds were selected and detailed growth inhibition (SRB) assays were done in paired BHD RCC cell lines (UOK257 derived from a patient with BHD). Selective sensitivity of FLCN-null over FLCN-wt UOK257 cells was observed in seven compounds. The most selective growth-inhibitory sensitivity was induced by mithramycin, which showed an approximately 10-fold difference in GI50 values between FLCN-null (64.2 ± 7.9 nmol/L, n = 3) and FLCN-wt UOK257 cells (634.3 ± 147.9 nmol/L, n = 4). Differential ability to induce caspase 3/7 activity by mithramycin was also detected in a dose-dependent manner. Clonogenic survival studies showed mithramycin to be approximately 10-fold more cytotoxic to FLCN-null than FLCN-wt UOK257 cells (200 nmol/L). Following mithramycin exposure, UOK257-FLCN-null cells were mainly arrested and blocked in S and G2-M phases of the cell cycle and low dose of rapamycin (1 nmol/L) potentiated mithramycin sensitivity (1.5-fold in G2-M population and 2-fold in G2-M period time, 2xGI50, 48 hours). These results provide a basis for further evaluation of mithramycin as a potential therapeutic drug for RCC associated with BHD. Mol Cancer Ther; 10(1); 80–9. ©2011 AACR.

Knockdown of Slingshot 2 (SSH2) serine phosphatase induces Caspase3 activation in human carcinoma cell lines with the loss of the Birt–Hogg–Dubé tumour suppressor gene ( FLCN )

Birt–Hogg–Dubé (BHD) syndrome, is a dominantly inherited familial cancer syndrome associated with susceptibility to renal cell carcinoma (RCC) caused by inactivating mutations in the folliculin (FLCN) gene. The precise functions of the FLCN gene product are still under investigation but RCC from BHD patients show loss of the wild-type allele consistent with a tumor suppressor gene function. In a search for potential synthetic-lethal targets for FLCN using a phosphatase siRNA library screening approach, we found that knockdown of SSH2 serine phosphatase (one of the three members of Slingshot family and previously implicated in actin reorganization) specifically induced Caspase3/7 activity in a dose-dependent manner (up to six-fold increase, 10 nM, 72 h) in two human FLCN-deficient cell lines (BHD-origin renal cell carcinoma UOK257 and thyroid carcinoma FTC133) but not in their folliculin expressing isogenic cell lines. SSH2 siRNA-induced knockdown was accompanied by increased expression of SSH1 and SSH3 (suggesting a compensatory regulatory mechanism among members of SSH family). FLCN-null cells exhibited evidence of dysregulated cofilin de/phosphorylation pathways. Knockdown of SSH2 in FLCN-null cells was associated with an alteration in cell cycle kinetics (20% increase in G1, 30% and 40% decrease in S and G2M, respectively). Combination treatment of multiple SSH family (SSH2 plus SSH1 and/or SSH3) siRNAs potentiated induction of Caspase3/7 activity and changes in the cell cycle kinetics. These data indicate that: (a) apoptotic cell death in FLCN-null cells can be triggered by SSH2 knockdown through cell cycle arrest; (b) SSH2 represents a potential therapeutic target for the development of agents for the treatment of BHD syndrome and, possibly, related tumors.

Late G1 accumulation after 2 Gy of gamma-irradiation is related to endogenous Raf-1 protein expression and intrinsic radiosensitivity in human cells.

We have previously reported a correlation between high endogenous expression of the protein product of the RAF-1 proto-oncogene, intrinsic cellular radiosensitivity and rapid exit from a G2/M delay induced by 2 Gy of gamma-irradiation. Raf1 is a positive serine/threonine kinase signal transduction factor that relays signals from the cell membrane to the MAP kinase system further downstream and is believed to be involved in an ionizing radiation signal transduction pathway modulating the G1/S checkpoint. We therefore extended our flow cytometric studies to investigate relationships between radiosensitivity, endogenous expression of the Raf1 protein and perturbation of cell cycle checkpoints, leading to alterations in the G1, S and G2/M populations after 2 Gy of gamma-irradiation. Differences in intrinsic radiosensitivity after modulation of the G1/S checkpoint have generally been understood to involve p53 function up to the present time. A role for dominant oncogenes in control of G1/S transit in radiation-treated cells has not been identified previously. Here, we show in 12 human in vitro cancer cell lines that late G1 accumulation after 2 Gy of radiation is related to both Raf1 expression (r = 0.91, P = 0.0001) and the radiosensitivity parameter SF2 (r = -0.71, P = 0.009).

Abstract LB-145: Folliculin interacts with p0071 (Plakophilin-4) and deficiency is associated with disordered RhoA signalling, epithelial polarization and cytokinesis

Inherited mutations in the folliculin gene (FLCN) cause the Birt-Hogg-Dube syndrome of familial hair follicle tumours (fibrofolliculomas), lung cysts and kidney tumours. Though folliculin has features of a tumour suppressor, the precise function of the FLCN gene product is not well characterised. We identified plakophilin-4 (PKP4, p0071) as a potential novel folliculin interacting protein by yeast-2-hybrid analysis. We confirmed the interaction of folliculin with p0071 by co-immunoprecipitation studies and, in view of previous studies linking p0071 to regulation of rho-signalling, cytokinesis and intercellular junction formation, we investigated the effect of cell folliculin status on p0071-related functions. Folliculin and p0071 partially colocalised at cell junctions and in mitotic cells, at the midbody during cytokinesis. Previously, p0071 has been reported to regulate RhoA signalling during cytokinesis and we found that folliculin deficiency was associated with increased expression and activity of RhoA and evidence of disordered cytokinesis. Treatment of folliculin deficient cells with a downstream inhibitor of RhoA signalling (the ROCK inhibitor Y-27632) reversed the increased cell migration phenotype observed in folliculin deficient cells. Deficiency of folliculin and of p0071 resulted in tight junction defects and mislocalisation of E-cadherin in IMCD-3 renal tubular cells. These findings suggest that aspects of folliculin tumour suppressor function are linked to interaction with p0071 and regulation of RhoA signalling. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-145. doi:1538-7445.AM2012-LB-145

Abstract 1129: FLCN-putative tumor suppressor

Birt-Hogg-Dube (BHD) is an autosomal dominantly inherited genodermatosis associated with the development of renal neoplasia, spontaneous pneumothorax and benign fibrofolliculomas. The BHD gene has been maps to chromosome 17p11.2 and encodes a protein called folliculin (FLCN). Although familial forms of renal cell carcinoma account for only about 3% of all cases, the elucidation of the molecular mechanisms of familial RCC syndromes has provided seminal insights into the pathogenesis of sporadic RCC. This is exemplified by the role of VHL tumor suppressor gene (TSG) inactivation in sporadic clear cell RCC. The FLCN TSG product has been linked to the mTOR/AMPK signaling pathways but the function of folliculin is much less well characterised than for other renal TSGs such as VHL. Among the multiple functions that have been associated with the VHL TSG product is a role in the regulation of apoptotic cell death. We have investigated whether folliculin also has a role in apoptosis. Comparison of isogenic FLCN null and replete cell lines demonstrated increased cell death in the latter. Flow cytometry revealed a 30 % shift in apoptotic index as determined by Annexin V vs PI staining which was demonstrated by a shift in the cell population towards an early apoptotic state. Expression of apoptosis-related proteins was investigated in FLCN null and replete cell lines using proteome array profiler. This demonstrated increased expression of SMAC, claspin HTRA2 and XIAP in FLCN replete cells. Further investigations to determine the precise role of folliculin in the regulation of apoptosis and the relationship between FLCN-induced apoptosis and p53 status are in progress. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1129.