The anti-proliferative effect of heat shock protein 90 inhibitor, 17-DMAG, on non-small-cell lung cancers being resistant to EGFR tyrosine kinase inhibitor.

Acquired resistance to epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs), gefitinib and erlotinib, is frequently observed after initiation of TKIs therapy. Non-small-cell lung cancers (NSCLC) with activating EGFR mutations were reported to be sensitive to heat shock protein 90 (Hsp90) inhibitors regardless of the secondary TKI-resistant T790M mutation. We established EGFR-TKI resistant clones for PC-9 cell lines, harboring EGFR exon 19 deletions, with or without the secondary T790M mutation. We examined the anti-proliferative effect of 17-dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG), an orally active Hsp90 inhibitor, on the growth of NSCLC cell lines in vitro and in vivo. In MTS assay, the IC(50) values of 17-DMAG for 13 EGFR-mutant cell lines including eight EGFR-TKI resistant cell lines ranged from 0.04 to 0.16 µM while those for seven EGFR-wild type cell lines ranged from 1.6 to 27.4 µM. Western blot analysis revealed that phospho-EGFR, phospho-Akt, phospho-MAPK, cdk4, and cyclin D1 were more readily depleted by 17-DMAG treatment in EGFR-mutant cell lines than in EGFR-wild type cell lines. Cleaved PARP expression confirmed apoptosis in response to 17-DMAG treatment in EGFR-mutant cell lines but not in EGFR-wild type cell lines. In mice xenograft models, 17-DMAG significantly reduced the growth of EGFR-mutant lines irrespective of T790M mutation. These results suggested that 17-DMAG is a potential novel therapeutic agent for NSCLC patients with EGFR mutations with or without EGFR-TKI resistance.

Primary extranodal non-Hodgkin's lymphoma of the common bile duct manifesting as obstructive jaundice: report of a case.

Primary non-Hodgkin's lymphoma (NHL) of the common bile duct (CBD) manifesting as obstructive jaundice is extremely rare: to our knowledge, only 22 cases of primary NHL arising from the CBD have been reported. The patient in this case report was a 63-year-old man who presented with obstructive jaundice. Abdominal sonography, positron emission tomography, and computed tomography showed a mass with abnormal 18-fluorodeoxyglucose uptake in pancreatic head. Magnetic resonance cholangiopancreatography demonstrated a strictured segment of the CBD with proximal bile duct dilatation. We performed pancreaticoduodenectomy for a presumptive diagnosis of pancreatic head carcinoma or cholangiocarcinoma of the CBD. However, the histological diagnosis was a primary, diffuse, large B-cell lymphoma of the CBD. He received three courses of combination chemotherapy, including rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP). The patient remains well, without evidence of tumor recurrence, 8 months after surgery. In summary, primary NHL of the CBD, despite its rarity, should be considered in the differential diagnosis of obstructive jaundice. An accurate histopathologic diagnosis and complete surgical resection, followed by combination chemotherapy plus rituximab may be effective.

Aberrant promoter methylation of insulin-like growth factor binding protein-3 gene in human cancers.

Insulin-like growth factor binding protein-3 (IGFBP-3) is postulated to be a mediator of growth suppression signals. Here, we examined the methylation status of IGFBP-3 to correlate to clinicopathological factors in human cancers. The methylation status of IGFBP-3 was determined by bisulfite DNA sequencing and was correlated with expression semi-quantified by real-time RT-PCR to develop a methylation-specific PCR (MSP) assay for IGFBP-3. Using the MSP assay, we examined the methylation status of IGFBP-3 in gastric cancer (GC), colorectal cancer (CRC), breast cancer (BC) and malignant mesothelioma (MM). IGFBP-3 methylation was detected in 6 of 13 (46%) and 16 of 24 (67%) GC cell lines and tumors, respectively; 4 of 8 (50%) and 15 of 26 (58%) CRC cell lines and tumors, respectively; 3 of 11 (27%) and 7 of 39 (18%) BC cell lines and tumors, respectively and 1 of 5 (20%) and 18 of 56 (32%) MM cell lines and tumors, respectively. Interestingly, the methylation status of MM specimens from Japanese patients (75%, 12 out of 16 patients) was significantly higher than those from the USA (15%, 6 out of 40 patients) (p < 0.0001), suggesting the presence of ethnic differences in the IGFBP-3 methylation status. We also found that IGFBP-3 methylation was preferentially present in GCs arising in the lower-third of the stomach (p = 0.079). In summary, our results showed that IGFBP-3 methylation played an important role in the silencing of its expression, suggesting that IGFBP-3 may act as a tumor suppressor gene in several human cancers examined.

Inhibition of hsp90 compromises the DNA damage response to radiation.

Inhibitors of the molecular chaperone Hsp90 have been shown to enhance tumor cell radiosensitivity. To begin to address the mechanism responsible, we have determined the effect of the Hsp90 inhibitor 17-(dimethylaminoethylamino)-17-demethoxygeldanamycin (17DMAG) on the DNA damage response to radiation. Exposure of MiaPaCa tumor cells to 17DMAG, which results in radiosensitization, inhibited the repair of DNA double-strand breaks according to gammaH2AX foci dispersal and the neutral comet assay. This repair inhibition was associated with reduced DNA-PK catalytic subunit (DNA-PKcs) phosphorylation after irradiation and a disruption of DNA-PKcs/ErbB1 interaction. These data suggest that the previously established 17DMAG-mediated reduction in ErbB1 activity reduces its interaction with DNA-PKcs and thus accounts for the attenuation of radiation-induced DNA-PK activation. 17DMAG was also found to abrogate the activation of the G(2)- and S-phase cell cycle checkpoints. Associated with these events was a reduction in radiation-induced ataxia-telangiectasia mutated (ATM) activation and foci formation in 17DMAG-treated cells. Although no interaction between ATM and Hsp90 was detected, Hsp90 was found to interact with the MRE11/Rad50/NBS1 (MRN) complex. 17DMAG exposure reduced the ability of the MRN components to form nuclear foci after irradiation. Moreover, 17DMAG exposure reduced the interaction between NBS1 and ATM, although no degradation of the MRN complex was detected. These results suggest that the diminished radiation-induced activation of ATM in 17DMAG-treated cells was the result of a compromise in the function of the MRN complex. These data indicate that Hsp90 can contribute to the DNA damage response to radiation affecting both DNA repair and cell cycle checkpoint activation.

ErbB3 expression predicts tumor cell radiosensitization induced by Hsp90 inhibition.

The ability to identify tumors that are susceptible to a given molecularly targeted radiosensitizer would be of clinical benefit. Towards this end, we have investigated the effects of a representative Hsp90 inhibitor, 17-(dimethylaminoethylamino)-17-demethoxygeldanamycin (17DMAG), on the radiosensitivity of a panel of human tumor cell lines. 17DMAG was previously shown to enhance the radiosensitivity of a number of human cell lines, which correlated with the loss of ErbB2. We now report on cell lines in which 17DMAG induced the degradation of ErbB2, yet had no effect on radiosensitivity. In a comparison of ErbB family members, ErbB3 protein was only detectable in cells resistant to 17DMAG-induced radiosensitization. To determine whether ErbB3 plays a casual role in this resistance, short interfering RNA (siRNA) was used to knockdown ErbB3 in the resistant cell line AsPC1. Whereas individual treatments with siRNA to ErbB3 or 17DMAG had no effect on radiosensitivity, the combination, which reduced both ErbB2 and ErbB3, resulted in a significant enhancement in AsPC1 radiosensitivity. In contrast to siRNA to ErbB3 or 17DMAG treatments only, AsPC1 cell exposure to the combination also resulted in a decrease in ErbB1 kinase activity. These results indicate that ErbB3 expression predicts for tumor cell susceptibility to and suggests that the loss of ErbB1 signaling activity is necessary for 17DMAG-induced radiosensitization. However, for cell lines sensitized by 17DMAG, treatment with siRNA to ErbB2, which reduced ErbB1 activity, had no effect on radiosensitivity. These results suggest that, whereas the loss of ErbB1 signaling may be necessary for 17DMAG-induced radiosensitization, it is not sufficient.

Enhancement of in vitro and in vivo tumor cell radiosensitivity by the DNA methylation inhibitor zebularine.

Aberrant DNA hypermethylation is a frequent finding in tumor cells, which has suggested that inhibition of DNA methylation may be an effective cancer treatment strategy. Because DNA methylation affects gene expression and chromatin structure, parameters considered to influence radioresponse, we investigated the effects of the DNA methylation inhibitor zebularine on the radiosensitivity of human tumor cells. Three human tumor cell lines were used in this study (MiaPaCa, DU145, and U251) and the methylation status of three genes frequently hypermethylated in tumor cells (RASSF1A, HIC-1, and 14-3-3sigma) was determined as a function of zebularine exposure. Zebularine resulted in DNA demethylation in a time-dependent manner, with the maximum loss of methylation detected by 48 hours. Treatment of cells with zebularine for 48 hours also resulted in an increase in radiosensitivity with dose enhancement factors of >1.5. As a measure of radiation-induced DNA damage, gammaH2AX expression was determined. Whereas zebularine had no effect on radiation-induced gammaH2AX foci at 1 hour, the number of gammaH2AX foci per cell was significantly greater in the zebularine-treated cells at 24 hours after irradiation, suggesting the presence of unrepaired DNA damage. Zebularine administration to mice reactivated gene expression in U251 xenografts; irradiation of U251 tumors in mice treated with zebularine resulted in an increase in radiation-induced tumor growth delay. These results indicate that zebularine can enhance tumor cell radiosensitivity in vitro and in vivo and suggest that this effect may involve an inhibition of DNA repair.

Aberrant promoter methylation of human DAB2 interactive protein (hDAB2IP) gene in lung cancers.

The human DOC-2/DAB2 interactive protein gene (hDAB2IP) is a novel member of the Ras GTPase-activating gene family that is known to act as a tumor suppressor gene and is inactivated by methylation in prostate and breast cancers. We established previously a methylation-specific PCR (MSP) for the promoter region (m2a and m2b regions) of hDAB2IP and examined hDAB2IP methylation status in breast cancers. We analyzed the methylation and expression status of hDAB2IP in lung cancers. The methylation status of hDAB2IP was examined in lung cancer cell lines using bisulfite sequencing and MSP. Expression was examined using conventional and real-time RT-PCR, and methylation was found to be inversely correlated with expression, confirming that the MSP can also be used to examine hDAB2IP methylation status in lung cancers. Aberrant methylation was detected at the m2a region in 19 of 47 lung cancer cell lines (40%) and 26 of 70 primary tumors (37%) and at the m2b in 16 lines (34%) and 25 of 70 tumors (36%). Gene expression was restored in methylated cell lines supplemented with 5-aza-2'-deoxycytidine, confirming that methylation was responsible for downregulation. We also examined the relationship between hDAB2IP methylation and clinico-pathological features of the lung cancers and found that hDAB2IP methylation was associated with advanced disease stage. Our results demonstrate that hDAB2IP methylation is frequently present in lung cancers and plays a key role in hDAB2IP silencing. hDAB2IP methylation could be used as a biomarker for disease stage, reflecting the degree of clinico-pathological malignancy of lung cancer.

Enhanced tumor cell radiosensitivity and abrogation of G2 and S phase arrest by the Hsp90 inhibitor 17-(dimethylaminoethylamino)-17-demethoxygeldanamycin.

PURPOSE: Because of the potential for affecting multiple signaling pathways, inhibition of Hsp90 may provide a strategy for enhancing tumor cell radiosensitivity. Therefore, we have investigated the effects of the orally bioavailable Hsp90 inhibitor 17-(dimethylaminoethylamino)-17-demethoxygeldanamycin (17-DMAG) on the radiosensitivity of human tumor cells in vitro and grown as tumor xenografts. EXPERIMENTAL DESIGN: The effect of 17-DMAG on the levels of three proteins (Raf-1, ErbB2, and Akt) previously implicated in the regulation of radiosensitivity was determined in three human solid tumor cell lines. A clonogenic assay was then used to evaluate cell survival after exposure to 17-DMAG followed by irradiation. For mechanistic insight, the G(2)- and S-phase checkpoints were evaluated in 17-DMAG-treated cells. Finally, the effect of in vivo administration of 17-DMAG in combination with radiation on the growth rate of xenograft tumors was determined. RESULTS: 17-DMAG exposure reduced the levels of the three radiosensitivity-associated proteins in a cell line-specific manner with ErbB2 being the most susceptible. Corresponding concentrations of 17-DMAG enhanced the radiosensitivity of each of the tumor cell lines. This sensitization seemed to be the result of a 17-DMAG-mediated abrogation of the G(2)- and S-phase cell cycle checkpoints. The oral administration of 17-DMAG to mice bearing tumor xenografts followed by irradiation resulted in a greater than additive increase in tumor growth delay. CONCLUSIONS: These data indicate that 17-DMAG enhances the in vitro and in vivo radiosensitivity of human tumor cells. The mechanism responsible seems to involve the abrogation of radiation-induced G(2)- and S-phase arrest.

Aberrant methylation of p57KIP2 gene in lung and breast cancers and malignant mesotheliomas.

The p57KIP2 gene belongs to the Cip/Kip family of CDK inhibitors and has been demonstrated to be a tumor suppressor gene, being inactivated in various types of human cancers. We analyzed the methylation and expression status of p57KIP2 in lung and breast cancers, and in malignant mesotheliomas (MMs). The promoter region of p57KIP2 was determined by methylation-specific PCR (MSP) in samples of lung and breast cancer, and of MM. The expression of the gene in the cell lines was determined by RT-PCR and correlated with the methylation status. Aberrant methylation was detected by MSP in 9 of 27 (33%) and 25 of 78 (32%) lung cancer cell lines and tumors, respectively, 11 of 18 (61%) and 17 of 38 (45%) breast cancer cell lines and tumors, respectively, and 1 of 25 (4%) MM tumors. DNA methylation was detected but rarely in the corresponding non-malignant tissues. In addition, the gene expression was restored in the methylated cell lines following 5-aza-2'-deoxycytidine treatment, confirming that the methylation was indeed responsible for the gene down-regulation. We also examined the relationship between the p57KIP2 methylation status and the clinicopathological features of the primary tumors, and found that there was no relationship between the p57KIP2 methylation status and any of the examined clinicopathological features. In summary, our results demonstrate that p57KIP2 methylation associated with the gene down-regulation is frequently present in lung and breast cancers and plays an important role at the molecular level in the pathogenesis of these cancers.

Less frequent promoter hypermethylation of DLC-1 gene in primary breast cancers.

Absence or low expression of DLC-1, a tumor suppressor gene, in breast cancers has been shown recently. LOH of 8p12-p22, on which DLC-1 is located, is frequent in breast cancers, but the correlation between low expression of DLC-1 and LOH has not been confirmed. To determine the implication of aberrant methylation, one of the most frequent mechanisms of silencing the tumor suppressor or cancer-related genes, we examined the methylation status of DLC-1 promoter region in breast cancer cell lines and primary breast tumors. The hypermethylation status was examined by MSP and 25% of cell lines harbored a methylated allele. The gene silencing by methylation was also confirmed by the re-expression of DLC-1 by the 5-aza-2'-deoxycytidine treatment in DLC-1 hypermethylated cell line. But the methylation of DLC-1 gene was less frequently shown in primary breast cancers (10%). These data suggest that hypermethylation is responsible for silencing of DLC-1 gene in a limited portion of breast cancers.

SYT-SSX fusion genes in synovial sarcoma of the thorax.

Synovial sarcoma (SS) is characterized by a chromosomal translocation resulting in the expression of an SYT-SSX chimeric transcript, usually SYT-SSX1 or SYT-SSX2. Synovial sarcoma typically originates in the limbs, and its location in the thorax is rare. Synovial sarcomas are usually classified into three histologic subtypes: biphasic, monophasic and poorly differentiated tumors. The detection of the characteristic chimeric transcript often contributes to a histopathological diagnosis, especially when the tumor arises in an unusual location. Previous studies have shown that SYT-SSX1 is the most common SYT-SSX fusion transcript in biphasic synovial sarcomas of the limbs. Here, we report two cases of synovial sarcoma originating in the thorax. The presence of SYT-SSX2 chimeric transcripts was confirmed by reverse transcript polymerase chain reaction (RT-PCR) and a direct sequencing analysis in both cases. The tumor in case 1 originated from the pericardium, which is an exceedingly rare site for primary synovial sarcoma; only three other cases of synovial sarcoma originating in the pericardium have been previously reported. Case 2 exhibited a biphasic synovial sarcoma of the mediastinum containing an SYT-SSX2 fusion transcript, which is a rare fusion type in biphasic synovial sarcomas of the limbs. We reviewed previous reports of thoracic synovial sarcomas containing an analysis of the SYT-SSX fusion transcript and found that case 2 in the present study was the first description of a biphasic synovial sarcoma of the thorax with an SYT-SSX2 fusion transcript. However, the number of reported cases was not sufficient to conclude that SYT-SSX2 fusion in biphasic synovial sarcoma of the thorax is, indeed, rare. Further genetic analysis is needed to fully understand the biological and clinical features of synovial sarcoma originating in the thorax.

Aberrant promoter methylation in human DAB2 interactive protein (hDAB2IP) gene in breast cancer.

PURPOSE: Human DOC-2/DAB2 interactive protein (hDAB2IP) gene is a novel member of the Ras GTPase-activating family and has been demonstrated to be a tumor suppressor gene inactivated by methylation in prostate cancer. We analyzed methylation and expression status of hDAB2IP in breast cancer. EXPERIMENTAL DESIGN: The promoter region of hDAB2IP was divided into two regions (m2a and m2b) following our previous report on prostate cancer, and methylation status was determined in breast cancer cell lines with bisulfited DNA sequencing. Expression was semiquantified with real-time reverse transcription-PCR to find that aberrant methylation showed the inverse relationship with expression. On the basis of sequence data, we developed methylation-specific PCR for m2a and m2b regions and applied to samples. RESULTS: Aberrant methylation was detected in 11 of 25 breast cancer cell lines (44%) and 15 of 39 primary tumors (38%) at the m2a region and in 12 of 25 cell lines (48%) and 13 of 39 tumors (33%) at the m2b region. In addition, gene expression was restored in methylated cell lines with 5-aza-2'-deoxycytidine, confirming that methylation caused gene down-regulation. We also examined the relationship between hDAB2IP methylation and clinicopathologic features in primary tumors and found that methylation in the m2b region was associated with progressive nodal status of tumors. CONCLUSIONS: We developed methylation-specific PCR for hDAB2IP and examined its methylation status in breast cancer. Our results demonstrate that hDAB2IP methylation frequently is present in breast cancer and plays a key role in hDAB2IP inactivation, suggesting the relationship between hDAB2IP methylation and lymph node metastasis of breast cancer.

Mutation analysis of the BRAF codon 599 in malignant pleural mesothelioma by enriched PCR-RFLP.

BRAF encodes a RAS-regulated serine/threonine kinase that mediates the pathway for cell growth and malignant transformation. Point mutations of BRAF were reported recently in 66% of melanomas, over 30% of thyroid papillary and low-grade ovarian cancers, and a smaller percentage of other human cancers. Mutations in malignant cells were reported to occur only in exons 11 and 15. Among these mutations, BRAF V599E is most frequent and proved to invert its transcript to the dominant active form. To exclude the interference of co-existing normal cells in clinical samples, we developed a new enriched PCR-RFLP assay for detecting mutations of BRAF codon 599 mutation. The sensitivity of this assay was examined to find that one mutant allele among 10(2) wild-type alleles could be detected. We applied this method for 53 cases of primary malignant pleural mesotheliomas (MPMs) and 6 cell lines and found no mutations in these samples. Our results demonstrate that the developed enriched PCR-RFLP is a sensitive assay to detect BRAF codon 599 mutation. However, it may be a rare type of mutation in MPMs. Our new assay is useful and can be applied for screening of BRAF codon 599 mutation in various kinds of clinical samples.