Tumor resistance to anti-mesothelin CAR-T cells caused by binding to shed mesothelin is overcome by targeting a juxtamembrane epitope.

Despite many clinical trials, CAR-T cells are not yet approved for human solid tumor therapy. One popular target is mesothelin (MSLN) which is highly expressed on the surface of about 30% of cancers including mesothelioma and cancers of the ovary, pancreas, and lung. MSLN is shed by proteases that cleave near the C terminus, leaving a short peptide attached to the cell. Most anti-MSLN antibodies bind to shed MSLN, which can prevent their binding to target cells. To overcome this limitation, we developed an antibody (15B6) that binds next to the membrane at the protease-sensitive region, does not bind to shed MSLN, and makes CAR-T cells that have much higher anti-tumor activity than a CAR-T that binds to shed MSLN. We have now humanized the Fv (h15B6), so the CAR-T can be used to treat patients and show that h15B6 CAR-T produces complete regressions in a hard-to-treat pancreatic cancer patient derived xenograft model, whereas CAR-T targeting a shed epitope (SS1) have no anti-tumor activity. In these pancreatic cancers, the h15B6 CAR-T replicates and replaces the cancer cells, whereas there are no CAR-T cells in the tumors receiving SS1 CAR-T. To determine the mechanism accounting for high activity, we used an OVCAR-8 intraperitoneal model to show that poorly active SS1-CAR-T cells are bound to shed MSLN, whereas highly active h15B6 CAR-T do not contain bound MSLN enabling them to bind to and kill cancer cells.

A novel amplification target, DUSP26, promotes anaplastic thyroid cancer cell growth by inhibiting p38 MAPK activity.

Anaplastic thyroid cancer (ATC) is one of the most lethal of all human tumors, but cytogenetic information concerning ATC is extremely limited. Using our in-house array-based comparative genomic hybridization and 14 ATC cell lines with further fluorescence in situ hybridization analysis, we demonstrated amplification of the DUSP26 gene, known by another report as MAP kinase phosphatase-8. DUSP26 was overexpressed in ATC cell lines and primary ATC tumor samples. When overexpressed, either exogenously or endogenously, DUSP26 promoted growth of the ATC cells. DUSP26 encodes a protein containing a dual-specificity phosphatase domain that can dephosphorylate itself. DUSP26 effectively dephosphorylates p38 and has a little effect on extracellular signal-regulated kinase in ATC cells. DUSP26 protein formed a physical complex with p38, and promoted survival of ATC cells by inhibiting p38-mediated apoptosis. Our findings suggest that DUSP26 may act as an oncogene in ATC, and might be a useful diagnostic marker and therapeutic target of this disease.

Alpha-1-antitrypsin deficiency (Siiyama) as indication for lung transplantation: proper timing for surgical intervention.

The authors report a new familial case of alpha-1- antitrypsin (AAT) deficiency with severe pulmonary emphysema and hemoptysis. A severely reduced serum AAT level of the proband, a 56-year-old farmer's wife and her sister were observed. Mutation analysis of the AAT gene was performed using allele-specific polymerase chain reaction (PCR) analysis followed by direct sequencing. The proband and her younger sister proved to be homozygous for PISiiyama. Although home oxygen therapy was induced in addition to previous medications including bronchodilators and cardiovascular conditioning, the proband's rate of decline of forced expiratory volume at one second (FEV1) was progressing. Lung transplantation was therefore advisable for the patient. Clinical analysis on Japanese cases reported in the literature shows that the rate of decline of FEV1 is one of the most convenient prognostic factors to find proper timing for surgical intervention. Lung transplantation is one of the best reliable current therapies to improve quality of life of severely impaired patients.

Down-regulation of an inhibitor of cell growth, transmembrane protein 34 (TMEM34), in anaplastic thyroid cancer.

PURPOSE: Ansaplastic thyroid cancer (ATC) is one of the most lethal malignancies, but the carcinogenic mechanism of ATC has not been clarified. Recently, we performed a cDNA microarray analysis and identified transmembrane protein 34 (TMEM34) that down-regulated in anaplastic thyroid cancer cell lines (ACL)s as compared to normal thyroid tissues. METHODS: To investigate the role of TMEM34 in ATC carcinogenesis, we examined expression levels of TMEM34 in ACLs as well as differentiated thyroid cancers (DTC)s and normal human tissues. To explore the effect of TMEM34 in ATC development, cell-growth assays with KTA2 cells were performed. RESULTS: Expression of TMEM34 was down-regulated in all 11 ACLs, as compared to either normal thyroid tissues or cell lines derived from papillary or follicular thyroid cancers. TMEM34 was expressed ubiquitously in normal human tissues tested. Transfection of TMEM34 into KTA2 cells led to inhibition of cell growth. CONCLUSIONS: Our findings suggest that TMEM34 might be a tumor suppressor gene, associated with the development of ATC from DTC.

Instability of X chromosome methylation in aberrant crypt foci of the human colon.

Aberrant crypt foci (ACF) in the colon have long been thought to be precancerous lesions and therefore monoclonal, but this is unresolved. Eleven ACF were isolated from five female patients. From these ACF, 178 individual aberrant crypts (ACs) were obtained and assessed for clonality using a method based on X chromosome inactivation of the polymorphic X-linked human androgen receptor (HUMARA) gene. Ten ACF were found to be mixtures of monoclonal and polyclonal types. The HUMARA analysis indicated that almost all ACF were polyclonal lesions. Simultaneously, we investigated K-ras mutations in each AC. We found that seven of the ACF harbored the K-ras mutation; strikingly, this was concordant for all of the ACs from a single ACF. These results, by contrast to the results of HUMARA analysis indicate that ACF lesions are monoclonal. This discrepancy suggests that ACF are apparently polyclonal because of de novo methylation on the active X chromosome. To confirm this possibility, we investigated the methylation status of the X chromosome in male ACF using a competitive PCR assay. One hundred nineteen individual ACs were isolated from eight ACF derived from four male patients. A total of 47 of 119 (39%) of male ACs showed de novo methylation of the HUMARA gene. We found that six of the eight male ACF harbored the K-ras mutation, and this was concordant for all of the ACs from a single ACF. We conclude that X chromosome methylation is unstable in ACF and that this might be an early event in colon carcinogenesis.

Overexpression of acidic and basic fibroblast growth factors in human pancreatic cancer correlates with advanced tumor stage.

Acidic fibroblast growth factor (aFGF) and basic FGF (bFGF) are mitogenic polypeptides that may contribute to cancer cell proliferation. In the present study we examined aFGF and bFGF expression in human pancreatic cancer. Northern blot analysis of total RNA isolated from 12 pancreatic cancers revealed elevated aFGF and bFGF mRNA levels in 12 and 10 samples, respectively, by comparison with the normal human pancreas. Immunostaining demonstrated the presence of aFGF and bFGF in many cancer cells and in the atrophic acini and ducts adjacent to the cancer cells, but to a much lesser extent in the surrounding fibroblasts. By in situ hybridization, both mRNA moieties colocalized with their respective proteins and were abundant in many cancer cells. Immunoblotting confirmed that cancer tissues with increased aFGF and bFGF immunoreactivity contained elevated levels of both proteins. To determine the significance of aFGF and bFGF expression in the pancreatic cancer cells, immunohistochemical analysis of 78 human pancreatic carcinomas was performed. aFGF and bFGF immunoreactivity was present in the cancer cells in 47 (60%) and 44 (56%) of the tumors, respectively. There was a significant correlation between the presence of either aFGF or bFGF in the cancer cells and advanced tumor stage, and the presence of bFGF and shorter patient survival. These data suggest that aFGF and bFGF are overexpressed in a significant proportion of human pancreatic carcinoma cells and that this overexpression may contribute to disease progression.

Lowering the isoelectric point of the Fv portion of recombinant immunotoxins leads to decreased nonspecific animal toxicity without affecting antitumor activity.

Recombinant immunotoxins are genetically engineered proteins in which the Fv portion of an antibody is fused to a toxin. Our laboratory uses a 38-kDa form of Pseudomonas exotoxin A termed PE38 for this purpose. Clinical studies with immunotoxins targeting CD25 and CD22 have shown that dose-limiting side effects are attributable to liver damage and other inflammatory toxicities. We recently showed that mutating exposed surface neutral residues to acidic residues in the framework region of the Fv portion of an immunotoxin targeting CD25 [anti-Tac(scFv)-PE38] lowered its isoelectric point (pI) and decreased its toxicity in mice without impairing its cytotoxic or antitumor activities. We have now extended these studies and made mutations that change basic residues to neutral or acidic residues. Initially the pI of the mutant Fv (M1) of anti-Tac(scFv)-PE38 was decreased further. Subsequently, mutations were made in two other immunotoxins, SS1(dsFv)-PE38 targeting ovarian cancer and B3(dsFv)-PE38 targeting colon and breast cancers. We have found that all these mutant molecules fully retained specific target cell cytotoxicity and antitumor activity but were considerably less toxic to mice. Therefore, lowering the pI of the Fv may be a general approach to diminish the nonspecific toxicity of recombinant immunotoxins and other Fv fusion proteins without losing antitumor activity.

Isolation of a candidate gene, CAB1, for cholesterol transport to mitochondria from the c-ERBB-2 amplicon by a modified cDNA selection method.

An improved cDNA selection method was established to isolate expressed genes efficiently from an amplified chromosome region in human cancer. Biotinylated yeast artificial chromosome DNA containing c-ERBB-2 was hybridized in solution with PCR-amplifiable cDNAs of an esophageal cancer cell line bearing the c-ERBB-2 amplification. After capturing the hybrids on avidin-coated magnetic beads, the cDNAs were amplified by PCR. Four new genes (A39, C51, CAB1, and GRB-7) coamplified with c-ERBB-2 were isolated from the enriched cDNA library. CAB1, GRB-7, and c-ERBB-2 were overexpressed in gastric and esophageal cancer cells in correspondence with the amplification. The deduced amino acid sequence of the CAB1 gene had significant homology to the recently discovered steroidogenic acute regulatory protein, StAR, which plays an essential role in cholesterol transport to mitochondria. It was established that multiple overexpressed genes are frequently present in a single amplicon.

Specific expression of the ret proto-oncogene in human neuroblastoma cell lines.

The expression of the ret proto-oncogene (proto-ret), which possibly encodes two isoforms of a receptor-type tyrosine kinase, was examined in human tumor cell lines. Expression of the proto-ret mRNA was detected in all 11 neuroblastoma cell lines examined. The level of mRNA varied more than 100-fold in these neuroblastoma cell lines and was particularly high in three of them. On the other hand, 19 non-neuroblastoma tumor cell lines derived from solid tumors and a human diploid fibroblast cell line did not express any detectable levels of proto-ret mRNA. No remarkable amplification of the proto-ret or gross structural changes in the coding region were found in these neuroblastoma cell lines. The specific expression of the proto-ret in neuroblastomas suggests that the proto-ret product may have a role in cellular functions specific to neuroblastoma cells.

Tob2, a novel anti-proliferative Tob/BTG1 family member, associates with a component of the CCR4 transcriptional regulatory complex capable of binding cyclin-dependent kinases.

Human cDNAs encoding a novel member of Tob/BTG1 anti-proliferative family proteins were cloned. The putative protein product termed Tob2 consisted of 344 amino acids with high similarity to the Tob protein. The tob2 mRNA was 4.1 kb long and was ubiquitously expressed in human adult tissues, as was revealed by Northern blot hybridization. However, further in situ hybridization analysis showed a characteristic expression of the tob2 mRNA in oocytes, suggesting a unique role of Tob2 in oogenesis. Like the Tob protein, Tob2 inhibited cell cycle progression from the G0/G1 to S phases. Intriguingly, the amino-terminal half of Tob2 as well as that of Tob was associated with a human homologue of yeast Caf1, a component of the CCR4 transcription factor complex. Moreover, Caf1 was associated with cyclin dependent kinases. These data suggested that both Tob and Tob2 were involved in cell cycle regulation through their interaction with Caf1. Finally, the tob2 gene was mapped to human chromosome 22q13.1-q13.31.

Human hst-2 (FGF-6) oncogene: cDNA cloning and characterization.

The hst-2 gene was previously identified by its close homology to the hst-1 gene. Cosmid clones containing the hst-2 gene were cloned from a normal human genomic library. Focus-forming activity was observed for the hst-2 cosmids when NIH3T3 transfection assay was performed in a serum-free medium, whereas induction of morphological transformation was difficult to detect in an ordinary serum-supplemented medium. The hst-2 cDNA was cloned from the NIH3T3 transformant. Nucleotide sequence analysis of the cDNA indicates that the hst-2 gene encodes a 198 amino acid transforming protein containing a signal peptide with the characteristics of a heparin-binding growth factor. The coding sequence was almost identical to the published portion of the exon sequence of the FGF-6 gene, indicating that hst-2 is identical to FGF-6. The hst-2 cDNA fragment, when inserted into an expression vector, was able to transform NIH3T3 cells effectively, and the resulting transformant formed a well-vascularized tumor in nude mice, thus suggesting an angiogenic property similar to some other members of the family. RNA blot analysis revealed the expression of the hst-2 gene in human leukemia cell lines with platelet/megakaryocytic differentiation potential.

Tob, a novel protein that interacts with p185erbB2, is associated with anti-proliferative activity.

We have molecularly cloned a cDNA for a novel protein termed Tob (Transducer of ErbB-2) that interacts with the c-erbB-2 gene product p185erbB2. Nucleotide sequencing reveals that the Tob protein is a 45 kDa protein that does not contain either SH2 (Src Homology 2) or SH3 domain but is homologous to the previously characterized anti-proliferative gene product BTG-1 at its amino-terminal half. The carboxyl-terminal half of Tob is characterized by the presence of a sequence rich in proline and glutamine and shows no homology to known proteins. Like BTG-1, exogenously expressed Tob is able to suppress growth of NIH3T3 cells, but the growth suppression is hampered by the presence of kinase-active p185erbB2. By using the GST-Tob protein that contains either full length or amino-terminal half of Tob, we show that the carboxyl-terminal half of Tob is relevant to its interaction with p185erbB2. Furthermore, we could co-immunoprecipitate the Tob protein with anti-ErbB-2 antibody, and reciprocally the p185erbB2 with anti-Tob antibodies. These data suggest that p185erbB2 negatively regulates the Tob-mediated anti-proliferative pathway through its interaction with Tob, resulting possibly in growth stimulation by p185erbB2. Finally, expression of the Tob mRNA is observed in various cell types and is not correlated with expression of c-erbB-2, suggesting that other receptor-type protein-tyrosine kinases are also involved in the Tob-mediated regulation of cell growth.

Identification of RAI3 as a therapeutic target for breast cancer.

We have been investigating gene-expression profiles in estrogen receptor (ER)-negative breast cancers to identify molecules involved in breast carcinogenesis and to select genes or gene products that might be useful as diagnostic markers or targets for new molecular therapies. Here we report evidence that the gene encoding retinoic acid-induced protein 3 (RAI3) is a potential molecular target for treatment of breast cancers. Using quantitative reverse transcription-PCR (RT-PCR), we documented increased expression of RAI3 in 19 of 25 primary breast cancers and in 6 of 11 breast-cancer cell lines examined, by comparison with normal mammary-gland tissue. Treatment of human embryonic kidney (HEK293) cells with siRNA against RAI3 suppressed expression of RAI3 and also suppressed cell growth. Transfection of siRNA into breast-cancer cell lines MCF7 and T47D also suppressed RAI3 mRNA and growth of the cancer cells. Because our data imply that up-regulation of RAI3 function is a frequent feature of breast carcinogenesis, we suggest that selective suppression of signal from RAI3 might hold promise for development of a new strategy for treating breast cancers.

A bivalent disulfide-stabilized Fv with improved antigen binding to erbB2.

We have used protein engineering to generate a stable bivalent Fv molecule of the anti-erbB2 monoclonal antibody e23. The VH and VL domains of the Fv are linked to each other by a disulfide bond and the two Fvs are connected by a flexible 15 amino acid residue (Gly4-Ser)3 linker. The e23 (dsFv)2 molecule is fused to a truncated form of Pseudomonas exotoxin to generate a bivalent disulfide-stabilized, (dsFv)2, immunotoxin. The immunotoxin was expressed in Escherichia coli, refolded in vitro and purified to about 95% purity. Binding studies demonstrated that the (dsFv)2 molecule has a much higher affinity for erbB2 than a monovalent dsFv molecule and a similar binding affinity as the parental antibody e23. The (dsFv)2 immunotoxin was 5 to 20-fold more cytotoxic to two e23 antigen-positive cell lines than the monovalent dsFv immunotoxin. The bivalent dsFv molecule is very stable, retaining 94% of its activity after a 24 hours incubation in human serum at 37 degreesC. Two other molecules with shorter linkers five and ten amino acid residues in length were produced and showed similar activities as the molecule containing a 15 amino acid residue linker. The bivalency, stability and the relative ease of purification makes these e23 (dsFv)2 molecules valuable reagents for cancer immunotherapy and diagnosis.

Escherichia coli mutM suppresses illegitimate recombination induced by oxidative stress.

DNA damage by oxidative stress is one of the causes of mutagenesis. However, whether or not DNA damage induces illegitimate recombination has not been determined. To study the effect of oxidative stress on illegitimate recombination, we examined the frequency of lambdabio transducing phage in the presence of hydrogen peroxide and found that this reagent enhances illegitimate recombination. To clarify the types of illegitimate recombination, we examined the effect of mutations in mutM and related genes on the process. The frequency of lambdabio transducing phage was 5- to 12-fold higher in the mutM mutant than in the wild type, while the frequency in the mutY and mutT mutants was comparable to that of the wild type. Because 7,8-dihydro-8-oxoguanine (8-oxoG) and formamido pyrimidine (Fapy) lesions can be removed from DNA by MutM protein, these lesions are thought to induce illegitimate recombination. Analysis of recombination junctions showed that the recombination at Hotspot I accounts for 22 or 4% of total lambdabio transducing phages in the wild type or in the mutM mutant, respectively. The preferential increase of recombination at nonhotspot sites with hydrogen peroxide in the mutM mutant was discussed on the basis of a new model, in which 8-oxoG and/or Fapy residues may introduce double-strand breaks into DNA.

Role of DNA ligase in the illegitimate recombination that generates lambdabio-transducing phages in Escherichia coli.

We studied the role of DNA ligase in illegitimate recombination in Escherichia coli. A temperature-sensitive mutation in the lig gene reduced the frequency with which lambdabio-transducing phages were generated to 10-14% of that of wild type under UV irradiation. Reintroduction of the lig gene into this mutant restored the frequency of recombinant phage generation to that of wild type. Furthermore, overexpression of DNA ligase enhanced illegitimate recombination by 10-fold with or without UV irradiation. In addition, when DNA ligase was present in only limited amounts, UV-induced or spontaneous illegitimate recombination occurred exclusively at hotspot sites that have relatively long sequences of homology (9 or 13 bp). However, when DNA ligase was overexpressed, most of the illegitimate recombination took place at non-hotspot sites having only short sequences of homology (<4 bp). Thus, the level of ligase activity affects the frequency of illegitimate recombination, the length of sequence homology at the recombination sites, and the preference for recombination at hotspots, at least after UV irradiation. These observations support our hypothesis that the illegitimate recombination that generates lambdabio-transducing phages is mediated by the DNA break-and-join mechanism.

Variations in 5-fluorouracil concentrations of colorectal tissues as compared with dihydropyrimidine dehydrogenase (DPD) enzyme activities and DPD messenger RNA levels.

Dihydropyrimidine dehydrogenase (DPD) is the initial key enzyme in 5-fluorouracil (5-FU) catabolism. We measured DPD activities represented as DPD protein levels (units/mg protein) and the associated mRNA levels in tumorous and normal tissues from 40 colorectal cancer patients, and we studied the relation to 5-FU concentrations in the same samples after treatment with doxifluridine, a prodrug of 5-FU. DPD mRNA levels were also measured in biopsy samples before treatment for comparison with those in surgical samples. 5-FU concentrations in tumors were higher than those in normal tissues (P < 0.05) and were inversely associated with DPD protein levels (r = -0.463; P < 0.05). DPD activities in tumorous and normal tissues showed a significant correlation (r = 0.527; P < 0.01). DPD protein levels correlated with their mRNA levels detected by semiquantitative reverse transcription-PCR in tumor tissues (r = 0.740; P < 0.01). DPD mRNA levels in tumor biopsy specimens correlated with those in surgical specimens (r = 0.366; P < 0.05). These results suggest DPD activities in tumors to be predictive of 5-FU levels in colorectal cancer tissues and are reflected by DPD mRNA levels as measured by reverse transcription-PCR.

Microsatellite instability in thyroid cancer: hot spots, clinicopathological implications, and prognostic significance.

PURPOSE: To determine whether microsatellite instability (MSI) in particular loci has clinicopathological significance in thyroid cancer. EXPERIMENTAL DESIGN: Seventy-six cases of surgically resected thyroid cancer were screened for MSI at nine microsatellites: THRA1, TSHR, D2S123, D11S912, D2S115, D2S399, p53, RET, or BAT-26. Multivariate analysis was performed to test for links between MSI and the clinical parameters of gender, age, histology, stage, nodal involvement, and prognosis. RESULTS: THRA1, residing in the thyroid hormone receptor alpha gene, displayed the highest levels of MSI at 36.5%. MSI in TSHR, located within the thyroid-stimulating hormone receptor gene, was found to be linked to cancer in the elderly (>70 years of age) and with high-grade (N 3, 4) nodal involvement. In follicular cancer, MSI in D2S123 occurred at a frequency of 100% (7/7) with no (0%) occurrence of MSI at the nearby D2S115, D2S399, or BAT-26 loci. Regarding prognosis, patients with MSI-positive cancer showed better long-term survival. BAT-26, which is an important marker in colorectal cancer, displayed the lowest frequency of MSI in our panel of thyroid tumors. CONCLUSION: Whereas patients with MSI-positive cancer showed better long-term survival, as is the case for colorectal cancer, our finding of the low frequency of MSI in BAT-26 suggests that the biochemical defects governing the spectrum of MSI in thyroid and colorectal cancer are different. MSI in THRA1, TSHR, and D2S123 appears to be an integral part of thyroid carcinogenesis, as evidenced by the high frequency of MSI and significant correlation to clinical data.

Tamoxifen-induced apoptosis in breast cancer cells relates to down-regulation of bcl-2, but not bax and bcl-X(L), without alteration of p53 protein levels.

Tamoxifen (TAM) has been shown to induce apoptosis in breast cancer cells. bcl-2 family genes, which can interact with each other, have been shown to interfere with apoptosis after various stimuli. In this study, we investigated the effects of TAM on bcl-2 family gene products bcl-2, bax, and bcl-X(L) and on p53 levels in estrogen receptor-positive MCF-7 breast cancer cells. We found that TAM induced time- and concentration-dependent down-regulation of bcl-2 at both the mRNA and protein level. Down-regulation of bcl-2 correlated with TAM-induced apoptosis. In addition, estradiol treatment significantly increased bcl-2 protein expression and blocked the reduction of bcl-2 by TAM. TAM did not, however, affect bax, bcl-X(L), or p53 expression at the mRNA or protein level. Our results demonstrate that TAM can induce apoptosis in a time- and dose-dependent manner by modulating bcl-2 levels in breast cancer cells, and down-regulation of bcl-2 induced by TAM was not accompanied by alterations in p53 levels.