Steven J. Weintraub

Identification of Trop-2 as an oncogene and an attractive therapeutic target in colon cancers

The cell surface protein Trop-2 is highly expressed in a wide variety of epithelial cancers. In contrast, there is little or no expression of Trop-2 in adult somatic tissue. Because it is a cell surface protein that is selectively expressed in tumor cells, Trop-2 is a potential therapeutic target. However, whether Trop-2 is actively involved in tumorigenesis and whether its targeting for treatment would be effective have not been examined. Here, we show that Trop-2 expression is necessary for tumorigenesis and invasiveness of colon cancer cells, as both are inhibited when Trop-2 expression is suppressed by RNA interference. Conversely, ectopic expression of Trop-2 in colon cancer cells enhances their capacity for anchorage-independent growth and ectopic expression of Trop-2 in NIH3T3 cells is sufficient to promote both anchorage-independent growth and tumorigenesis. Importantly, we show that an antibody against the extracellular domain of Trop-2 reduces tumor cell invasiveness. Therefore, we have identified Trop-2 as an oncogene that has potential as a therapeutic target. Given the restricted expression of Trop-2 in normal tissue, anti–Trop-2 therapeutics would be predicted to have limited toxicity. [Mol Cancer Ther 2008;7(2):280–5]

Chronoregulation by asparagine deamidation.

Every asparagine in every protein undergoes nonenzymatic deamidation to aspartate or isoaspartate at a rate determined by the surrounding protein structure and cellular environment. Under physiologic conditions, the deamidation half-life of individual asparagines in proteins is proposed to range from less than a day to several centuries. More than 200 proteins have been shown to undergo deamidation to a meaningful degree, and modeling predicts that hundreds more undergo deamidation at rates that have the potential to be of biological consequence. Because deamidation converts an asparagine into an aspartate or isoaspartate, it introduces a negative charge into a protein and results in the isomerization of a residue. Therefore, deamidation has the potential to change protein function. Additionally, deamidation is thought to render some proteins more susceptible to degradation. In most instances in which asparagine deamidation has been identified in vivo, it is involved in pathology. Hence, deamidation has been viewed primarily as a form of protein damage. However, the pervasiveness and evolutionary persistence of these unstable asparagines suggest that they may have a beneficial role. Notably, the change of even a single neighboring amino acid can have a marked effect on the rate of deamidation of an asparagine. Therefore, the underlying rate of deamidation of any asparagine is genetically programmable. This characteristic, combined with the wide range of deamidation rates that can be programmed, imparts to asparagines the potential to serve as molecular timers that regulate protein function and stability. The deamidation of an asparagine is a common posttranslational modification. When an asparagine in a protein undergoes deamidation, its amide side chain is nonenzymatically hydrolyzed, which results in the replacement of the asparagine residue with either an aspartate or an isoaspartate. By introducing a negative charge and isomerization into a protein in this manner, the deamidation of an asparagine has the potential to alter protein function. Asparagine deamidation is widely thought to be an unregulated process that is nothing more than a form of protein damage. However, it has been demonstrated that the inherent deamidation rate of an asparagine is actually tightly regulated by its surrounding sequence and that a wide range of deamidation rates can be genetically programmed into a protein simply by altering the amino acids in the sequence surrounding an asparagine. Because of this property, it was proposed 40 years ago that asparagine deamidation could serve as molecular timer that is genetically programmed to time biological processes. This STKE review, with 4 figures and 49 references, discusses the evidence in support of the molecular timer hypothesis.

Control of Cellular Bcl-xL Levels by Deamidation-Regulated Degradation

Deamidation of two asparagines activates a conditional PEST sequence to target Bcl-xL for degradation.

Interaction of a common factor with ATF, Sp1, or TATAA promoter elements is required for these sequences to mediate transactivation by the adenoviral oncogene E1a.

The adenovirus protein E1a stimulates transcription of both viral and cellular genes. Unlike most other transcription factors, it induces transactivation through several different promoter elements. The mechanism by which elements of diverse sequence mediate the effect of E1a is the focus of this study. Three E1a-responsive elements (an ATF site, an Sp1 site, and a TATA box containing the sequence TATAA) were studied to determine whether their interaction with a common factor is necessary for transactivation. In transfection assays, each element was used as a competitor against promoter constructs containing the other elements. The elements as competitors had no effect on basal transcription, but each competitor completely inhibited transactivation by E1a. Competitors that were not E1a responsive failed to inhibit transactivation. Therefore, either E1a itself or an E1a-inducible factor interacts with each of the elements to cause transactivation, most likely though an association with each elements specific binding protein.

Vitamin D and the racial difference in the genotype 1 chronic hepatitis C treatment response

BACKGROUND African Americans with genotype 1 chronic hepatitis C attain a sustained virologic response (SVR) at only approximately one-half the rate of whites after peginterferon and ribavirin treatment. The serum concentration of 25-hydroxyvitamin D [25(OH)D] has recently been established as a predictor of treatment response. Therefore, the low serum concentrations of 25(OH)D found among African Americans may contribute to the low response rate; however, to our knowledge, none of the studies of vitamin D in chronic hepatitis C treatment have included a significant number of black patients. OBJECTIVE The objective was to compare the relation between the 25(OH)D concentration and genotype 1 chronic hepatitis C treatment response in African Americans with that in whites. DESIGN This cross-sectional analysis included 106 African American and 65 white patients with genotype 1 chronic hepatitis C. RESULTS Consistent with previous studies, we found that the SVR rate in the white patients increased significantly with an increasing serum concentration of 25(OH)D [SVR rates were 20%, 46%, and 70% for 25(OH)D serum concentrations <20, 20-35, and >35 ng/mL, respectively; P-trend = 0.008]; however, there was no relation between the SVR rate and 25(OH)D serum concentration in the African American patients [SVR rates were 32%, 28%, and 33% for 25(OH)D serum concentrations <20, 20-35, and >35 ng/mL, respectively; P-trend = 0.832]. We also found an analogous racial difference in the relation between the extent of liver fibrosis and the 25(OH)D concentration. CONCLUSION Racial differences in vitamin D physiology or race-specific factors that modify the effects of vitamin D may affect the immune response to genotype 1 hepatitis C virus.

Resistance to antineoplastic therapy: The oncogenic tyrosine kinase-Bcl-xL axis

The discovery two decades ago that the Philadelphia chromosome encodes an oncogenic fusion of Bcr and Abl remains among the most important contributions to our understanding of the process of malignant transformation. We now know that Bcr-Abl is one of more than 30 aberrantly activated tyrosine kinases that are expressed in a variety of tumors. Conventional treatment of the tumors in which these proteins are expressed is usually doomed to failure because the activated tyrosine kinases render the tumor cells stubbornly resistant to apoptosis. In this context, it is notable that Zhao and coworkers have uncovered a novel weapon in the resistance armamentarium of these rogue kinases, the suppression of the inactivating deamidation of Bcl-xL (this issue of Cancer Cell).

Stepwise Recruitment of Components of the Preinitiation Complex by Upstream Activators In Vivo

ABSTRACT Recently, it was found that if either the TATA binding protein or RNA polymerase II holoenzyme is artificially tethered to a promoter, transcription is activated. This finding provided presumptive evidence that upstream activating proteins function by recruiting components of the preinitiation complex (PIC) to the promoter. To date, however, there have been no studies demonstrating that upstream factors actually recruit components of the PIC to the promoter in vivo. Therefore, we have studied the mechanism of action of two disparate transactivating domains. We present a series of in vivo functional assays that demonstrate that each of these proteins targets different components of the PIC for recruitment. We show that, by targeting different components of the PIC for recruitment, these activating domains can cooperate with each other to activate transcription synergistically and that, even within one protein, two different activating subdomains can activate transcription synergistically by cooperating to recruit different components of the PIC. Finally, considering our work together with previous studies, we propose that certain transcription factors both recruit components of the PIC and facilitate steps in transcriptional activation that occur subsequent to recruitment.

Diazepam in the Treatment of Moderate to Severe Alcohol Withdrawal.

Benzodiazepines ameliorate or prevent the symptoms and complications of moderate to severe alcohol withdrawal, which can include autonomic hyperactivity, agitation, combativeness, hallucinations, seizures, delirium, and death. The benzodiazepines most commonly used for this purpose are lorazepam, chlordiazepoxide, oxazepam, and diazepam. It is widely asserted that no member of this group is superior to the others for treatment of alcohol withdrawal. However, of these, diazepam has the shortest time to peak effect, which facilitates both rapid control of symptoms and accurate titration to avoid over-sedation. Furthermore, diazepam and its active metabolite, desmethyldiazepam, have the longest elimination half-lives, so their levels decrease in a gradual, self-tapering manner, resulting in a smoother withdrawal, i.e., a lower incidence and severity of both breakthrough symptoms and rebound phenomena, including a possibly decreased seizure risk. Importantly, the fear of increased risk of over-sedation with diazepam compared with other benzodiazepines is based on a misunderstanding of its pharmacokinetics and is unfounded. Similarly, the notion that diazepam should be avoided in patients with liver disease and elderly patients to avoid prolonged over-sedation is based on no more than conjecture. In fact, there is clinical evidence that diazepam is safe for the treatment of alcohol withdrawal in these patients when administered using a simple symptom-based approach. There is one instance in which diazepam should not be used: when intramuscular administration is the only option, the lipophilicity of diazepam can result in slow absorption—either lorazepam or, when rapid control of symptoms is required, midazolam should be used. The comparative pharmacokinetics of the benzodiazepines used in the treatment of alcohol withdrawal together with a comprehensive review of the literature on their use strongly suggest that diazepam should be the preferred benzodiazepine for the treatment of patients experiencing moderate to severe alcohol withdrawal under most circumstances.

Vitamin D‐binding protein gene polymorphisms may contribute to the racial disparity in genotype 1 chronic hepatitis C treatment outcome

We read with great interest the study by Falleti et al. in which the authors report that the rs7041 G>T and rs4588 C>A single nucleotide polymorphisms (SNPs) of the vitamin D-binding protein gene are predictors of the treatment outcome of patients with chronic hepatitis C. The authors found that patients with any combination of three or more rs7041 G and rs4588 C alleles (wild type [WT1]) achieve a sustained virologic response (SVR) at a greater rate than patients with other genotypes (WT2). Additionally, they found that the relative frequency of the rs7041 T allele was increased significantly among the patients when compared with its frequency among healthy controls, raising the possibility that rs7041 T alleles are associated with increased susceptibility to infection or a lower rate of spontaneous viral clearance. Importantly, there is biologic plausibility for these findings in that each of the protein isoforms encoded by the variants of the vitamin D-binding protein gene studied by Falleti et al. differentially affect certain components of the immune response. In light of these findings, we sought to determine if there are racial differences in the distribution of the alleles of these SNPs among patients with chronic hepatitis C that could potentially contribute to the racial disparity in treatment outcome. We genotyped the rs7041 G>T and rs4588 C>A SNPs in 48 Caucasian and 95 African American genotype 1 chronic hepatitis C patients. Most notable were our findings concerning the rs7041 G>T SNP. The frequency of the rs7041 G>T alleles among the Caucasians was G 5 0.542 and T 5 0.458. This is similar to the frequency among the patients studied by Falleti et al., all of whom were Caucasian, of G 5 0.553 and T 5 0.447. However, the rs7041 G>T allele frequency among the African Americans we studied was G 5 0.147 and T 5 0.853, which is significantly different than that of the Caucasians we studied (P< 0.0001) and the patients studied by Falleti et al. (P< 0.0001). We then grouped our patients into the WT1 and WT2 categories as defined by Falleti et al. (Table 1). Of the Caucasian patients we studied, 26/48 (54.2%) were WT1. This is similar to the finding by Falleti et al. that 100/206 (48.5%) of their patients were WT1. In contrast, however, only 25/95 (26.3%) of the African American patients in our study were WT1, which is significantly less than the frequency of the WT1 genotype in both our group of Caucasian patients (P 5 0.0016) and the patients examined by Falleti et al. (P 5 0.0003). Although our study was not sufficiently powered to confirm that vitamin D-binding protein genotype predicts antiviral treatment outcome independently of race, when considered with the findings of Falleti et al. our findings raise the possibility that racial variations in distribution of vitamin D-binding protein gene SNP alleles contribute to the racial disparity in treatment outcome in patients with chronic hepatitis C. Further studies are warranted.

Abstract C167: Trop2 as a target for breast cancer treatment

Trop2 is expressed on many epithelial tumors and stem cells of the prostate and liver. Its association with aggressive cancer has led to the speculation that it might be a therapeutic target. We have previously shown a role for Trop2 in colon tumorigenesis and also found that antibodies against Trop2 can block tumor cell invasion. Consistent with this data, a monoclonal antibody against Trop2 is in pre‐clinical development against several tumor types. However, the expression pattern and role of Trop2 in breast cancer has not been examined. Here, we show heterogeneous expression of Trop2 in normal mammary gland, primary breast tumors and cancer cells lines. After purification of Trop2 expressing tumor cell populations by fluorescence activated cell sorting, we find that Trop2 hi cells are preferentially invasive and tumorigenic compared to the Trop2 lo . RNAi mediated suppression of Trop2 in breast cancer cells arrests their proliferation. Even more importantly, a monoclonal antibody against Trop2 shows anti‐tumor activity in vivo . Therefore, Trop2 represents a novel and unexplored therapeutic target in this common malignancy. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C167.