Molecular biology of neuroendocrine tumors: from pathways to biomarkers and targets.

Neuroendocrine tumors (NETs) represent a heterogeneous group of diseases with varied natural history and prognosis depending upon the organ of origin and grade of aggressiveness. The most widely used biomarker to determine disease burden and monitor response to treatment is chromogranin A (CgA), but it is far from being the optimal predictive and prognostic biomarker in NETs. Biological understanding and derived treatment options for NETs have changed markedly in recent years. Over the last decade, the genomic landscape of these tumors has been extensively investigated. This has resulted in the discovery of mutations and expression anomalies in genes and pathways such as the PI3K/Akt/mTOR, DAXX/ATRX, and MEN1, which are promising predictive and prognostic biomarkers and future candidates for targeted therapies. Additionally, the study of tumor stroma and environment are one of the most promising fields for discovery of potential new targets and biomarkers.

Circulating leukocyte telomere length and risk of overall and aggressive prostate cancer.

BACKGROUND: Recent large-scale prospective studies suggest that long telomeres are associated with an increase cancer risk, counter to conventional wisdom. METHODS: To further clarify the association between leukocyte telomere length (LTL) and prostate cancer, and assess genetic variability in relation to both LTL and prostate cancer, we performed a nested case-control study (922 cases and 935 controls). The participants provided blood in 1993-1995 and were followed through August 2004 (prostate cancer incidence) or until 28 February 2013 (lethal or fatal prostate cancer). Relative LTL was measured by quantitative PCR and was calculated as the ratio of telomere repeat copy number to a single gene (36B4) copy number (T/S). Genotyping was performed using the TaqMan OpenArray SNP Genotyping Platform. Logistic regression was used to estimate odds ratios (ORs) and 95% confidence intervals (CIs) of all prostate cancer and subtypes defined by Gleason grade, stage and lethality (metastasis or death). RESULTS: We observed a positive association between each s.d. increase in LTL and all (multivariable-adjusted OR 1.11, 95% CI: 1.01-1.22), low-grade (OR 1.13, 95% CI:1.01-1.27), and localised (OR 1.12, 95% CI:1.01-1.24) prostate cancer. Associations for other subtypes were similar, but did not reach statistical significance. In subgroup analyses, associations for high grade and advanced stage (OR=2.04, 95% CI 1.00-4.17; Pinteraction=0.06) or lethal disease (OR=2.37, 95% CI 1.19-4.72; Pinteraction=0.01) were stronger in men with a family history of the disease compared with those without. The minor allele of SNP, rs7726159, which has previously been shown to be positively associated with LTL, showed an inverse association with all prostate cancer risk after correction for multiple testing (P=0.0005). CONCLUSION: In this prospective study, longer LTL was modestly associated with higher risk of prostate cancer. A stronger association for more aggressive cancer in men with a family history of the disease needs to be confirmed in larger studies.

Prostatic preneoplasia and beyond.

Prostate cancer is a heterogeneous neoplasm both with regard to its development, molecular abnormalities and clinical course. For example, in the United States, 1 in 6 men is diagnosed with prostate cancer whilst only 1 in 34 dies of metastatic disease [A. Jemal, R. Siegel, E. Ward, T. Murray, J. Xu, M.J. Thun, Cancer Statistics, 2007, CA Cancer J. Clin. 57 (2007) 43-66]. In this review, we summarise novel understandings of the early molecular events in prostatic carcinogenesis that may underlie both the molecular and clinical heterogeneity. Issues covered include those related to stem cells and embryonic signalling, oncogene/tumor suppressor abnormalities, androgen signalling, apoptosis and the nature of tumor-stromal interactions. Emphasis is placed on signalling pathway abnormalities, their causation, consequences and interactions. For example, genomic abnormalities involving the TMPRSS2-ETS and PTEN loci and the resulting signalling effects suggest the importance of genomic instability as a crucial factor in the emergence of this neoplasm. Together with new insights into signalling pathways consequent to abnormalities such as these, a greater understanding of the pathophysiology involved in prostatic carcinogenesis will lead to targeted approaches for both therapy and chemoprevention in the future.

In vivo immunologic selection of class I major histocompatibility complex gene deletion variants from the B16-BL6 melanoma.

The mechanism by which tumor allografts escape host immunologic attack was investigated. B16-BL6 cells (the bladder 6 subline of the B16 melanoma) (H-2b) were transfected with a gene (Dd) encoding an allogeneic class I major histocompatibility complex antigen. Clones that expressed Dd antigen were injected into the footpads of nonimmune syngeneic mice, syngeneic immune mice, and nude mice. Under conditions of immunologic selection a clone that contained multiple copies of the transfected gene formed variants that lacked the transfected gene. Primary tumors and pulmonary metastases of immunized mice and pulmonary metastases of nonimmunized mice had lost the Dd gene and, in most cases, all of the associated plasmid. In contrast, in immunodeficient nude mice, primary tumors and pulmonary metastases retained the Dd gene and the associated plasmid. Deletion of genes encoding cell surface antigens may be one of the mechanisms by which allogeneic tumors escape immunologic attack.

Dissociation of therapeutic and toxic effects of polyinosinic-polycytidylic acid admixed with poly-L-lysine and solubilized with carboxymethyl cellulose in tumor-bearing mice.

In this paper, we describe a study of the therapeutic parameters (dose and schedule) and immunomodulatory activity (macrophage, natural killer cell, and T-cell number and function) of polyinosinic-polycytidylic acid admixed with poly-L-lysine and solubilized with carboxymethyl cellulose [poly(I,C)-LC] in the treatment of MBL-2 tumor ascites. Tumor-bearing mice received an optimal therapeutic protocol [100 micrograms poly(I,C)-LC administered twice a wk], a maximum tolerated dose [50 micrograms poly(I,C)-LC administered daily], or the optimal immunomodulatory protocol for normal mice [10 micrograms poly(I,C)-LC administered daily]. The percentage of tumor-associated macrophages and their cytotoxic activity correlated with host survival. In addition, splenic T-cell activity correlated with host survival, and splenic natural killer cell function had a near significant correlation with host survival. These results indicate that the optimal dose and schedule of poly(I,C)-LC for immunomodulation in tumor-bearing animals are also the optimal therapeutic protocol but have less toxicity than the maximum tolerated dose.

Telomerase activity: a prevalent marker of malignant human prostate tissue.

We urgently need biochemical markers to detect the malignant nature and pathological states of the human prostate. We report that telomerase activity is associated with prostate cancer but absent in the benign disease and normal gland. Telomerase is, therefore, a potential diagnostic marker for prostate cancer. Twenty-five human prostates resected at the time of radical prostatectomy were dissected to obtain matched adjacent areas of normal, central zone benign prostatic hyperplasia (BPH), and pathologically confirmed cancer tissue. These matched tissue samples were assayed for telomerase activity using a sensitive PCR technique. None of the normal tissues exhibited telomerase activity. In contrast, 21 of the 25 (84%) cancers were strongly positive. At the time of prostatectomy, four lymph nodes were positive for metastases and all were strongly positive for telomerase activity. In adjacent BPH tissues taken from the cancerous prostates, only 3 of the 25 tissues (12%) were weakly positive. Telomerase activity was not detected in ten BPH samples recovered from patients who underwent open surgery solely for BPH. All five available cell lines of human prostate cancer (DU145, LNCaP, PC3, PPC1, and TSU) were strongly positive. Short telomere lengths have been observed in several human cancers. We also measured the telomere lengths in 27 matched samples of normal, BPH, and cancer tissue taken from nine radical prostatectomies. The telomeres from cancer tissue were significantly and consistently shorter than either the adjacent normal or adjacent BPH tissues. Our results indicate that telomerase activity, as well as telomere lengths, may be markers for distinguishing prostate cancer from normal and benign prostate tissues.

Analysis of potential response predictors to capecitabine/temozolomide in metastatic pancreatic neuroendocrine tumors.

The capecitabine and temozolomide (CAPTEM) regimen is active in the treatment of metastatic pancreatic neuroendocrine tumors (pNETs), with response rates ranging from 30 to 70%. Small retrospective studies suggest that O(6)-methylguanine DNA methyltransferase (MGMT) deficiency predicts response to temozolomide. High tumor proliferative activity is also commonly perceived as a significant predictor of response to cytotoxic chemotherapy. It is unclear whether chromosomal instability (CIN), which correlates with alternative lengthening of telomeres (ALT), is a predictive factor. In this study, we evaluated 143 patients with advanced pNET who underwent treatment with CAPTEM for radiographic and biochemical response. MGMT expression (n=52), grade (n=128) and ALT activation (n=46) were investigated as potential predictive biomarkers. Treatment with CAPTEM was associated with an overall response rate (ORR) of 54% by RECIST 1.1. Response to CAPTEM was not influenced by MGMT expression, proliferative activity or ALT pathway activation. Based on these results, no biomarker-driven selection criteria for use of the CAPTEM regimen can be recommended at this time.

Evidence for strained interactions between side-chains and the polypeptide backbone.

In most proteins, a small but significant fraction of residues adopt phi,psi angles that generate unfavorable steric interactions between side-chain atoms and the peptide backbone. For the small protein staphylococcal nuclease, the X-ray structure reveals that 18 of 133 residues occupy unusual and, presumably, energetically unfavorable backbone conformations. To quantify the amount of strain energy generated by these local interactions, we have analyzed the changes in stability that accompany replacement of the wild-type side-chain with glycine, a residue that can access a much larger set of phi,psi angles without energy penalty. To correct for the many other sources of stability loss that might accompany this mutation, the glycine mutant was compared to an alanine mutant at the same position and the resulting free energy difference delta delta GG-->A was then compared to the average delta delta GG-->A at all other, unstrained positions in the nuclease occupied by similar amino acid types. In addition, potential steric clashes were introduced by substituting alanine at each of six positions occupied in the wild-type by glycine with phi,psi angles that are unfavorable for all other residue types. The data suggest that residues with phi,psi angles outside the preferred alpha-helical and beta-sheet regions represent sites of local strain energy that lower the stability of the native state by 1 to 2 kcal/mol and, in some cases, as much as 3 to 4 kcal/mol. Given that 10 to 20% of residues in globular proteins adopt phi,psi angles outside the preferred alpha-helical and beta-sheet regions, this implies that there is on the order of 20 kcal/mol of strain energy in a protein of 100 residues that may be relieved by appropriate mutations.

Paucity of PD-L1 expression in prostate cancer: innate and adaptive immune resistance.

BACKGROUND: Primary prostate cancers are infiltrated with programmed death-1 (PD-1) expressing CD8+ T-cells. However, in early clinical trials, men with metastatic castrate-resistant prostate cancer did not respond to PD-1 blockade as a monotherapy. One explanation for this unresponsiveness could be that prostate tumors generally do not express programmed death ligand-1 (PD-L1), the primary ligand for PD-1. However, lack of PD-L1 expression in prostate cancer would be surprising, given that phosphatase and tensin homolog (PTEN) loss is relatively common in prostate cancer and several studies have shown that PTEN loss correlates with PD-L1 upregulation--constituting a mechanism of innate immune resistance. This study tested whether prostate cancer cells were capable of expressing PD-L1, and whether the rare PD-L1 expression that occurs in human specimens correlates with PTEN loss. METHODS: Human prostate cancer cell lines were evaluated for PD-L1 expression and loss of PTEN by flow cytometry and western blotting, respectively. Immunohistochemical (IHC) staining for PTEN was correlated with PD-L1 IHC using a series of resected human prostate cancer samples. RESULTS: In vitro, many prostate cancer cell lines upregulated PD-L1 expression in response to inflammatory cytokines, consistent with adaptive immune resistance. In these cell lines, no association between PTEN loss and PD-L1 expression was apparent. In primary prostate tumors, PD-L1 expression was rare, and was not associated with PTEN loss. CONCLUSIONS: These studies show that some prostate cancer cell lines are capable of expressing PD-L1. However, in human prostate cancer, PTEN loss is not associated with PD-L1 expression, arguing against innate immune resistance as a mechanism that mitigates antitumor immune responses in this disease.

Telomerase is activated in the prostate and seminal vesicles of the castrated rat.

Telomeres, the repetitive non-coding DNA sequences found at the ends of all eukaryotic chromosomes, shorten with each cell division. It has been proposed that telomere shortening may be the counting element of a mitotic clock that keeps track of cell divisions; with shortening to a critical length acting as a senescence signal underlying cellular aging. The enzyme telomerase functions to maintain telomere length, thus allowing unlimited cell division, and has been associated with cellular immortalization and cancer. Stem cells have large, perhaps unlimited, replicative capacities. Since these cells are potentially immortal, we reasoned that they might posses active telomerase. We therefore assayed for telomerase activity in the stem cell enriched pools of the androgen-depleted sex accessory tissues in the castrated male rat. Following castration, the ventral prostate and seminal vesicles of the rat involute, losing approximately 90% of their cells by 21 days. These residual glands persist, and are enriched for stem cells, being capable of fully regenerating these glands if testosterone is re-introduced into the animal. We assayed telomerase activity in extracts from normal, involuted, and regenerating ventral prostate and seminal vesicles. Normal glands were found to be telomerase negative, whereas telomerase activity appeared as these glands involuted following castration. Conversely, telomerase activity disappeared during testosterone-induced regeneration of these residual glands. These results provide strong evidence for the ability of androgen to negatively-regulate telomerase activity in stem cell populations of the rat ventral prostate and seminal vesicles. and represent the first in vivo model system for the modulation of telomerase activity.

Telomerase as drug and drug target for the treatment of thyroid cancer.

Telomerase is known to be activated in almost all cancer cells and is quiescent in almost all normal cells. Therefore, it follows that therapeutic strategies directed against cancer would include the targeting of telomerase, as well as the use of telomerase. Several approaches have been used both in vitro and in vivo and include the following: 1) antisense; 2) immunotherapy directed against hTERT; and 3) the use of telomerase promoter to direct cytotoxic therapy. Herein we review these approaches and discuss their potential applicability against thyroid cancer.

Use of surface-enhanced laser desorption ionization protein chip system to analyze streptococcal exotoxin B activity secreted by Streptococcus pyogenes.

Ciphergen surface-enhanced laser desorption ionization (SELDI) protein chip technology was used to analyze the secretion and autoactivation of the Streptococcus pyogenes cysteine protease SpeB. This method allowed rapid identification of both the zymogen form of the protein Mr approximately 41,000 and the fully active enzyme Mr approximately 28,500. SpeB production in culture supernatants was demonstrated to be growth-phase regulated and SpeB positive and negative variants of a blood passaged S. pyogenes isolate could readily be distinguished. In kinetic studies of the autoactivation of the zymogen form of SpeB, the sequential generation of four intermediates was detected before the accumulation of the fully active enzyme. The methods described enabled enhanced speed, use of lower sample volumes and concentrations, and a more complete molecular characterization of SpeB than allowed by existing methods of analysis using SDS-PAGE and Western immunoblotting.

Prospective payment. An idea whose time has come.

The home care industry has experience with Medicaid per-visit rates and hospice daily and hourly rates capped by an overall patient limit. But no extensive home care prospective rates have been developed. Is this gap an opening for the industry to provide some valuable input?

Significant human beta-cell turnover is limited to the first three decades of life as determined by in vivo thymidine analog incorporation and radiocarbon dating.

AIMS: Diabetes mellitus results from an absolute or relative deficiency of insulin-producing pancreatic ß-cells. The turnover rate of adult human ß-cells remains unknown. We employed two techniques to examine adult human islet ß-cell turnover and longevity in vivo. METHODS: Subjects enrolled in National Institutes of Health clinical trials received thymidine analogs [iododeoxyuridine (IdU) or bromodeoxyuridine (BrdU)] 8 d to 4 yr prior to death. Archival autopsy samples from 10 patients (aged 17-74 yr) were employed to assess ß-cell turnover by scoring nuclear analog labeling within insulin-staining cells. Human adult ß-cell longevity was determined by estimating the cells' genomic DNA integration of atmospheric (14)C. DNA was purified from pancreatic islets isolated from cadaveric donors; whole islet prep DNA was obtained from a 15-yr-old donor, and purified ß-cell DNA was obtained from two donors (ages 48 and 80 yr). (14)C levels were then determined using accelerator mass spectrometry. Cellular "birth date" was determined by comparing the subject's DNA (14)C content relative to a well-established (14)C atmospheric prevalence curve. RESULTS: In the two subjects less than 20 yr of age, 1-2% of the ß-cell nuclei costained for BrdU/IdU. No ß-cell nuclei costained in the eight patients more than 30 yr old. Consistent with the BrdU/IdU turnover data, ß-cell DNA (14)C content indicated that the "birth date" of cells occurred within the subject's first 30 yr of life. CONCLUSIONS: Under typical circumstances, human ß-cells and their cellular precursors are established by young adulthood.

Residual structure in large fragments of staphylococcal nuclease: effects of amino acid substitutions.

In an attempt to develop a model of the denatured state of staphylococcal nuclease that can be analyzed experimentally under physiological conditions, a series of four large fragments of this small protein which extend from residues 1 to 103, 1 to 112, 1 to 128, and 1 to 136 have been generated through the overexpression of nuclease genes containing stop codons at defined positions. Large amounts of protein fragments were accumulated in induced cells and were purified by carrying out all fractionation steps in the presence of 6 M urea. The far-ultraviolet circular dichroism spectra of all four fragments suggested the presence of small to moderate amounts of residual structure. When the CD spectra were monitored as a function of concentrations of the tight-binding ligands Ca2+ and thymidine 3',5'-bisphosphate and the known affinity constants for wild-type nuclease (1-149) were used, apparent equilibrium constants of 160 and 2000 for the reversible denaturation reaction for fragments 1-136 and 1-128, respectively, were estimated. Four single and two double mutations, all of which exhibit unusual behavior in the full-length protein on solvent denaturation [Shortle, D., & Meeker, A. K. (1986) Proteins: Struct., Funct., Genet. 1, 81-89] and thermal denaturation [Shortle, D., Meeker, A. K., & Freire, E. (1988) Biochemistry 27, 4761-4768], were recombined into the 1-136 and 1-128 fragment expression vectors, and purified mutant fragments were characterized.(ABSTRACT TRUNCATED AT 250 WORDS)

Telomerase: a promising marker of biological immortality of germ, stem, and cancer cells. A review.

This review will describe the current state of knowledge of telomerase as it relates to human malignancies, focusing primarily on published measurements of this enzymes activity in benign and malignant neoplasms and their normal tissue counterparts. Key questions concerning the potential clinical utility of assaying for telomerase activity will be addressed and the implications of recent findings discussed.

A fusion protein between serum amyloid A and staphylococcal nuclease--synthesis, purification, and structural studies.

We developed a recombinant DNA system to overexpress a fusion protein between the small, minimally soluble acute phase serum protein, serum amyloid A (SAA), and the bacterial enzyme staphylococcal nuclease (SN). This fusion protein is very soluble and is immunoreactive to polyclonal anti-SAA antibodies. Tryptophan fluorescence shows smooth denaturation curves for the fusion protein in guanidinium HCl or potassium thiocyanate. Fluorescence also indicates that only a single tryptophan residue (of the four present) is accessible to iodide quenching and, presumably, is exposed on the surface of the fusion protein. Circular dichroism (CD) shows a significant signal indicating alpha-helix, which can be attributed to the SAA portion of the molecule; these are the first CD spectral data available for SAA. pH titration shows persistence of helix domains for the fusion protein at pH 3.0, in contrast to the denaturation of SN under the same conditions. (The entire fusion protein shows a random coil pattern below pH 3.0.) By exploiting the structural and solubility properties of SN, this fusion protein has provided the first structural data about SAA-the precursor of the amyloid deposits in secondary amyloidosis. This fusion protein should be useful for further physical and physiologic studies of SAA.

Mutant forms of staphylococcal nuclease with altered patterns of guanidine hydrochloride and urea denaturation.

Eleven mutant forms of staphylococcal nuclease with one or more defined amino acid substitutions have been analyzed by solvent denaturation by using intrinsic fluorescence to follow the denaturation reaction. On the basis of patterns observed in the value of m--the rate of change of log Kapp (the apparent equilibrium constant between the native and denatured states) with denaturant concentration--these proteins can be grouped into two classes. For class I mutants, the value of m with guanidine hydrochloride is less than the wild-type value and is either constant or increases slightly with increasing denaturant; the value of m with urea is also less than wild type but shows a marked increase with increasing denaturant concentration, often approaching but never exceeding the wild-type value. For class II mutants, m is constant and is greater than wild type in both denaturants, with the increase being consistently larger in guanidine hydrochloride than in urea. When double or triple mutants are constructed from members of the same mutant class, the change in m is usually the sum of the changes produced by each mutation in isolation. One plausible explanation for these altered patterns of denaturation is that chain-chain or chain-solvent interactions in the denatured state have been modified--interactions which appear to involve hydrophobic groups.

Effects of denaturants at low concentrations on the reversible denaturation of staphylococcal nuclease.

Three very unstable mutant forms of staphylococcal nuclease were used to quantitate the change in the apparent equilibrium constant for reversible denaturation (Kapp) as a function of denaturant concentration for a variety of different denaturing solutes. The value of this equilibrium constant in the absence of denaturant (Kapp,0) was determined by renaturation of the mutant proteins with a combination of glycerol and calcium ion, the latter of which binds at the active site in the native conformation. Because Kapp,0 fell in the easily measurable range between 0.1 and 1, the change in Kapp, and thus the change in free energy (delta Gapp), at very low concentrations of denaturants could be accurately measured. With guanidine hydrochloride (GuHCl), the rate of change of the apparent free energy of denaturation with respect to denaturant concentration (d(delta Gapp)/dCGuHCl or mGuHCl) was found to be remarkably constant down to zero denaturant concentration, even though this value was different for each of the three proteins. Unlike GuHCl, urea exhibited a slightly reduced value of d delta Gapp/dCurea at low concentrations. Results with a number of thiocyanate, perchlorate, and iodide salts confirmed that the Hofmeister series holds for concentrations below 0.1 M; that is, with regard to efficacy as a denaturant SCN- greater than ClO4- greater than I- and Li+,NH4+ greater than Na+,K+. However, all of the chaotropic salts analyzed exhibited markedly increased values of d(delta Gapp)/dCsalt at concentrations below 0.2 M. One possible explanation for these large deviations from a linear relationship between delta Gapp and salt concentration is that weak binding or adsorption of chaotropic anions is occurring at a saturable number of sites in hydrophobic regions of the denatured state.