Alternative splicing of pre-mRNA in cancer: focus on G protein-coupled peptide hormone receptors

Through alternative splicing, multiple different transcripts can be generated from a single gene. Alternative splicing represents an important molecular mechanism of gene regulation in physiological processes such as developmental programming as well as in disease. In cancer, splicing is significantly altered. Tumors express a different collection of alternative spliceoforms than normal tissues. Many tumor-associated splice variants arise from genes with an established role in carcinogenesis or tumor progression, and their functions can be oncogenic. This raises the possibility that products of alternative splicing play a pathogenic role in cancer. Moreover, cancer-associated spliceoforms represent potential diagnostic biomarkers and therapeutic targets. G protein-coupled peptide hormone receptors provide a good illustration of alternative splicing in cancer. The wild-type forms of these receptors have long been known to be expressed in cancer and to modulate tumor cell functions. They are also recognized as attractive clinical targets. Recently, splice variants of these receptors have been increasingly identified in various types of cancer. In particular, alternative cholecystokinin type 2, secretin, and growth hormone-releasing hormone receptor spliceoforms are expressed in tumors. Peptide hormone receptor splice variants can fundamentally differ from their wild-type receptor counterparts in pharmacological and functional characteristics, in their distribution in normal and malignant tissues, and in their potential use for clinical applications.

Glucose-dependent insulinotropic polypeptide receptors in most gastroenteropancreatic and bronchial neuroendocrine tumors

Gastrointestinal peptide hormone receptors overexpressed in neuroendocrine tumors (NET), such as somatostatin or glucagon-like peptide-1 (GLP-1) receptors, are used for in vivo tumor targeting. Unfortunately, not all NET express these receptors sufficiently.

Re-evaluating adjuvant breast cancer trials: assessing hormone receptor status by immunohistochemical versus extraction assays

BACKGROUND: Tumor levels of steroid hormone receptors, a factor used to select adjuvant treatment for early-stage breast cancer, are currently determined with immunohistochemical assays. These assays have a discordance of 10%-30% with previously used extraction assays. We assessed the concordance and predictive value of hormone receptor status as determined by immunohistochemical and extraction assays on specimens from International Breast Cancer Study Group Trials VIII and IX. These trials predominantly used extraction assays and compared adjuvant chemoendocrine therapy with endocrine therapy alone among pre- and postmenopausal patients with lymph node-negative breast cancer. Trial conclusions were that combination therapy provided a benefit to pre- and postmenopausal patients with estrogen receptor (ER)-negative tumors but not to ER-positive postmenopausal patients. ER-positive premenopausal patients required further study. METHODS: Tumor specimens from 571 premenopausal and 976 postmenopausal patients on which extraction assays had determined ER and progesterone receptor (PgR) levels before randomization from October 1, 1988, through October 1, 1999, were re-evaluated with an immunohistochemical assay in a central pathology laboratory. The endpoint was disease-free survival. Hazard ratios of recurrence or death for treatment comparisons were estimated with Cox proportional hazards regression models, and discriminatory ability was evaluated with the c index. All statistical tests were two-sided. RESULTS: Concordance of hormone receptor status determined by both assays ranged from 74% (kappa = 0.48) for PgR among postmenopausal patients to 88% (kappa = 0.66) for ER in postmenopausal patients. Hazard ratio estimates were similar for the association between disease-free survival and ER status (among all patients) or PgR status (among postmenopausal patients) as determined by the two methods. However, among premenopausal patients treated with endocrine therapy alone, the discriminatory ability of PgR status as determined by immunohistochemical assay was statistically significantly better (c index = 0.60 versus 0.51; P = .003) than that determined by extraction assay, and so immunohistochemically determined PgR status could predict disease-free survival. CONCLUSIONS: Trial conclusions in which ER status (for all patients) or PgR status (for postmenopausal patients) was determined by immunohistochemical assay supported those determined by extraction assays. However, among premenopausal patients, trial conclusions drawn from PgR status differed--immunohistochemically determined PgR status could predict response to endocrine therapy, unlike that determined by the extraction assay.

Secretin receptors in the human liver: expression in biliary tract and cholangiocarcinoma, but not in hepatocytes or hepatocellular carcinoma

BACKGROUND/AIMS: Gut hormone receptors are over-expressed in human cancer and allow receptor-targeted tumor imaging and therapy. A novel promising receptor for these purposes is the secretin receptor. The secretin receptor expression was investigated in the human liver because the liver is a physiological secretin target and because novel diagnostic and treatment modalities are needed for liver cancer. METHODS: Nineteen normal livers, 10 cirrhotic livers, 35 cholangiocarcinomas, and 45 hepatocellular carcinomas were investigated for secretin receptor expression by in vitro receptor autoradiography using (125)I-[Tyr(10)] rat secretin and, in selected cases, for secretin receptor mRNA by RT-PCR. RESULTS: Secretin receptors were present in normal bile ducts and ductules, but not in hepatocytes. A significant receptor up-regulation was observed in ductular reaction in liver cirrhosis. Twenty-two (63%) cholangiocarcinomas were positive for secretin receptors, while hepatocellular carcinomas were negative. RT-PCR revealed wild-type receptor mRNA in the non-neoplastic liver, wild-type and spliced variant receptor mRNAs in cholangiocarcinomas found receptor positive in autoradiography experiments, and no receptor transcripts in autoradiographically negative cholangiocarcinomas. CONCLUSIONS: The expression of secretin receptors in the biliary tract is the molecular basis of the secretin-induced bicarbonate-rich choleresis in man. The high receptor expression in cholangiocarcinomas may be used for in vivo secretin receptor-targeting of these tumors and for the differential diagnosis with hepatocellular carcinoma.

NPY receptors in human cancer: a review of current knowledge

Many peptide hormone receptors are over-expressed in human cancer, permitting an in vivo targeting of tumors for diagnostic and therapeutic purposes. NPY receptors are novel and promising candidates in this field. Using in vitro receptor autoradiography, Y1 and Y2 receptors have been found to be expressed in breast carcinomas, adrenal gland and related tumors, renal cell carcinomas, and ovarian cancers in both tumor cells and tumor-associated blood vessels. Pathophysiologically, tumoral NPY receptors may be activated by endogenous NPY released from intratumoral nerve fibers or tumor cells themselves, and mediate NPY effects on tumor cell proliferation and tumoral blood supply. Clinically, tumoral NPY receptors may be targeted with NPY analogs coupled with adequate radionuclides or cytotoxic agents for a scintigraphic tumor imaging and/or tumor therapy.

Targeting CCK receptors in human cancers

Gut hormone receptors can be over-expressed in several human cancers and represent the basis for receptor-targeted tumor imaging and therapy. A promising receptor for such clinical applications is the cholecystokinin receptor. Cholecystokinin receptors are expressed in numerous neuroendocrine tumors, in particular medullary thyroid carcinomas and neuroendocrine gut tumors, as well as in stromal tumors. Moreover, several radiolabeled CCK or gastrin analogs have been developed allowing to detect these tumors and their metastases in patients using in vivo cholecystokinin receptor scintigraphy, proving the feasibility of targeting CCK receptors in human tumors in vivo.

Wild-type and splice-variant secretin receptors in lung cancer: overexpression in carcinoid tumors and peritumoral lung tissue

Gastrointestinal peptide hormone receptors, like somatostatin receptors, are often overexpressed in human cancer, allowing receptor-targeted tumor imaging and therapy. A novel candidate for these applications is the secretin receptor recently identified in pancreatic and cholangiocellular carcinomas. In the present study, secretin receptors were assessed in a non-gastrointestinal tissue, the human lung. Non-small-cell lung cancers (n=26), small-cell lung cancers (n=10), bronchopulmonary carcinoid tumors (n=29), and non-neoplastic lung (n=46) were investigated for secretin receptor protein expression with in vitro receptor autoradiography, using (125)I-[Tyr(10)] rat secretin and for secretin receptor transcripts with RT-PCR. Secretin receptor protein expression was found in 62% of bronchopulmonary carcinoids in moderate to high density, in 12% of non-small cell lung cancers in low density, but not in small cell lung cancers. In tumors found to be secretin receptor positive by autoradiography, RT-PCR revealed transcripts for the wild-type secretin receptor and for novel secretin receptor splice variants. In the non-neoplastic lung, secretin receptor protein expression was observed in low density along the alveolar septa in direct tumor vicinity in cases of acute inflammation, but not in histologically normal lung. In the autoradiographically positive peritumoral lung, RT-PCR showed transcripts for the wild-type secretin receptor and for a secretin receptor spliceoform different from those occurring in lung and gut tumors. In conclusion, secretin receptors are new markers for bronchopulmonary carcinoid tumors, and represent the molecular basis for an in vivo targeting of carcinoid tumors for diagnosis and therapy. Furthermore, secretin receptors may play a role in peritumoral lung pathophysiology. Secretin receptor mis-splicing specifically occurs in tumor and non-tumor lung pathology.

Estrogen receptor β expression and androgen receptor phosphorylation correlate with a poor clinical outcome in hormone-naive prostate cancer and are elevated in castration-resistant disease

Patients with advanced prostate cancer (PC) are usually treated with androgen withdrawal. While this therapy is initially effective, nearly all PCs become refractory to it. As hormone receptors play a crucial role in this process, we constructed a tissue microarray consisting of PC samples from 107 hormone-naïve (HN) and 101 castration-resistant (CR) PC patients and analyzed the androgen receptor (AR) gene copy number and the protein expression profiles of AR, Serin210-phosphorylated AR (pAR(210)), estrogen receptor (ER)β, ERα and the proliferation marker Ki67. The amplification of the AR gene was virtually restricted to CR PC and was significantly associated with increased AR protein expression (P<0.0001) and higher tumor cell proliferation (P=0.001). Strong AR expression was observed in a subgroup of HN PC patients with an adverse prognosis. In contrast, the absence of AR expression in CR PC was significantly associated with a poor overall survival. While pAR(210) was predominantly found in CR PC patients (P<0.0001), pAR(210) positivity was observed in a subgroup of HN PC patients with a poor survival (P<0.05). Epithelial ERα expression was restricted to CR PC cells (9%). ERβ protein expression was found in 38% of both HN and CR PCs, but was elevated in matched CR PC specimens. Similar to pAR(210), the presence of ERβ in HN patients was significantly associated with an adverse prognosis (P<0.005). Our results strongly suggest a major role for pAR(210) and ERβ in HN PC. The expression of these markers might be directly involved in CR tumor growth.

FOXA1 Promotes Tumor Progression in Prostate Cancer and Represents a Novel Hallmark of Castration-Resistant Prostate Cancer

Forkhead box protein A1 (FOXA1) modulates the transactivation of steroid hormone receptors and thus may influence tumor growth and hormone responsiveness in prostate cancer. We therefore investigated the correlation of FOXA1 expression with clinical parameters, prostate-specific antigen (PSA) relapse-free survival, and hormone receptor expression in a large cohort of prostate cancer patients at different disease stages. FOXA1 expression did not differ significantly between benign glands from the peripheral zone and primary peripheral zone prostate carcinomas. However, FOXA1 was overexpressed in metastases and particularly in castration-resistant cases, but was expressed at lower levels in both normal and neoplastic transitional zone tissues. FOXA1 levels correlated with higher pT stages and Gleason scores, as well as with androgen (AR) and estrogen receptor expression. Moreover, FOXA1 overexpression was associated with faster biochemical disease progression, which was pronounced in patients with low AR levels. Finally, siRNA-based knockdown of FOXA1 induced decreased cell proliferation and migration. Moreover, in vitro tumorigenicity was inducible by ARs only in the presence of FOXA1, substantiating a functional cooperation between FOXA1 and AR. In conclusion, FOXA1 expression is associated with tumor progression, dedifferentiation of prostate cancer cells, and poorer prognosis, as well as with cellular proliferation and migration and with AR signaling. These findings suggest FOXA1 overexpression as a novel mechanism inducing castration resistance in prostate cancer.

Controversies of adjuvant endocrine treatment for breast cancer and recommendations of the 2007 St Gallen conference

Endocrine treatment for breast cancer was introduced more than a century ago. The discovery of hormone receptors has allowed targeting of endocrine treatment to patients whose primary tumours express these receptors. In the adjuvant setting, different approaches are used in premenopausal or postmenopausal women. In premenopausal patients, suppression of ovarian function and the use of tamoxifen are the most important therapeutic options, even though questions on timing, duration, and combination of these compounds remain unanswered. The use of aromatase inhibitors in combination with ovarian-function suppression is currently under investigation in the premenopausal setting. In postmenopausal patients, aromatase inhibitors given after 2-3 years or 5 years of tamoxifen have shown a significant benefit over tamoxifen alone. However, questions on this treatment also remain unanswered. For example, whether all patients should receive an aromatase inhibitor or whether some subgroups of patients might be optimally treated by tamoxifen alone is yet to be established. In this paper we review the published work on adjuvant endocrine treatment in breast cancer and provide recommendations from the 2007 St Gallen International Conference on Primary Therapy of Early Breast Cancer.

GLP-1 receptor expression in human tumors and human normal tissues: potential for in vivo targeting

Peptide hormone receptors overexpressed in human tumors, such as somatostatin receptors, can be used for in vivo targeting for diagnostic and therapeutic purposes. A novel promising candidate in this field is the GLP-1 receptor, which was recently shown to be massively overexpressed in gut and lung neuroendocrine tumors--in particular, in insulinomas. Anticipating a major development of GLP-1 receptor targeting in nuclear medicine, our aim was to evaluate in vitro the GLP-1 receptor expression in a large variety of other tumors and to compare it with that in nonneoplastic tissues. METHODS: The GLP-1 receptor protein expression was qualitatively and quantitatively investigated in a broad spectrum of human tumors (n=419) and nonneoplastic human tissues (n=209) with receptor autoradiography using (125)I-GLP-1(7-36)amide. Pharmacologic competition experiments were performed to provide proof of specificity of the procedure. RESULTS: GLP-1 receptors were expressed in various endocrine tumors, with particularly high amounts in pheochromocytomas, as well as in brain tumors and embryonic tumors but not in carcinomas or lymphomas. In nonneoplastic tissues, GLP-1 receptors were present in generally low amounts in specific tissue compartments of several organs--namely, pancreas, intestine, lung, kidney, breast, and brain; no receptors were identified in lymph nodes, spleen, liver, or the adrenal gland. The rank order of potencies for receptor binding--namely, GLP-1(7-36)amide = exendin-4 >> GLP-2 = glucagon(1-29)--provided proof of specific GLP-1 receptor identification. CONCLUSION: The GLP-1 receptors may represent a novel molecular target for in vivo scintigraphy and targeted radiotherapy for a variety of GLP-1 receptor-expressing tumors. For GLP-1 receptor scintigraphy, a low-background signal can be expected, on the basis of the low receptor expression in the normal tissues surrounding tumors.

High variability and non-neutral evolution of the mammalian avpr1a gene

BACKGROUND: The arginine-vasopressin 1a receptor has been identified as a key determinant for social behaviour in Microtus voles, humans and other mammals. Nevertheless, the genetic bases of complex phenotypic traits like differences in social and mating behaviour among species and individuals remain largely unknown. Contrary to previous studies focusing on differences in the promotor region of the gene, we investigate here the level of functional variation in the coding region (exon 1) of this locus. RESULTS: We detected high sequence diversity between higher mammalian taxa as well as between species of the genus Microtus. This includes length variation and radical amino acid changes, as well as the presence of distinct protein variants within individuals. Additionally, negative selection prevails on most parts of the first exon of the arginine-vasopressin receptor 1a (avpr1a) gene but it contains regions with higher rates of change that harbour positively selected sites. Synonymous and non-synonymous substitution rates in the avpr1a gene are not exceptional compared to other genes, but they exceed those found in related hormone receptors with similar functions. DISCUSSION: These results stress the importance of considering variation in the coding sequence of avpr1a in regards to associations with life history traits (e.g. social behaviour, mating system, habitat requirements) of voles, other mammals and humans in particular.

Insulin-like growth factor (IGF)-binding protein-3 (IGFBP-3) is closely associated with the chondrocyte nucleus in human articular cartilage

OBJECTIVE: Insulin-like growth factor-I (IGF-I) is critically involved in the control of cartilage matrix metabolism. It is well known that IGF-binding protein-3 (IGFBP-3) is increased during osteoarthritis (OA), but its function(s) is not known. In other cells, IGFBP-3 can regulate IGF-I action in the extracellular environment and can also act independently inside the cell; this includes transcriptional gene control in the nucleus. These studies were undertaken to localize IGFBP-3 in human articular cartilage, particularly within cells. DESIGN: Cartilage was dissected from human femoral heads derived from arthroplasty for OA, and OA grade assessed by histology. Tissue slices were further characterized by extraction and assay of IGFBPs by IGF ligand blot (LB) and by enzyme-linked immunosorbent assay (ELISA). Immunohistochemistry (IHC) for IGF-I and IGFBP-3 was performed on cartilage from donors with mild, moderate and severe OA. Indirect fluorescence and immunogold-labeling IHC studies were included. RESULTS: LBs of chondrocyte lysates showed a strong signal for IGFBP-3. IHC of femoral cartilage sections at all OA stages showed IGF-I and IGFBP-3 matrix stain particularly in the top zones, and closely associated with most cells. A prominent perinuclear/nuclear IGFBP-3 signal was seen. Controls using non-immune sera or antigen-blocked antibody showed negative or strongly reduced stain. In frozen sections of human ankle cartilage, immunofluorescent IGFBP-3 stain co-localized with the nuclear 4',6-diamidino-2-phenyl indole (DAPI) stain in greater than 90% of the cells. Immunogold IHC of thin sections and transmission electron immunogold microscopy of ultra-thin sections showed distinct intra-nuclear staining. CONCLUSIONS: IGFBP-3 in human cartilage is located in the matrix and within chondrocytes in the cytoplasm and nuclei. This new finding indicates that the range of IGFBP-3 actions in articular cartilage is likely to include IGF-independent roles and opens the door to studies of its nuclear actions, including the possible regulation of hormone receptors or transcriptional complexes to control gene action.

Does somatostatin or gastric inhibitory peptide receptor expression correlate with tumor grade and stage in gut neuroendocrine tumors?

BACKGROUND/AIMS Important characteristics of neuroendocrine neoplasms (NEN) for prognosis and therapeutic decisions are the MIB-1 proliferative index (tumor grade) and tumor stage. Moreover, these tumors express peptide hormone receptors like somatostatin and gastric inhibitory peptide (GIP) receptors which represent important established and potential future targets, respectively, for molecular imaging and radiotherapy. However, the interrelation between tumor proliferation, stage, and peptide receptor amounts has never been assessed. METHODS In 114 gastrointestinal and bronchopulmonary NEN, the proliferative rate assessed with MIB-1 immunohistochemistry and tumor stage were compared with the somatostatin type 2 receptor (sst2) and GIP receptor expression measured quantitatively with in vitro receptor autoradiography. RESULTS NEN generally showed high sst2 and GIP receptor expression. GIP receptor but not sst2 expression correlated with the MIB-1 index. GIP receptor levels gradually increased in a subset of insulinomas and nonfunctioning pancreatic NEN, and decreased in ileal and bronchopulmonary NEN with increasing MIB-1 rate. MIB-1 levels were identified, above which GIP receptor levels were consistently high or low. These MIB-1 levels were clearly different from those defining tumor grade. In grade 3 NEN, GIP receptor levels were always low, while sst2 levels were variable and sometimes extremely high. Conversely, sst2 expression correlated more frequently with tumor stage than GIP receptor expression, with metastasized NEN showing higher sst2 levels than localized tumors. CONCLUSIONS sst2, a clinically crucial molecular target, shows variable and unpredictable expression in NEN irrespective of tumor grade. Therefore, each NEN should be tested for sst2 if clinical applications with somatostatin analogs are considered. Conversely, the potential future role of GIP receptors as molecular targets in NEN may be dependent on the MIB-1 level.

Thyroid disruption in zebrafish (Danio rerio) larvae: Different molecular response patterns lead to impaired eye development and visual functions

The vertebrate thyroid system is important for multiple developmental processes, including eye development. Thus, its environmentally induced disruption may impact important fitness-related parameters like visual capacities and behaviour. The present study investigated the relation between molecular effects of thyroid disruption and morphological and physiological changes of eye development in zebrafish (Danio rerio). Two test compounds representing different molecular modes of thyroid disruption were used: propylthiouracil (PTU), which is an enzyme-inhibitor of thyroid hormone synthesis, and tetrabromobisphenol A (TBBPA), which interacts with the thyroid hormone receptors. Both chemicals significantly altered transcript levels of thyroid system-related genes (TRα, TRβ, TPO, TSH, DIO1, DIO2 and DIO3) in a compound-specific way. Despite these different molecular response patterns, both treatments resulted in similar pathological alterations of the eyes such as reduced size, RPE cell diameter and pigmentation, which were concentration-dependent. The morphological changes translated into impaired visual performance of the larvae: the optokinetic response was significantly and concentration-dependently decreased in both treatments, together with a significant increase of light preference of PTU-treated larvae. In addition, swimming activity was impacted. This study provides first evidence that different modes of molecular action of the thyroid disruptors can be associated with uniform apical responses. Furthermore, this study is the first to show that pathological eye development, as it can be induced by exposure to thyroid disruptors, indeed translates into impaired visual capacities of zebrafish early life stages.