Jose S. Subauste

RACK1 downregulates levels of the pro-apoptotic protein Fem1b in apoptosis-resistant colon cancer cells.

Evasion of apoptosis plays an important role in colon cancer progression. Following loss of the Apc tumor suppressor gene in mice, the gene encoding Fem1b is up-regulated early in neoplastic intestinal epithelium. Fem1b is a pro-apoptotic protein that interacts with Fas, TNFR1, and Apaf-1, and increased expression of Fem1b induces apoptosis of cancer cells. Fem1b is a homolog of FEM-1, a protein in Caenorhabditis elegans that is negatively regulated by ubiquitination and proteasomal degradation. To study Fem1b regulation in colon cancer progression, we used apoptotis-sensitive SW480 cells, derived from a primary colon cancer, and their isogenic, apoptosis-resistant counterparts SW620 cells, derived from a subsequent metastatic lesion in the same patient. Treatment with proteasome inhibitor increased Fem1b protein levels in SW620 cells, but not in SW480 cells. In SW620 cells we found that endogenous Fem1b co-immunoprecipitates in complexes with RACK1, a protein known to mediate ubiquitination and proteasomal degradation of other pro-apoptotic proteins and to be up-regulated in colon cancer. Full-length Fem1b, or the N-terminal region of Fem1b, associated with RACK1 when co-expressed in HEK293T cells, and RACK1 stimulated ubiquitination of Fem1b. RACK1 over-expression in SW620 cells led to down-regulation of Fem1b protein levels. Conversely, down-regulation of RACK1 led to up-regulation of Fem1b protein levels, associated with induction of apoptosis, and this apoptosis was inhibited by blocking Fem1b protein up-regulation. In conclusion, RACK1 down-regulates levels of the pro-apoptotic protein Fem1b in metastatic, apoptosis-resistant colon cancer cells, which may promote apoptosis-resistance during progression of colon cancer.

Abnormal Glucose Homeostasis and Pancreatic Islet Function in Mice with Inactivation of the Fem1b Gene

ABSTRACT Type 2 diabetes mellitus is a disorder of glucose homeostasis involving complex gene and environmental interactions that are incompletely understood. Mammalian homologs of nematode sex determination genes have recently been implicated in glucose homeostasis and type 2 diabetes mellitus. These are the Hedgehog receptor Patched and Calpain-10, which have homology to the nematode tra-2 and tra-3 sex determination genes, respectively. Here, we have developed Fem1b knockout (Fem1b-KO) mice, with targeted inactivation of Fem1b, a homolog of the nematode fem-1 sex determination gene. We show that the Fem1b-KO mice display abnormal glucose tolerance and that this is due predominantly to defective glucose-stimulated insulin secretion. Arginine-stimulated insulin secretion is also affected. The Fem1b gene is expressed in pancreatic islets, within both β cells and non-β cells, and is highly expressed in INS-1E cells, a pancreatic β-cell line. In conclusion, these data implicate Fem1b in pancreatic islet function and insulin secretion, strengthening evidence that a genetic pathway homologous to nematode sex determination may be involved in glucose homeostasis and suggesting novel genes and processes as potential candidates in the pathogenesis of diabetes mellitus.

Calcium abnormalities in hospitalized patients.

Abstract Depending upon the method of measurement, hypocalcemia occurs in 15% to 88% and hypercalcemia occurs in 15% of hospitalized patients. Ionized calcium should be measured in place of total serum calcium to avoid errors related to hypoalbuminemia, which is seen commonly in acutely ill patients. Symptomatic hypocalcemia requires prompt intravenous calcium administration. Symptomatic hypercalcemia (most often caused by hyperparathyroidism or malignancy) warrants aggressive intravenous hydration to correct volume depletion and, usually, additional therapy with diuretics, bisphosphonates, calcitonin, or corticosteroids. Identification and treatment of the underlying cause of the calcium derangement must be addressed after the acute electrolyte abnormality is stabilized.

11Beta-hydroxylase deficiency and other syndromes of mineralocorticoid excess as a rare cause of endocrine hypertension.

Hypertension represents a major public and global health problem, most of which likely can be improved by lifestyle changes including changing dietary habits with less consumption of processed and preserved foods, which generally contain higher amounts of salt than freshly prepared food items. Among causes for endocrine hypertension are syndromes of mineralocorticoid excess. This group of mostly monogenic and acquired disorders typically causes hypertension through activation of the mineralocorticoid receptor either directly or indirectly via hormonal mediators and from overactive amiloride-sensitive epithelial sodium channels located in the distal tubule and collecting ducts of the kidneys. Apart from primary aldosteronism, mineralocorticoid excess can be caused by congenital adrenal hyperplasia (CAH) due to mutations of the 11beta-hydroxylase and 17alpha-hydroxylase genes, by inactivating mutations of the glucocorticoid receptor gene (Chrousos syndrome), endogenous hypercortisolism (Cushings syndrome), by mutations of the 11beta-hydroxysteroid dehydrogenase type 2 gene (apparent mineralocorticoid excess/AME) or licorice/carbenoxolone intake, mutations of the epithelial sodium channel genes (Liddle syndrome), mutations of the mineralocorticoid receptor gene (Geller syndrome), and by mutations in the WNK1, WNK4, KLHL3, CUL3 genes (pseudohypoaldosteronism type 2 or Gordon syndrome). Most of these conditions are treated by restricting dietary salt intake. However, some require special therapies including dexamethasone/hydrocortisone (CAH), spironolactone/eplerenone (AME), epithelial sodium channel inhibitors amiloride/triamterene (Liddle and Gordon syndrome), while in others spironolactone and MR antagonists may be contraindicated due to an abnormally structured MR (Geller syndrome). We here review the pathophysiology, diagnosis, and therapy of these rare conditions including the presentation of a patient with 11beta-hydroxylase deficiency.

Dimerization Interfaces of v-ErbA Homodimers and Heterodimers with Retinoid X Receptor α

The oncoprotein v-ErbA, a member of the zinc finger transcription factor superfamily, is a mutated version of thyroid hormone receptor α1 that is virtually incapable of binding T3. v-ErbA and other members of this family can bind as homodimers and heterodimers with retinoid X receptors to specific DNA sequences arranged as direct, inverted, or everted repeats. At least two regions in the C-terminal domain, the I box (10 and 11 helices in v-ErbA and thyroid hormone receptors) and the 20-amino acid region are involved in dimerization. However, it has not been entirely understood how these receptors dimerize on differently oriented core motifs and whether the domain(s) responsible for homodimerization and heterodimerization are identical. Therefore, deletions of the entire 20-amino acid region, the 10 helix, the 11 helix, and point mutations within these regions of v-ErbA were made by site-directed mutagenesis. The mutant proteins were tested for their ability to form v-ErbA homodimers and heterodimers with retinoid X receptor α on differently oriented core motifs by electrophoretic mobility shift assay. Transient transfections were performed to determine the dominant negative activity of the v-ErbA mutants. The data indicate that different dimerization interfaces are used for v-ErbA homodimerization and heterodimerization with retinoid X receptor α, and different dimerization interfaces are used on differently oriented core motifs. The data are of general interest because the information improves our understanding of the role of these dimerization interfaces in the mechanism of action not only of v-ErbA but also of other members of the superfamily.

FEM1A is a candidate gene for polycystic ovary syndrome.

Polycystic ovary syndrome (PCOS) is the most common endocrine disorder among women of reproductive age, and is characterized by infertility, hyperandrogenism and insulin resistance in skeletal muscle. There is evidence for a PCOS gene localized to chromosome 19p13.3. The FEMIA gene maps to chromosome 19p13.3 and is highly expressed in skeletal muscle. FEMIA is a homolog of fem-1, a sex-determination gene of Caenorhabditis elegans that controls masculinization. In a pilot study of Caucasian PCOS patients from our local clinic, we found that one of these five patients exhibited a heterozygous germline missense mutation in FEM1A, designated FEM1A*H500Y. This mutation alters an amino acid conserved from human to C. elegans, and was not found in any of 198 control chromosomes. This missense allele was not found in any of a separate group of 30 PCOS patients from a different regional/ethnic background. Immunostaining of mouse ovary demonstrated that the mouse homolog of FEM1A is expressed in androgen-producing secondary interstitial cells, with a marked increase in expression after puberty, consistent with a key feature of PCOS – ovarian hyperandrogenism. In conclusion, FEM1A should be considered a candidate gene for PCOS, and more extensive analysis of FEM1A, both coding and regulatory sequences, is warranted in patients and families with PCOS.

Retinoic acid regulates several genes in bile acid and lipid metabolism via upregulation of small heterodimer partner in hepatocytes.

Retinoic acid (RA) affects multiple aspects of development, embryogenesis and cell differentiation processes. The liver is a major organ that stores RA suggesting that retinoids play an important role in the function of hepatocytes. In our previous studies, we have demonstrated the involvement of small heterodimer partner (SHP) in RA-induced signaling in a non-transformed hepatic cell line AML 12. In the present study, we have identified several critical genes in lipid homeostasis (Apoa1, Apoa2 and ApoF) that are repressed by RA-treatment in a SHP dependent manner, in vitro and also in vivo with the use of the SHP null mice. In a similar manner, RA also represses several critical genes involved in bile acid metabolism (Cyp7a1, Cyp8b1, Mdr2, Bsep, Baat and Ntcp) via upregulation of SHP. Collectively our data suggest that SHP plays a major role in RA-induced potential changes in pathophysiology of metabolic disorders in the liver.

The Fem1a Gene Is Downregulated in Rhabdomyosarcoma

Rhabdomyosarcoma (RMS) is the most common soft tissue neoplasm of children, and those metastatic at presentation have a poor prognosis. RMS development is related to defective skeletal muscle differentiation, involving a variety of cell signaling and transcriptional control pathways, including aberrant hedgehog signaling. Here we evaluate Fem1a, a gene highly expressed in skeletal muscle, as a candidate for involvement in RMS. Fem1a is a homolog of fem-1, which controls cell fate decisions in the sex determination pathway of Caenorhabditis elegans, a pathway with homology to mammalian hedgehog signaling. We show that Fem1a expression is activated during myocyte differentiation of C2C12 myoblasts, and this expression is largely confined to the terminally differentiating pool, not to the satellite-cell-like quiescent reserve cell pool. We find that the human homolog, FEM1A, is downregulated in all of 8 different human RMS cell lines, including those derived from embryonal and alveolar RMS. Using mouse genetic models of RMS development, we further show that Fem1a is consistently downregulated in primary RMS from Ptch1+/– mice, from p53–/– mice, from p53+/–; Ptch1+/– mice, and from HGF/SF-Ink4a/Arf–/– mice. Therefore, Fem1a downregulation may be involved in, and/or a marker of, an early cell fate defect fundamental to RMS pathogenesis.

Clustering of sebaceous gland carcinoma, papillary thyroid carcinoma and breast cancer in a woman as a new cancer susceptibility disorder: a case report

IntroductionMultiple distinct tumors arising in a single individual or within members of a family raise the suspicion of a genetic susceptibility disorder.Case presentationWe present the case of a 52-year-old Caucasian woman diagnosed with sebaceous gland carcinoma of the eyelid, followed several years later with subsequent diagnoses of breast cancer and papillary carcinoma of the thyroid. Although the patient was also exposed to radiation from a pipe used in the oil field industry, the constellation of neoplasms in this patient suggests the manifestation of a known hereditary susceptibility cancer syndrome. However, testing for the most likely candidates such as Muir-Torre and Cowden syndrome proved negative.ConclusionWe propose that our patients clustering of neoplasms either represents a novel cancer susceptibility disorder, of which sebaceous gland carcinoma is a characteristic feature, or is a variant of the Muir-Torre syndrome.

Modulation of expression of RA-regulated genes by the oncoprotein v-erbA

Retinoic acid (RA) modulates the expression of genes involved in embryogenesis, development and differentiation processes in vertebrates. The v-erbA oncogene is known to exert a dominant-negative effect on the expression of RA-responsive genes. v-erbA belongs to a superfamily of transcription factors called nuclear receptors, which includes the retinoic acid receptors (RARs) responsible for mediating the effects of retinoic acid. While RA inhibits cell proliferation and promotes cell differentiation and apoptosis in a variety of tissues, v-erbA seems to play a role in oncogenesis, namely in the development of hepatocellular carcinoma (HCC) in a transgenic mouse model. In order to study the effect of v-erbA on RA-responsive genes, we used microarray analysis to identify genes differentially expressed in murine hepatocytes in culture (AML12 cells) stably transfected with v-erbA and exposed to RA for 3 h or 24 h. We have identified RA-responsive genes that are affected by v-erbA, as well as genes that are regulated by v-erbA alone. We have found that v-erbA can affect gene expression in the presence of RA and at the level of basal transcription. We have also identified a number of v-erbA-responsive genes that are known to be involved in carcinogenesis and which may play a role in the development of HCC.