A.G. Rodriguez-Gabin

Role of rRAB22b, an Oligodendrocyte Protein, in Regulation of Transport of Vesicles From Trans Golgi to Endocytic Compartments

Intracellular membrane trafficking plays an essential role in the biogenesis and maintenance of myelin. Members of the Rab protein family are important components of the systems that regulate intracellular vesicle transport. We examine the function of rRab22b, a novel rat Rab protein cloned from an oligodendrocyte cDNA library, by visualizing and identifying in living Hela cells the organelles that contain rRab22b. Our results show that rRab22b is present in the trans Golgi/TGN and endocytic compartments. Trafficking of membranes from trans Golgi to endocytic compartments takes place via small tubulo vesicular organelles containing rRab22b. The formation of vesicles in the trans Golgi also appears to be regulated by rRab22b. Additionally, our results suggest that rRab22b controls the transport of vesicles from the trans Golgi to endocytic compartments that localize in oligodendrocyte processes. That rRab22b is involved in the transport of certain proteins from trans Golgi to myelin is suggested by the evidence that certain proteins being targeted to the plasma membrane are first transported from trans Golgi to endocytic compartments. J Neurosci. Res. 66:1149–1160, 2001.

Vesicle transport in oligodendrocytes: probable role of Rab40c protein.

Intracellular membrane trafficking plays an essential role in the structural and functional organization of oligodendrocytes, which synthesize a large amount of membrane to form myelin. Rab proteins are key components in intracellular vesicular transport. We cloned a novel Rab protein from an oligodendrocyte cDNA library, designating it Rab40c because of its homology with Rab40a and Rab40b. The DNA sequence of Rab40c shows an 843‐base pair open reading frame. The deduced amino acid sequence is a protein with 281 amino acids, with a molecular weight of 31,466 Da and an isoelectric point of 9.83. Rab40c presents a number of distinct structural features including a carboxyl terminal extension and amino acid substitutions in the consensus sequence of the GTP‐binding motifs. The carboxyl terminal region contains motifs that permit isoprenylation and palmitoylation. Binding studies indicate that Rab40c binds guanosine 5′‐0‐(3‐thiotriphosphate) (GTPγS) with a Kd of 21 μM and has a higher affinity for guanosine triphosphate (GTP) than for guanosine diphosphate (GDP). Rab40c is localized in the perinuclear recycling compartment, suggesting its involvement in endocytic events such as receptor recycling. The importance of this recycling in myelin formation is suggested by the increase in both Rab40c mRNA and Rab40c protein as oligodendrocytes differentiate.

Transport of mannose-6-phosphate receptors from the trans-Golgi network to endosomes requires Rab31

Rab31, a protein that we originally cloned from a rat oligodendrocyte cDNA library, localizes in the trans-Golgi network (TGN) and endosomes. However, its function has not yet been established. Here we show the involvement of Rab31 in the transport of mannose 6-phosphate receptors (MPRs) from TGN to endosomes. We demonstrate the specific sorting of cation-dependent-MPR (CD-MPR), but not CD63 and vesicular stomatitis virus G (VSVG) protein, to Rab31-containing trans-Golgi network carriers. CD-MPR and Rab31 containing carriers originate from extending TGN tubules that also contain clathrin and GGA1 coats. Expression of constitutively active Rab31 reduced the content of CD-MPR in the TGN relative to that of endosomes, while expression of dominant negative Rab31 triggered reciprocal changes in CD-MPR distribution. Expression of dominant negative Rab31 also inhibited the formation of carriers containing CD-MPR in the TGN, without affecting the exit of VSVG from this compartment. Importantly, siRNA-mediated depletion of endogenous Rab31 caused the collapse of the Golgi apparatus. Our observations demonstrate that Rab31 is required for transport of MPRs from TGN to endosomes and for the Golgi/TGN organization.

Interaction of Rab31 and OCRL-1 in oligodendrocytes: Its role in transport of mannose 6-phosphate receptors

Rab31, a protein that we cloned from an oligodendrocyte cDNA library, is required for transport of mannose 6‐phosphate receptors (MPRs) from the trans‐Golgi network (TGN) to endosomes and for Golgi/TGN organization. Here we extend the knowledge of the mechanism of action of Rab31 by demonstrating its interaction with OCRL‐1, a phosphatidylinositol 4,5‐diphosphate 5‐phosphatase (PI(4,5)P2 5‐phosphatase) that regulates the levels of PI(4,5)P2 and PI(4)P, molecules involved in transport and Golgi/TGN organization. We show that Rab31 interacts with OCRL‐1 in a yeast two‐hybrid system, GST‐Rab31 pull‐down experiments, and coimmunoprecipitation of OCRL‐1 using oligodendrocyte culture lysates. Rab31 and OCRL‐1 colocalize in the TGN, post‐TGN carriers, and endosomes. Cation‐dependent MPR (CD‐MPR) is sorted to OCRL‐1‐containing carriers, but CD63 and vesicular stomatitis virus G (VSVG) are not. siRNA‐mediated depletion of endogenous Rab31 causes collapse of the TGN apparatus and markedly decreases the levels of OCRL‐1 in the TGN and endosomes. Our observations indicate that the role of Rab31 in the Golgi/TGN structure and transport of MPRs depends on its capability to recruit OCRL‐1 to domains of the TGN where the formation of carriers occurs. The importance of our observations is highlighted by the fact that mutation of OCRL‐1 causes demyelination in humans.

Molecular analysis of the monomeric GTP-binding proteins of oligodendrocytes.

Vesicle transport plays an important role in the formation of myelin. Transport of proteins, including proteolipid protein and myelin associated glycoprotein, from their site of synthesis in the endoplasmic reticulum in the perikaryon of the oligodendrocytes, to myelin, takes place via carrier vesicles. The mechanisms that regulate vesicle transport in oligodendrocytes are largely unknown. The presence of monomeric GTP-binding proteins in myelin and oligodendrocytes suggested the hypothesis that these proteins participate in the regulation of vesicle transport. In an attempt to identify the Rab and Rho GTP-binding proteins present in oligodendrocytes, a cDNA library specific for these proteins was generated using a reverse transcriptase-polymerase chain reaction (RT-PCR) approach. Twelve different clones containing sequences that coded for members of the Rab and Rho families of GTP-binding proteins were isolated. This group includes Rab1, -1b, -2, -5b, -5c, -7, -8, -12, -14, -23 and Rho A. One additional clone revealed a novel cDNA sequence. Analysis of the effector loop motif indicated that this sequence encodes for a member of the Rab family. We refer to this new sequence as Rab0. Comparison of Rab0 with the most similar rat Rab sequences, Rab 14 and Rab 22, and with a recently cloned human Rab22b, showed a 71%, 72% and 94% identity, respectively. By RT-PCR analysis the Rab0 mRNA was found to be mainly expressed in oligodendrocytes and to a lesser extent in oligodendrocyte precursors, astrocytes and microglia. Moreover, the highest levels of Rab0 mRNA were observed in areas of the brain that are heavily myelinated. Rab0 mRNA was also detected in other tissues such as kidney, liver, skeletal muscle. These data provide initial evidence regarding signal transduction pathways that regulate intracellular transport in oligodendrocytes.

Exploiting MEK Inhibitor-Mediated Activation of ERα for Therapeutic Intervention in ER-Positive Ovarian Carcinoma

While the clinical benefit of MEK inhibitor (MEKi)-based therapy is well established in Raf mutant malignancies, its utility as a suppressor of hyperactive MAPK signaling in the absence of mutated Raf or Ras, is an area of ongoing research. MAPK activation is associated with loss of ERα expression and hormonal resistance in numerous malignancies. Herein, we demonstrate that MEKi induces a feedback response that results in ERα overexpression, phosphorylation and transcriptional activation of ER-regulated genes. Mechanistically, MEKi-mediated ERα overexpression is largely independent of erbB2 and AKT feedback activation, but is ERK-dependent. We subsequently exploit this phenomenon therapeutically by combining the ER-antagonist, fulvestrant with MEKi. This results in synergistic suppression of tumor growth, in vitro and potentiation of single agent activity in vivo in nude mice bearing xenografts. Thus, we demonstrate that exploiting adaptive feedback after MEKi can be used to sensitize ERα-positive tumors to hormonal therapy, and propose that this strategy may have broader clinical utility in ERα-positive ovarian carcinoma.

A Novel Indication for Panobinostat as a Senolytic Drug in NSCLC and HNSCC

Panobinostat (pano) is an FDA-approved histone deacetylase inhibitor. There is interest in evaluating alternate dosing schedules and novel combinations of pano for the treatment of upper aerodigestive and lung malignancies; thus we evaluated it in combination with Taxol, a chemotherapeutic with activity in both diseases. Dose-dependent synergy was observed in Non-Small Cell Lung Cancer (NSCLC) and Head and Neck Squamous Cell Carcinoma (HNSCC) cell lines and was due to senescence rather than potentiation of cell death. Senescence occurred following cisplatin- or Taxol-treatment in cell lines from both cancer types and was associated with decreased histone 3 (H3) acetylation and increased Bcl-xL expression: the latter a biomarker of senescence and target of anti-senescence therapeutics, or senolytics. Since H3 acetylation and Bcl-xL expression were altered in senescence, we subsequently evaluated pano as a senolytic in chemotherapy-treated cancer cells enriched for senescent cells. Pano caused cell death at significantly higher rates compared to repeat dosing with chemotherapy. This was associated with decreased expression of Bcl-xL and increased acetylated H3, reversing the expression patterns observed in senescence. These data support evaluating pano as a post-chemotherapy senolytic with the potential to kill persistent senescent cells that accumulate during standard chemotherapy in NSCLC and HNSCC.

Expression and signal transduction of the glucagon receptor in βTC3 cells

The expression and signal transduction of the glucagon receptor (GR) have been studied in betaTC3 cells. Northern blot and RT-PCR analysis indicated the expression of the GR gene in betaTC3 cells. One-5 nM glucagon stimulated a 2.5-fold increase in the IP(S) production. At glucagon concentrations higher than 5 nM, the production of IP(S) was blunted but not abolished. The accumulation of intracellular cAMP was observed following the stimulation with 5 nM of glucagon. A maximal 4.5-fold increase in cAMP was observed using 250 nM glucagon and higher. Comparative studies using a glucagon anatogonist, des-His1[Glu]9glucagon, showed no effect on intracellular cAMP and IPs in betaTC3 cells. Our data shows that the GR gene is expressed in betaTC3 cells. The GR in betaTC3 cells transmits its intracellular signal by causing the accumulation of both IP(S) and cAMP.

Characterization of a human βV‐tubulin antibody and expression of this isotype in normal and malignant human tissue

There are seven distinct β‐tubulin isotypes and eight α‐tubulin isotypes in mammals that are hypothesized to have tissue‐ and cell‐specific functions. There is an interest in the use of tubulin isotypes as prognostic markers of malignancy. βV‐tubulin, like βIII‐tubulin, has been implicated in malignant transformation and drug resistance, however little is known about its localization and function. Thus, we generated for the first time, a rabbit polyclonal antibody specific for human βV‐tubulin. The antibody did not cross‐react with mouse βV‐tubulin or other human β‐tubulin isotypes and specifically labeled βV‐tubulin by immunoblotting, immunofluorescence and immunohistochemistry. Immunohistochemistry of various human normal tissues revealed that βV‐tubulin was expressed in endothelial cells, myocytes and cells with muscle differentiation, structures with transport and/or secretory function such as renal tubules, pancreatic ducts and bile ducts, and epithelium with secretory function such as prostate. βV‐tubulin was also specifically expressed in pancreatic islets and intratubular germ cell neoplasia, where it may have diagnostic utility. Initial studies in breast, lung and ovarian cancers indicated aberrant expression of βV‐tubulin, suggesting that this isoform may be associated with tumorigenesis. Thus, βV‐tubulin expression is a potentially promising prognostic marker of malignancy.

Germline mutations of the DNA repair pathways in uterine serous carcinoma

OBJECTIVE Treatment options are limited for patients with uterine serous carcinoma (USC). Knowledge of USCs somatic mutation landscape is rapidly increasing, but its role in hereditary cancers remains unclear. We aim to evaluate the frequency and characteristics of germline mutations in genes commonly implicated in carcinogenesis, including those within homologous recombination (HR) and mismatch repair (MMR) pathways in patients with pure USC. METHODS By using targeted capture exome sequencing, 43 genes were analyzed in a cohort of 7 consecutive patients with paired tumor and non-tumor USC samples in our institutional tumor repository. Mutations predicted to have damaging effects on protein function are validated by Sanger Sequencing. RESULTS We found 21 germline mutations in 11 genes in our USC cohort. Five patients harbored 7 germline mutations (33.3%) within genes involved in the HR pathway, RAD51D being the most common. Four patients had 9 (42.8%) germline mutations in hereditary colon cancer genes, most commonly MLH. All patients (42.7%) who are platinum-sensitive had HR germline mutations (RAD50, NBN, ATM). Patients with HER2 overexpression (2/7, 28.6%) had germline HR mutations and were platinum-sensitive. Three patients in our cohort reported a personal history of breast cancer, one with HR germline mutation, and 2 in patients with germline mutations in HCC genes. In addition, 5 out of 7 patients had germline mutations in genes associated with growth factor signaling pathway. CONCLUSIONS A significant proportion of our cohort harbor germline mutations in DNA repair genes. This may be associated with the high rate of breast cancer in our patients and their family, and suggests a targeted cohort for genetic counseling. If validated in a larger cohort, our findings may allow clinicians to expand therapeutic options to include targeted therapies and inclusion of USC patient in preventative and genetic counseling.