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The natural product (+)-discodermolide (DDM) is a microtubule stabilizing agent and potent inducer of senescence. We refined the structure of DDM and evaluated the activity of novel congeners in triple negative breast and ovarian cancers, malignancies that typically succumb to taxane resistance. Previous structure-activity analyses identified the lactone and diene as moieties conferring anticancer activity, thus identifying priorities for the structural refinement studies described herein. Congeners possessing the monodiene with a simplified lactone had superior anticancer efficacy relative to taxol, particularly in resistant models. Specifically, one of these congeners, B2, demonstrated 1) improved pharmacologic properties, specifically increased maximum response achievable and area under the curve, and decreased EC(50); 2) a uniform dose-response profile across genetically heterogeneous cancer cell lines relative to taxol or DDM; 3) reduced propensity for senescence induction relative to DDM; 4) superior long-term activity in cancer cells versus taxol or DDM; and 5) attenuation of metastatic characteristics in treated cancer cells. To contrast the binding of B2 versus DDM in tubulin, X-ray crystallography studies revealed a shift in the position of the lactone ring associated with removal of the C2-methyl and C3-hydroxyl. Thus, B2 may be more adaptable to changes in the taxane site relative to DDM that could account for its favorable properties. In conclusion, we have identified a DDM congener with broad range anticancer efficacy that also has decreased risk of inducing chemotherapy-mediated senescence. SIGNIFICANCE STATEMENT: Here, we describe the anticancer activity of novel congeners of the tubulin-polymerizing molecule (+)-discodermolide. A lead molecule is identified that exhibits an improved dose-response profile in taxane-sensitive and taxane-resistant cancer cell models, diminished risk of chemotherapy-mediated senescence, and suppression of tumor cell invasion endpoints. X-ray crystallography studies identify subtle changes in the pose of binding to β-tubulin that could account for the improved anticancer activity. These findings support continued preclinical development of discodermolide, particularly in the chemorefractory setting.

Objective Treatment options are limited for patients with uterine serous carcinoma (USC). Knowledge of USC's 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.

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. © 2001 Wiley-Liss, Inc.

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.

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.

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, muscle and cells with muscle differentiation, structures with transport and/or secretary function such as renal tubules, pancreatic ducts and bile ducts, and epithelium with secretary function such as prostate. βV-tubulin was also specifically expressed in pancreatic islets and intratubular germ cell neoplasia, where it may have diagnostic utility. In a small number of malignancies, breast, lung and ovarian cancers, βV-tubulin was aberrantly expressed, suggesting that this isoform may be associated with tumorigenesis. Thus, βV-tubulin expression is a potentially promising prognostic marker of malignancy.

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)P(2) 5-phosphatase) that regulates the levels of PI(4,5)P(2) 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.

Background: Activated MAPK and PI3K/AKT/mTOR pathway signaling are associated with poor prognosis in triple negative Breast cancer (TNBC). Therefore, suppression of both arms of the MAPK/PI3K/AKT/mTOR network is a promising strategy for targeting TNBC. Here we explore the anti-tumor effects of combinations of MEK inhibitor with PI3K, AKT, or mTOR inhibitors with a focus on cell fate and biomarker development in two major subtypes of TNBC, basal-like and mesenchymal. Methods: Combinations of a MEK inhibitor with a PI3K inhibitor, AKT inhibitor, dual mTORC 1/2 inhibitor, or rapalog were evaluated in TNBC cell lines and an orthotopically implanted patient-derived xenograft (PDX) model of TNBC. We utilized reverse-phase protein array (RPPA) and quantitative real-time PCR to interrogate the signaling architecture and transcriptional activity of the treated cell lines. Results: All combinations synergistically suppress the growth of basal-like and mesenchymal TNBC models. RPPA confirms that all combinations suppress common network targets including pERK Th202/T204, pPRAS40 T246, pS6rp S235/236, and p4E-BP1 S65 in all cell lines. Notably, however, comparable repression of MAPK/PI3K/AKT/mTOR signaling produces distinct fates in basal-like and mesenchymal subtypes. Basal-like cell lines preferentially undergo delayed cell death with surviving cells displaying profound growth arrest. In contrast, mesenchymal cell lines respond with uniform quiescence exhibiting little or no cell death. Transcriptional analysis corroborates these phenotypic effects demonstrating differential modulation of genes regulating apoptosis and proliferation in basal-like versus mesenchymal cell lines. Drug treated mesenchymal cells exhibit transcriptional ‘reprogramming’ of epithelial-mesenchymal status as evidenced by reduced mesenchymal gene expression inferring potential effects on invasion and metastasis. In a PDX model of basal-like TNBC that is paclitaxel resistant, early and sustained suppression of tumor growth was achieved by treating once daily with the MEK inhibitor, GDC-0973, in combination with either the PI3K inhibitor, GDC-0941, or the rapalog, Temsirolimus. Concurrent dosing on this intense schedule was associated with some toxicity that could likely be diminished with dose modifications. Conclusions: These data highlight the therapeutic potential of combined MEK and PI3K/AKT/mTOR inhibition in chemo-resistant TNBC. Importantly, they demonstrate innate differences in the response of basal-like and mesenchymal subtypes of TNBC to these combinations, supporting the concept that molecular subtype will be an important predictor of response both in this setting and for other targeted therapies. The combination of GDC-0973 and GDC-0941 performed particularly well in a taxane resistant PDX model and deserves further evaluation in clinical trials for the treatment of TNBC. Citation Format: Sarah J. Schweber, Alicia Rodriguez-Gabin, Jinghang Zhang, Valerie Calvert, Huiping Liu, Emanuel M. Petricoin, III, Eleni Andreopoulou, Susan Band Horwitz, Hayley M. McDaid. Proteomic and transcriptional profiling reveal differential responses to combined MEK and PI3K-mTOR network inhibition in basal-like and mesenchymal subtypes of triple negative breast cancer. [abstract]. In: Proceedings of the AACR Special Conference: Targeting the PI3K-mTOR Network in Cancer; Sep 14-17, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(7 Suppl):Abstract nr A35.

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. © 2004 Wiley-Liss, Inc.

Objectives: Our objective is to determine the effect of PI3K activity in mediating ligand-independent estrogen signaling and endocrine sensitivity in Uterine Papillary Serous Carcinoma (UPSC). Methods: Drug effect on cell proliferation was calculated via Calcusyn in patient-derived UPSC cell lines ARK 1 and 2. PI3K mutation was analyzed after laser capture microdissection of tumor DNA in 7 consecutive patients with UPSC. Protein expression, via Western Blot, was correlated prospectively with clinical parameters. Results: Table 1 shows cells line baseline characteristics and IC50 to drugs. Fulvestrant, an ER antagonist, rendered the cells significantly more resistant to taxol in ARK1 and 2(p=0.035, 0.021 respectively). The concomitant decrease in pERS167 and pAKTS473 with Fulvestrant treatment is independent of baseline ER expression and PI3K mutation. In ARK2, a cell line that is ER-, disrupting AKT signaling via the PI3K inhibitor GDC-0941, or with the lapatinib is synergistic with Fulvestrant with Combination Index (CI)50 of 0.441 and 0.229, respectively. Independent of HER2 amplification or PI3K mutation, lapatinib and GDC-0941 exhibited synergistic cytotoxicity in both UPSC cell lines (CI50 of 0.577, ARK 1 and 0.233, ARK2). Finally, PI3K mutation and pAKTS473 expression was analyzed in 7 tumor samples. While none harbored PI3K mutation, patients who are chemotherapy resistant had significantly lower expression of baseline pAKTS473 in their tumor samples than those who are chemosensitive, similar to our cell line data. Conclusions: PI3K pathway dysregulation, either via upstream erbB amplification, or downstream constitutive activation of AKT, may be important in mediating endocrine and taxol sensitivity in UPSC. pAKTS473 may be an important biomarker of drug sensitivity. The combination of PI3K inhibitor and lapatinib is synergistic and warrant further therapeutic investigation. Baseline characteristics and drug sensitivities in UPSC cell linesARK1ARK2PI3KCA mutationfExon 9NoERα (WB)YesNopERα167 (WB)YesNopHER (WB)NoYesIC50 nM (mean ± STD)GDC-094193.1±5.9271±116.5LapatinibNo effect135.6±29.2FulvestrantNo effectNo effectTaxol4.6±1.97.8±5.6Cisplatin1103±65.72632.6±1797.9 Citation Format: Kelly S. Levano, Alicia Rodriguez-Gabin, Gary L. Goldberg, Susan B. Horwitz, June Y. Hou. The PI3K inhibitor GDC-0941 is synergistic with lapatinib, and mediates endocrine sensitivity in uterine papillary serous carcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4687. doi:10.1158/1538-7445.AM2014-4687

Intracellular membrane trafficking plays an essential role in the biogenesis and maintenance of myelin and regulation is necessary to maintain the structural and functional organization of the oligodendrocytes. Exocytic events are involved in the targeting of proteins synthesized in the endoplasmic reticulum such as proteolipid protein, DM20 and MAGs. Endocytic events also play an important part in the formation and maintenance of myelin as indicated by endocytosis in oligodendrocyte processes and myelin domains. Rab proteins are key components in mechanisms that regulate intracellular membrane trafficking. We cloned a novel oligodendrocyte Rab protein, designated Rab40c from its homology with Rab40. The cDNA sequence of Rab40c and the deduced amino acid sequence show an 843 base open reading frame indicating a protein with 281 amino acids. This protein has a deduced molecular of weight 31 466 Da and an isoelectric point of 9.83. Rab40c presents a number of distinct structural features including a carboxyl terminal extension and the occurrence of amino acid substitutions in the consensus sequence of the GTP-binding motifs. The carboxyl terminal region contains motifs that indicated possible isoprenylation and palmitoylation. Rab40c is localized in the perinuclear endocytic recycling compartment suggesting that it is involved in the regulation of endocytic events such as the recycling of receptors. The importance of this recycling in myelin formation is indicated by the increase in Rab40c mRNA with differentiation of oligodendrocytes.

The expression and signal transduction of the glucagon receptor (GR) have been studied in β TC3 cells. Northern blot and RT-PCR analysis indicated the expression of the GR gene in β TC3 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-His 1 [Glu] 9 glucagon, showed no effect on intracellular cAMP and IPs in β TC3 cells. Our data shows that the GR gene is expressed in β TC3 cells. The GR in β TC3 cells transmits its intracellular signal by causing the accumulation of both IP s and cAMP.