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PDF file - 515K, Members of the ErbB4 pathway. Genes found to be differentially expressed in one or more of the MCF-7 cell lines are shaded

Mouse mammary tumor virus (MMTV) is associated primarily with mammary carcinomas and lymphomas. The signal peptide of the MMTV envelope precursor is uniquely targeted to nucleoli of cells that harbor the virus, where it can function as a nuclear export factor for intron-containing transcripts. Antibodies to this signal peptide, which we refer to as p14, were previously shown to label nucleoli in a subset of human breast cancers. To look for additional cellular functions of p14, different mutants were ectopically expressed in the MCF-7 human breast cancer cell line. This approach identified motifs responsible for its nucleolar targeting, nucleocytoplasmic shuttling, target protein (B23, nucleophosmin) binding, and phosphorylation at serine 18 and 65 both in situ and in vitro. To test the role of these phosphorylation sites, we carried out in vivo tumorigenesis studies in severe combined immunodeficient mice. The findings show that the p14-Ser65Ala mutation is associated with impaired tumorigenicity, whereas the p14-Ser18Ala mutation is associated with enhanced tumorigenicity. Microarray analysis suggests that phosphorylation at serine 18 or at serine 65 is associated with transcriptional regulation of the L5 nucleolar ribosomal protein (a p14 target) and the Erb-B signal transduction pathway. Taken together, these results show that the phosphorylation status of p14 determines whether it functions as a pro-oncogenic or antioncogenic modulator. Mol Cancer Res; 10(8); 1077–86. ©2012 AACR.

XLS file - 23K, 142 genes which are transcriptionally regulated (up, down) by the PKC- vs CK2- mutant p14s

PDF file - 69K, Supplemental Table 1 Primers used to construct plasmids expressing point mutations in the p14 sequence Supplemental Table 2 Primers used to construct plasmids expressing deletions in the p14 sequence

Excessive RTK signaling, often caused by activating mutations in Ras, Raf and/or MEK, occurs in most human tumors. Intriguingly, confirmed cancer-driver mutations in the downstream effector kinase, ERK, have not been reported. To test if... Receptor tyrosine kinase signaling plays prominent roles in tumorigenesis, and activating oncogenic point mutations in the core pathway components Ras, Raf, or MEK are prevalent in many types of cancer. Intriguingly, however, analogous oncogenic mutations in the downstream effector kinase ERK have not been described or validated in vivo. To determine if a point mutation could render ERK intrinsically active and oncogenic, we have assayed in Drosophila the effects of a mutation that confers constitutive activity upon a yeast ERK ortholog and has also been identified in a few human tumors. Our analyses indicate that a fly ERK ortholog harboring this mutation alone (RolledR80S), and more so in conjunction with the known sevenmaker mutation (RolledR80S+D334N), suppresses multiple phenotypes caused by loss of Ras-Raf-MEK pathway activity, consistent with an intrinsic activity that is independent of upstream signaling. Moreover, expression of RolledR80S and RolledR80S+D334N induces tissue overgrowth in an established Drosophila cancer model. Our findings thus demonstrate that activating mutations can bestow ERK with pro-proliferative, tumorigenic capabilities and suggest that Drosophila represents an effective experimental system for determining the oncogenicity of ERK mutants and their response to therapy.

Receptor tyrosine kinase signaling plays prominent roles in tumorigenesis, and activating oncogenic point mutations in the core pathway components Ras, Raf, or MEK are prevalent in many types of cancer. Intriguingly, however, analogous oncogenic mutations in the downstream effector kinase ERK have not been described or validated in vivo. To determine if a point mutation could render ERK intrinsically active and oncogenic, we have assayed in Drosophila the effects of a mutation that confers constitutive activity upon a yeast ERK ortholog and has also been identified in a few human tumors. Our analyses indicate that a fly ERK ortholog harboring this mutation alone (Rolled(R80S)), and more so in conjunction with the known sevenmaker mutation (Rolled(R80S+D334N)), suppresses multiple phenotypes caused by loss of Ras-Raf-MEK pathway activity, consistent with an intrinsic activity that is independent of upstream signaling. Moreover, expression of Rolled(R80S) and Rolled(R80S+D334N) induces tissue overgrowth in an established Drosophila cancer model. Our findings thus demonstrate that activating mutations can bestow ERK with pro-proliferative, tumorigenic capabilities and suggest that Drosophila represents an effective experimental system for determining the oncogenicity of ERK mutants and their response to therapy.

Central nervous system acute lymphoblastic leukemia (CNS-ALL) is a major clinical problem. Prophylactic therapy is neurotoxic, and a third of the relapses involve the CNS. Increased expression of interleukin 15 (IL-15) in leukemic blasts is associated with increased risk for CNS-ALL. Using in vivo models for CNS leukemia caused by mouse T-ALL and human xenografts of ALL cells, we demonstrate that expression of IL-15 in leukemic cells is associated with the activation of natural killer (NK) cells. This activation limits the outgrowth of leukemic cells in the periphery, but less in the CNS because NK cells are excluded from the CNS. Depletion of NK cells in NOD/SCID mice enabled combined systemic and CNS leukemia of human pre-B-ALL. The killing of human leukemia lymphoblasts by NK cells depended on the expression of the NKG2D receptor. Analysis of bone marrow (BM) diagnostic samples derived from children with subsequent CNS-ALL revealed a significantly high expression of the NKG2D and NKp44 receptors. We suggest that the CNS may be an immunologic sanctuary protected from NK-cell activity. CNS prophylactic therapy may thus be needed with emerging NK cell-based therapies against hematopoietic malignancies.

Multi-drug resistance (MDR) is still a major cause of the eventual failure of chemotherapy in cancer treatment. Different approaches have been taken to render these cells drug sensitive. Here, we attempted sensitizing drug-resistant cells from within, using a translocating immune conjugate approach. To that effect, a monoclonal antibody, C219, directed against the intracellular ATP-binding site of the membrane-anchored MDR transporter ABCB1 [P-glycoprotein (P-gp), MDR1], was conjugated to human immunodeficiency virus [HIV(37-72)Tat] translocator peptide through a disulfide bridge. Fluorescence-labelled IgG-Tat conjugates accumulated in drug resistant Chinese hamster ovary (CHO) cells within less than 20 min. Preincubation with C219-S-S-(37-72)Tat conjugate augmented calcein accumulation in drug-resistant CHO and mouse lymphoma cells, indicating reduction in ABCB1 transporter activity. A thioether conjugate C219-S-(37-72)Tat was ineffective, as were disulfide and thioether conjugates of an irrelevant antibody. Furthermore, in the presence of C219-S-S-(37-72)Tat, drug resistant cells were sensitized to colchicine and doxorubicin. Taken together, these findings demonstrate, as proof of principle, a novel approach for the reversal of MDR from within cells, by delivery of translocating immune conjugates as sensitizing agents towards chemotherapy.

Mouse Mammary Tumor Virus (MMTV) causes mammary carcinoma or lymphoma in mice. An increasing body of evidence in recent years supports its involvement also in human sporadic breast cancer. It is thus of importance to develop new strategies to impair the development, growth and metastasis of MMTV-associated cancers. The signal peptide of the envelope precursor protein of this virus: MMTV-p14 (p14) is an excellent target for such strategies, due to unique characteristics distinct from its regular endoplasmic reticulum targeting function. These include cell surface expression in: murine cancer cells that harbor the virus, human breast cancer (MCF-7) cells that ectopically express p14, as well as cultured human cells derived from an invasive ductal breast carcinoma positive for MMTV sequences. These findings support its use in signal peptide-based immune targeting. Indeed, priming and boosting mice with p14 elicits a specific anti-signal peptide immune response sufficient for protective vaccination against MMTV-associated tumors. Furthermore, passive immunization using a combination of anti-p14 monoclonal antibodies or the transfer of T-cells from immunized mice (Adoptive Cell Transfer) is also therapeutically effective. With reports demonstrating involvement of MMTV in human breast cancer, we propose the immune-mediated targeting of p14 as a strategy for prevention, treatment and diagnosis of MMTV-associated cancers.

We have previously described two nucleolar proteins, named p14 and p21, in MMTV-induced T cell lymphomas. These proteins were identified by a monoclonal antibody (M-66) generated from a nontumorigenic, immunogenic variant of S49 T cell lymphoma. While p14 was common to several MMTV-derived T cell lymphomas, p21 was found only in highly tumorigenic variants of S49 cells. Here we report that p14 is the leader peptide of the MMTV env precursor. The epitope recognized by M-66 contains a putative nuclear localization signal. Actinomycin D was found to induce redistribution of p14/p21 from the nucleolus to the nucleoplasm. p14 coimmunoprecipitated and colocalized with the cellular protein, B23. Association with B23 has been previously reported for other auxiliary nucleolar retroviral proteins, such as Rev (HIV) and Rex (HTLV).

Mouse mammary tumor virus (MMTV) is associated primarily with mammary carcinomas and lymphomas. The signal peptide of the MMTV envelope precursor is uniquely targeted to nucleoli of cells that harbor the virus, where it can function as a nuclear export factor for intron-containing transcripts. Antibodies to this signal peptide, which we refer to as p14, were previously shown to label nucleoli in a subset of human breast cancers. To look for additional cellular functions of p14, different mutants were ectopically expressed in the MCF-7 human breast cancer cell line. This approach identified motifs responsible for its nucleolar targeting, nucleocytoplasmic shuttling, target protein (B23, nucleophosmin) binding, and phosphorylation at serine 18 and 65 both in situ and in vitro. To test the role of these phosphorylation sites, we carried out in vivo tumorigenesis studies in severe combined immunodeficient mice. The findings show that the p14-Ser65Ala mutation is associated with impaired tumorigenicity, whereas the p14-Ser18Ala mutation is associated with enhanced tumorigenicity. Microarray analysis suggests that phosphorylation at serine 18 or at serine 65 is associated with transcriptional regulation of the L5 nucleolar ribosomal protein (a p14 target) and the Erb-B signal transduction pathway. Taken together, these results show that the phosphorylation status of p14 determines whether it functions as a pro-oncogenic or antioncogenic modulator. Mol Cancer Res; 10(8); 1077–86. ©2012 AACR.

The receptor regulated adenylyl cyclase system is a multiprotein complex which is a member of the family of the receptor-effector systems whose signal is transduced by heterotrimeric GTP-binding proteins. The system consists of stimulatory and inhibitory receptors (Rs and Ri), stimulatory and inhibitory G proteins (Gs and Gi) and the adenylyl cyclase enzyme (C). While quite specific in situ, receptors (stimulatory or inhibitory) from one source can activate the appropriate G protein from other cell types or species which in turn can act on C from other sources. Studies with chimeric proteins have shown that the various specificities (stimulatory or inhibitory) can be mapped to defined domains in both receptors and G proteins. The mechanism by which the heterotrimeric G proteins couple to the stimulatory and inhibitory signals is discussed in detail. Specifically, the data supporting collision coupling vs the shuttle mechanism is reviewed, as well as the role of beta gamma subunits in both the stimulatory and inhibitory signals.

In addition to its nutritional value, cranberry juice has been effective in treating urinary tract infections. Various reports have also demonstrated its potential for inhibiting in vitro growth of transformed cell lines. Here we show that a fraction [nondialyzable material (NDM) of a molecular weight range 12,000-30,000 (NDM 12-30K)] derived from cranberry juice impairs in vitro growth and invasion through extracellular matrix of Rev-2-T-6 murine lymphoma cells. Furthermore, intraperitoneal injection of this fraction at nontoxic doses both inhibits the growth of Rev-2-T-6 tumors in vivo and enhances the generation of antilymphoma antibodies. These findings demonstrate the in vivo efficacy of cranberry components against malignant lymphoma in immune competent hosts.

Signal peptides (SPs) are N-terminal extensions on nascent secretory and membrane proteins that mediate insertion into, or translocation across the membrane of the endoplasmic reticulum (ER). A growing number of SPs have been shown to carry out post-ER targeting functions. A case in point is the SP of the envelope precursor protein of Mouse Mammary Tumor Virus (MMTV), a beta retrovirus known to cause lymphoma and mammry carcinoma in laboratory mice. This signal peptide (named MMTV-p14 , or p14 for short) is uniquely localized to nucleoli of cells that harbor the virus where it binds to specific protein targets and functions as a nuclear export factor for viral transcripts. P14 is also a phosphoprotein tumor modulator, the phosphorylation status of which determines whether it will function, in vivo, in a tumor promoting or tumor suppressing capacity. Here we report that p14 is expressed also on the surface of a variety of cells that contain MMTV (murine lymphoma and mammary carcinoma, and human breast cancer cells ectopically expressing p14). Furthermore p14 is immunogenic and vaccination with purified recombinant p14 (using different adjuvants) is sufficient to immunize mice against malignant cells that harbor MMTV. This can be adoptively transferred into naïve mice. Furthermore, newly derived monoclonal anti-p14 antibodies with different epitope specificity are now available to study their effect on MMTV associated tumors in vivo as well as for targeted drug delivery to these tumors In view of reports suggesting involvement of MMTV in about 30% of human breast cancers we propose p14 and anti-p14 antibodies as candidates for immunization against MMTV associated malignancies, and as a functional example for other viral signal peptides. Citation Format: Ori Braitbard, Tamar Gross, Maayan Roniger, Allan Bar-Sinai, Nira Hochman, Jacob Hochman. Signal peptide –mediated immunization against mouse mammary tumor virus (MMTV)-associated tumors. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr B30.

We have previously shown that the leader peptide (p14) of the Env-precursor of mouse mammary tumor virus is translocated into the nucleoli of murine T cell lymphomas that harbor this virus. Using a polyclonal antibody against recombinant p14, we show here that p14 is also localized to the nucleoli of murine mammary carcinomas and some human breast cancer samples. Affinity purification studies define a number of proteins, mostly nucleolar, that bind p14. Taken together, these findings point towards a more general involvement of p14 in lymphomagenesis and mammary carcinogenesis.

Tumorigenic S49 mouse lymphoma cells (T-25) were compared to their nontumorigenic (immunogenic) substrate-adherent descendants (T-25-Adh), using the differential display technique. A 784-bp fragment with 92% sequence homology to the intracisternal A-particle (IAP) element family was isolated from the latter cells. IAP sequences are endogenous, noninfectious retroviral elements that can undergo transpositions and act as mutagens. Expression of IAP transcripts (as detected by the isolated fragment) was 5- to 10-fold higher in T-25-Adh cells than in T-25 cells. IAP RT-PCR cDNA clones derived from the immunogenic T-25-Adh cells, but not from T-25 cells, contain two distinctive motifs: (i) a motif characteristic of IAP elements expressed in lymphoid cells (lymphocyte specific, LS); (ii) a nonapeptide sequence known to stimulate cytotoxic T lymphocytes in a leukemia cell line expressing IAP sequences. In addition, expression of transcripts containing these motifs is enhanced in the immunogenic cells as opposed to the tumorigenic cells. Furthermore, one of the IAP elements (belonging to the LS1 subfamily) is specifically hypomethylated in the DNA of the immunogenic cells. The above-mentioned relationship was strengthened when tumorigenic revertants derived from T-25-Adh cells, as well as independently selected tumorigenic and immunogenic S49 sublines, were studied. In all cases, enhanced immunogenicity was linked to the up-regulation of specific IAP elements. No transpositions of LS1 elements were observed among the different sublines studied. These findings suggest that, in the S49 lymphoma, selectively expressed IAP retroviral elements may function in a tumor suppressive capacity by affecting the immunogenic potential of these cells.

Acute lymphoblastic leukemia (ALL) is the most common childhood malignancy accounting for 80% of leukemias. The involvement of the central nervous system (CNS) by ALL is a major clinical problem and occurs in about 50% of the children without adequate treatment. The introduction of CNS-directed therapy consisting of intrathecal and high dose systemic chemotherapy and, occasionally, cranial irradiation, reduced relapse rate to less than 5% and has become a prerequisite for treating children with ALL. However, substantial neurotoxicity associated with this therapy is a major concern. Moreover the CNS is involved in up to a third from all relapses. To date very little is known about the pathogenesis of CNS leukemia. Our research was promoted by the previous observation that high mRNA expression of interleukin 15 (IL15) in leukemic blasts is associated with increased risk for CNS involvement (Cario et al JCO 2007;25:4813-20). As IL15 is a strong stimulant of Natural Killer (NK) cells, we hypothesized that the increased expression of IL15 may activate NK cells which, in turn, will control residual ALL cells in the peripheral blood but not in the relatively protected central nervous system. To investigate this hypothesis, we utilized two mouse models, a S49-derived T lymphoblastic leukemia syngeneic model and a novel human xenograft ALL model in immune-deficient mice. We found that constitutive expression of IL15 in mouse T lymphoblastic leukemia cells transplanted in neonatal Balb/c mice markedly slowed the development of systemic disease and caused CNS leukemia characterized by pronounced clinical CNS symptoms and subarachnoid infiltration of leukemia cells. This phenotype was accompanied by increase in activated natural killer (NK) cells (from 0.16% to 18.6% P Taken together we show here, for the first time, a crucial role for NK cells in the control of CNS leukemia. We suggest that the association between IL15 expression in ALL blasts and isolated CNS relapse might be explained by activation of NK cells leading to increased surveillance of residual leukemia in the bone marrow but not in the CNS which serve as a sanctuary site for tumor growth. Disclosures: No relevant conflicts of interest to declare.

Mouse Mammary Tumor Virus (MMTV) causes mammary carcinoma and lymphoma in mice. An increasing body of evidence from different laboratories suggests an association between MMTV and up to 38% of human breast cancers. This seems related to severity of the disease and geographical location. Signal peptides are N-terminal extensions of nascent secretory and membrane proteins (typically including 15-25 amino acid residues). They mediate translocation across, or insertion into the membrane of the endoplasmic reticulum (ER). The signal peptide of Mouse Mammary Tumor Virus (MMTV) envelop precursor (named MMTV-14 or p14 for short), in addition to being unusually long (98 a.a) demonstrates some unique characteristics: 1) It localizes to nucleoli; 2) It functions as a nuclear export factor for intron-containing viral transcripts; 3) It can function in a pro-oncogenic or anti-oncogenic capacity depending on its phosphorylation by the kinases CK2 (at serine 65) or PKC (at serine 18), respectively. We report that, in addition to its nucleolar localization, p14 is expressed on the cell surface of murine lymphomas and mammary carcinomas that harbor MMTV, as well as on the surface of MCF-7 human breast cancer cells ectopically expressing p14. Cell surface expression allows p14 to play the role of a tumor associated antigen. Indeed, priming and boosting of mice with recombinant p14, using different adjuvants, induces specific anti-p14 antibodies, as well as immunizes these mice against malignant tumor cells that harbor MMTV. This immunity is adoptively transferred to naïve mice. Thus, MMTV-p14 is a multi-faceted signal peptide with immune modulating characteristics that can be used for developing both preventive and therapeutic approaches towards MMTV-associated breast cancers. Supported in part by a Kamin grant from the Office of the Chief Scientist. Citation Format: Ori Braitbard, Allan Bar-Sinai, Maayan Roniger, Dana Rajchman, Nira Hochman, Jacob Hochman. A signal peptide as a putative tool against MMTV-associated breast cancers. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr A109.

Cranberry fruit and juice have been reported to exert a multitude of health-related benefits. NDM is a 12-30K molecular weight fraction, which we have derived from cranberry juice and which, as we have previously reported, impairs in vitro growth and invasion through extracellular matrix of murine lymphoma cells [N.Hochman et al. Nutrition and Cancer 60(4), 511-517,2008]. Furthermore, intraperitoneal inoculation of NDM inhibited the in vivo growth of these tumors in syngeneic hosts, demonstrating its in vivo efficacy as an anti-tumorigenic agent. MMTV-p14 (p14 for short) is the signal peptide of the env-precursor of Mouse Mammary Tumor Virus. This peptide was found by us to localize in the nucleoli of every MMTV-containing lymphoma or mammary carcinoma tested, (as well as in some cases of human breast cancer). Here we report that priming and boosting of mice with purified recombinant p14 +NDM results in enhanced generation of anti-p14 antibodies in the sera of inoculated mice. Furthermore, mice primed and boosted with p14+NDM are immunized against a subsequent challenge with malignant lymphoma cells that contain MMTV. Mice, primed and boosted separately with p14 or NDM were not immunized against a lymphoma challenge. These findings demonstrate that NDM can be used as adjuvant in a protein vaccination protocol against malignant lymphoma cells that harbor the virus MMTV. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 763. doi:10.1158/1538-7445.AM2011-763

In this study we describe an extension of our previous studies on cis-benzylidenemalononitrile tyrphostins. We have introduced S-aryl substituents in the 5 position (meta vis-a-vis the malononitrile moiety). We find that these compounds are potent blockers of EGFR kinase and its homolog HER-2 kinase. Interestingly, we find that certain S-aryltryphostins discriminate between EGFR and HER-2 kinase in favor of the HER-2 kinase domain by almost 2 orders of magnitude. When examined in intact cells it was found that these selective S-aryltrphostins are equipotent in inhibiting EGF dependent proliferation of NIH 3T3 harboring either the EGF receptor or the chimera EGF/neu (HER1-2). These findings suggest that the antiproliferative activity of these tyrphostins is mainly due to the inhibition of a mitogenic signaling element downstream to the growth receptor kinase.