Outgrowth In Mice Research Articles

Astrocyte-induced Cdk5 expedites breast cancer brain metastasis by suppressing MHC-I expression to evade immune recognition.

Brain metastases (BrMs) evade the immune response to develop in the brain, yet the mechanisms of BrM immune evasion remains unclear. This study shows that brain astrocytes induce the overexpression of neuronal-specific cyclin-dependent kinase 5 (Cdk5) in breast cancer-derived BrMs, which facilitates BrM outgrowth in mice. Cdk5-overexpressing BrMs exhibit reduced expression and function of the class I major histocompatibility complex (MHC-I) and antigen-presentation pathway, which are restored by inhibiting Cdk5 genetically or pharmacologically, as evidenced by single-cell RNA sequencing and functional studies. Mechanistically, Cdk5 suppresses MHC-I expression on the cancer cell membrane through the Irf2bp1-Stat1-importin α-Nlrc5 pathway, enabling BrMs to avoid recognition by T cells. Treatment with roscovitine-a clinically applicable Cdk5 inhibitor-alone or combined with immune checkpoint inhibitors, significantly reduces BrM burden and increases tumour-infiltrating functional CD8+ lymphocytes in mice. Thus, astrocyte-induced Cdk5 overexpression endorses BrM immune evasion, whereas therapeutically targeting Cdk5 markedly improves the efficacy of immune checkpoint inhibitors and inhibits BrM growth.

The role of p63 in embryonic external genitalia outgrowth in mice.

Embryonic external genitalia (genital tubercle [GT]) protrude from the cloaca and outgrow as cloacal development progresses. Individual gene functions and knockout phenotypes in GT development have been extensively analyzed; however, the interactions between these genes are not fully understood. In this study, we investigated the role of p63, focusing on its interaction with the Shh-Wnt/Ctnnb1-Fgf8 pathway, a signaling network that is known to play a role in GT outgrowth. p63 was expressed in the epithelial tissues of the GT at E11.5, and the distal tip of the GT predominantly expressed the ΔNp63α isoform. The GTs in p63 knockout embryos had normal Shh expression, but CTNNB1 protein and Fgf8 gene expression in the distal urethral epithelium was decreased or lost. Constitutive expression of CTNNB1 in p63-null embryos restored Fgf8 expression, accompanied by small bud structure development; however, such bud structures could not be maintained by E13.5, at which point mutant GTs exhibited severe abnormalities showing a split shape with a hemorrhagic cloaca. Therefore, p63 is a key component of the signaling pathway that triggers Fgf8 expression in the distal urethral epithelium and contributes to GT outgrowth by ensuring the structural integrity of the cloacal epithelia. Altogether, we propose that p63 plays an essential role in the signaling network for the development of external genitalia.

Critical Role for Cap-Independent c-MYC Translation in Progression of Multiple Myeloma.

Dysregulated c-myc is a determinant of multiple myeloma progression. Translation of c-myc can be achieved by an mTOR-mediated, cap-dependent mechanism or a cap-independent mechanism where a sequence in the 5'UTR of mRNA, termed the internal ribosome entry site (IRES), recruits the 40S ribosomal subunit. This mechanism requires the RNA-binding factor hnRNP A1 (A1) and becomes critical when cap-dependent translation is inhibited during endoplasmic reticulum (ER) stress. Thus, we studied the role of A1 and the myc IRES in myeloma biology. A1 expression correlated with enhanced c-myc expression in patient samples. Expression of A1 in multiple myeloma lines was mediated by c-myc itself, suggesting a positive feedback circuit where myc induces A1 and A1 enhances myc translation. We then deleted the A1 gene in a myc-driven murine myeloma model. A1-deleted multiple myeloma cells demonstrated downregulated myc expression and were inhibited in their growth in vivo. Decreased myc expression was due to reduced translational efficiency and depressed IRES activity. We also studied the J007 inhibitor, which prevents A1's interaction with the myc IRES. J007 inhibited myc translation and IRES activity and diminished myc expression in murine and human multiple myeloma lines as well as primary samples. J007 also inhibited tumor outgrowth in mice after subcutaneous or intravenous challenge and prevented osteolytic bone disease. When c-myc was ectopically reexpressed in A1-deleted multiple myeloma cells, tumor growth was reestablished. These results support the critical role of A1-dependent myc IRES translation in myeloma.

In This Issue

In This Issue

Augmented antibody-based anticancer therapeutics boost neutrophil cytotoxicity.

Most clinically used anticancer mAbs are of the IgG isotype, which can eliminate tumor cells through NK cell-mediated antibody-dependent cellular cytotoxicity and macrophage-mediated antibody-dependent phagocytosis. IgG, however, ineffectively recruits neutrophils as effector cells. IgA mAbs induce migration and activation of neutrophils through the IgA Fc receptor (FcαRI) but are unable to activate NK cells and have poorer half-life. Here, we combined the agonistic activity of IgG mAbs and FcαRI targeting in a therapeutic bispecific antibody format. The resulting TrisomAb molecules recruited NK cells, macrophages, and neutrophils as effector cells for eradication of tumor cells in vitro and in vivo. Moreover, TrisomAb had long in vivo half-life and strongly decreased B16F10gp75 tumor outgrowth in mice. Importantly, neutrophils of colorectal cancer patients effectively eliminated tumor cells in the presence of anti-EGFR TrisomAb but were less efficient in mediating killing in the presence of IgG anti-EGFR mAb (cetuximab). The clinical application of TrisomAb may provide potential alternatives for cancer patients who do not benefit from current IgG mAb therapy.

Preclinical Development of Inducible MyD88/CD40 (iMC)-Enhanced Chimeric Antigen Receptor Natural Killer (GoCAR-NK) Cells to Target BCMA+ Tumors

Background: Natural Killer (NK) cells possess potent innate anti-tumor cytotoxicity that can be augmented and focused by engineering with chimeric antigen receptors (CARs). Because NK cells do not express T cell receptors that can direct alloreactivity, they have potential as an off-the-shelf (OTS) cell therapy for the treatment of cancer. We recently demonstrated that a drug-inducible co-activation molecule (inducible MyD88/CD40; iMC) synergizes with transgenic IL-15 to boost CAR-NK cell proliferation, survival and anti-tumor cytotoxic effects (Blood Adv.4:1950 [2020]). Here, we describe the pre-clinical development of an OTS iMC/IL-15-enhanced CAR-NK cell platform targeting B cell maturation antigen (BCMA) for the treatment of multiple myeloma. Methods: NK cells were isolated from peripheral blood mononuclear cells by CD56+ selection, activated with IL-15 and microparticles conjugated with IL-21 and 4-1BB ligand. Activated NK cells were transduced with retrovirus encoding an optimized iMC and IL-15-expressing BCMA CAR construct (iMC-BCMA.z-IL15) where iMC signaling could be activated by exposure to rimiducid (Rim), a small molecule dimerizing ligand. Anti-tumor cytotoxicity and cytokine production was assessed using co-culture assays with control or modified CAR-NK cells against BCMA-expressing myeloma cells (NCIH929, RPMI8226, MM1S, U266 and NALM-6-BCMA). Additional experiments were performed with BCMA-edited cell lines (CRISPR/Cas9) to evaluate the innate cytotoxic potential of GoCAR-NK cells. In vivo anti-tumor efficacy and NK cell expansion was measured using immunodeficient NSG mice engrafted with 1.5 x 106 NCIH929-GFPffluc, MM1S-GFPffluc or THP1-GFPffluc cells followed by i.v. treatment with up to 1 x 107 BCMA GoCAR-NK cells. Tumor and NK cells were tracked via bioluminescence imaging. Results: Following IL-15 and IL-21/4-1BBL microparticle stimulation, NK cells were efficiently transduced (40-70%) and exhibited rapid ex vivo expansion (200-fold in 13 days). iMC-BCMA.CAR-IL15-modified NK cells exhibited potent cytotoxicity against BCMA+target cells compared with mock-transduced NK cells (MM1S, 58±4% versus 17±2%; Nalm-6-BCMA, 61±2% versus 19±6%) after 24 hours. Long-term (7 day) co-culture assays revealed the effect of iMC/IL-15 enhancement on NK cell potency, proliferation and cytokine production where iMC-BCMA.z-IL15-modified NK cells stimulated with Rim showed a >70% increase in tumor-specific killing compared to cells without iMC activation. Further, rimiducid-induced activation led to NK cell persistence and proliferation, 8.1±4.0-fold expansion compared to the start of the coculture. In comparison, there was an 80% reduction mock transduced NK cells or GoCAR-NK cells in cocultures without rimiducid. Induced-MC signaling also drove production of cytokines such as TNF-α, IFN-g (6.6X stimulation with 1 nM Rim relative to no drug), GMCSF, IP-10, and IL-13. In addition, activation of the iMC co-activation protein in combination with IL-15 secretion prevented NK cell exhaustion and led to retained functional activity of the modified GoCAR-NK cells for over 4-weeks in culture. In contrast, unmodified NK cells or modified GoCAR-NK cells without Rim exposure became functionally deficient. Of interest, a comparison of NK and T cells modified with the iMC/IL-15 BCMA CAR construct indicated that CAR-NK cells display more rapid target killing, which is further augmented by iMC-mediated cell signaling in the presence of Rim. Furthermore, GoCAR-NK cells were capable of lysis of BCMA-null target cells due to their innate anti-tumor activity. In vivo efficacy studies showed that neither iMC activation nor IL-15 secretion alone were sufficient to support CAR-NK cell engraftment in NSG mice but, in combination, they resulted in CAR-NK cell expansion and persistence. iMC/IL-15-enhanced BCMA GoCAR-NK cells proliferation was associated with improved control of tumor outgrowth in mice challenged with BCMA+ myeloma cells. Summary: These results indicate that the synergistic activity of iMC signaling combined with transgenic IL-15 production can enhance BCMA-specific CAR-NK cytotoxicity, cytokine production, long-term proliferation and persistence and may improve overall anti-tumor efficacy of a potential OTS cell therapy for the treatment of myeloma. Disclosures Wang: Bellicum Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. Duong:Bellicum Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. Guerrero:Bellicum Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. Mahendravada:Bellicum Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. Sharp:Bellicum Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. Brandt:Bellicum Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. Gagliardi:Bellicum Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. Foster:Bellicum Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. Bayle:Bellicum Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company.

The Antidiabetic Agent Acarbose Improves Anti-PD-1 and Rapamycin Efficacy in Preclinical Renal Cancer.

Simple SummaryAlthough immune-stimulatory and targeted therapies benefit many patients with metastatic kidney cancer, a sizeable proportion of patients fail to respond. Recent studies in mice demonstrate that nutrient-limiting dietary interventions can improve responses to chemotherapy. However, these studies did not investigate effects on metastasis, and the impact of these interventions on the response to immunotherapy or targeted therapies in kidney cancer is unknown. We therefore studied the effects of a glucose-limiting drug called acarbose, which is used to treat type 2 diabetes, in a spontaneously-metastasizing mouse model of kidney cancer. We found that acarbose slowed kidney cancer growth and promoted protective immune responses. In combination with either an immunotherapy or a targeted therapy used clinically to treat kidney cancer, acarbose led to improved treatment outcomes and reduced lung metastases. Our findings contribute to the emerging idea of using nutrition-based interventions to enhance responses to cancer treatments. Although immune checkpoint inhibitors and targeted therapeutics have changed the landscape of treatment for renal cell carcinoma (RCC), most patients do not experience significant clinical benefits. Emerging preclinical studies report that nutrition-based interventions and glucose-regulating agents can improve therapeutic efficacy. However, the impact of such agents on therapeutic efficacy in metastatic kidney cancer remains unclear. Here, we examined acarbose, an alpha-glucosidase inhibitor and antidiabetic agent, in a preclinical model of metastatic kidney cancer. We found that acarbose blunted postprandial blood glucose elevations in lean, nondiabetic mice and impeded the growth of orthotopic renal tumors, an outcome that was reversed by exogenous glucose administration. Delayed renal tumor outgrowth in mice on acarbose occurred in a CD8 T cell-dependent manner. Tumors from these mice exhibited increased frequencies of CD8 T cells that retained production of IFNγ, TNFα, perforin, and granzyme B. Combining acarbose with either anti-PD-1 or the mammalian target of rapamycin inhibitor, rapamycin, significantly reduced lung metastases relative to control mice on the same therapies. Our findings in mice suggest that combining acarbose with current RCC therapeutics may improve outcomes, warranting further study to determine whether acarbose can achieve similar responses in advanced RCC patients in a safe and likely cost-effective manner.

A Multifunctional Therapy Approach for Cancer: Targeting Raf1- Mediated Inhibition of Cell Motility, Growth, and Interaction with the Microenvironment.

Prostate cancer cells move from their primary site of origin, interact with a distant microenvironment, grow, and thereby cause death. It had heretofore not been possible to selectively inhibit cancer cell motility. Our group has recently shown that inhibition of intracellular activation of Raf1 with the small-molecule therapeutic KBU2046 permits, for the first time, selective inhibition of cell motility. We hypothesized that simultaneous disruption of multiple distinct functions that drive progression of prostate cancer to induce death would result in advanced disease control. Using a murine orthotopic implantation model of human prostate cancer metastasis, we demonstrate that combined treatment with KBU2046 and docetaxel retains docetaxel's antitumor action, but provides improved inhibition of metastasis, compared with monotherapy. KBU2046 does not interfere with hormone therapy, inclusive of enzalutamide-mediated inhibition of androgen receptor (AR) function and cell growth inhibition, and inclusive of the ability of castration to inhibit LNCaP-AR cell outgrowth in mice. Cell movement is necessary for osteoclast-mediated bone degradation. KBU2046 inhibits Raf1 and its downstream activation of MEK1/2 and ERK1/2 in osteoclasts, inhibiting cytoskeleton rearrangement, resorptive cavity formation, and bone destruction in vitro, with improved effects observed when the bone microenvironment is chemically modified by pretreatment with zoledronic acid. Using a murine cardiac injection model of human prostate cancer bone destruction quantified by CT, KBU2046 plus zoledronic exhibit improved inhibitory efficacy, compared with monotherapy. The combined disruption of pathways that drive cell movement, interaction with bone, and growth constitutes a multifunctional targeting strategy that provides advanced disease control.

LTX-315 sequentially promotes lymphocyte-independent and lymphocyte-dependent antitumor effects.

LTX-315 is an oncolytic peptide that has antitumor efficacy in mice grafted with various tumor cell lines and is currently being tested in phase II clinical trials. Here we aimed to further evaluate LTX-315 in conditional genetic mouse models of cancer that typically resist current treatment options and to better understand the drug's mode of action in vivo. We report LTX-315 mediates profound antitumor effects against Braf- and Pten-driven melanoma and delays the progression of Kras- and P53-driven soft tissue sarcoma in mice. Additionally, we show in melanoma that LTX-315 triggers two sequential phases of antitumor response. The first phase of response, which begins within minutes of drug delivery into tumors, is defined by disrupted tumor vasculature and decreased tumor burden and occurs independently of lymphocytes. The second phase of response, which continues over weeks, is defined by long-term alteration of the tumor microenvironment; the changes induced by LTX-315 are most notably characterized by CD8+ T cell infiltration. We further show that these CD8+ T cells are involved in suppressing melanoma outgrowth in mice and report similar CD8+ T cell infiltration following LTX-315 treatment in melanoma and sarcoma patients. Taken together, these findings reveal LTX-315's multiple antitumor effects, including disrupting the tumor vasculature and promoting the conversion of poorly immunogenic tumors into ones that display antitumor T cell immunity.

Abstract 4010: Identification of signaling pathways involved in colorectal adenoma-to-carcinoma progression using phosphoproteomics

Abstract Background Colorectal cancer (CRC) develops in a multi-step-process from normal epithelium, through a pre-malignant lesion (adenoma), into a malignant lesion (adenocarcinoma). A minority of about 5% of adenomas will ultimately progress into cancer. To improve clinical practice it is important to identify the subgroup of colorectal adenomas that is at risk of progressing to cancer. By extensive genomic analysis we have shown that specific copy number alterations are associated with risk of progression. An example of such an alteration is the gain of chromosome arm 20q and we identified AURKA and TPX2 as major drivers of this amplicon. Yet, the activation status of these and other genes during colorectal carcinogenesis and the downstream signaling pathways affected with gene (in)activation are not fully known. In order to better understand the biology of adenoma to carcinoma transition, we performed a comprehensive analysis of phosphoproteomes at different stages of colorectal carcinogenesis. Methods Phosphotyrosine containing peptides were immunoprecipitated from 5 mg of colorectal adenoma (n=81) and colorectal carcinoma tissues (n=50) using agarose bead-coupled phosphotyrosine antibody P-Tyr-1000. A Q Exactive HF mass spectrometer was used to perform NanoLC-MS/MS. Spectral counts of phosphoproteins and ion intensities of phosphopeptides were defined by MaxQuant for relative quantitation of protein phosphorylation. Results Phosphotyrosine-based phosphoproteomics of the 131 colorectal tissue samples yielded 6056 phosphopeptides, corresponding to 2745 unique phosphoproteins including 183 phosphokinases. Data analysis to identify regulated phosphorylation states and inference of kinase activity is ongoing. Conclusions and future plans This is the first large phosphoproteomics dataset of colorectal adenomas and carcinomas. Preliminary data analysis shows promising differences in kinase activities between adenomas and carcinomas. After further analysis, we will manipulate the activity of the driver kinases in adenoma-derived organoids and monitor tumor outgrowth in mice. Citation Format: Sanne Martens-de Kemp, Alex Henneman, Richard de Goeij-de Haas, Sander Piersma, Thang Pham, Gerrit Meijer, Beatriz Carvalho, Connie Jimenez. Identification of signaling pathways involved in colorectal adenoma-to-carcinoma progression using phosphoproteomics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4010.

PO-117 Role of NFE2L3 in colon cancer by regulating cancer cells proliferation

IntroductionT-ALL is a malignancy characterised by aberrant Notch signalling, sustained by activating mutations in Notch1 as well as over-expression of Notch3, a Notch paralog physiologically subjected to lysosome-dependent degradation in human cancer cells. Given some limitations of existing drugs blocking Notch signalling, it is important to get new insights into the biology of Notch3 to further stimulate the development of Notch-targeted therapies in cancer.Material and methodsT-ALL cell lines and cells obtained from patient-derived xenografts (PDX) were treated in vitro with HDAC inhibitors and other drugs or HDAC6-specific shRNA and effects on Notch expression and activity were investigated by standard methods. Confocal microscopy was used for intracellular localization studies. PDX models were also utilised to investigate effects of HDAC6 silencing on leukaemia growth.Results and discussionsWe initially found that treatment with the pan-HDAC inhibitor Trichostatin A (TSA) strongly decreases Notch3 full-length protein levels in T-ALL cell lines and primary human T-ALL cells xenografted in mice without substantially reducing NOTCH3 mRNA levels. Moreover, TSA markedly reduced the levels of Notch target genes, including pTa, CR2 and DTX-1, and induced apoptosis of T-ALL cells. We further observed that Notch3 was post-translationally regulated following TSA treatment, with reduced Notch3 surface levels and increased accumulation of Notch3 protein in the lysosomal compartment. Surface Notch3 levels were rescued by inhibition of Dynein with Ciliobrevin D. Pharmacologic studies with HDAC1, 6 and 8-specific inhibitors disclosed that these effects were largely due to inhibition of HDAC6 in T-ALL cells. HDAC6 silencing by specific shRNA was followed by reduced Notch3 expression and increased apoptosis of T-ALL cells. Finally, HDAC6 silencing impaired leukaemia outgrowth in mice, associated with reduction of Notch3 full-length protein in vivo.ConclusionThese results connect HDAC6 activity to regulation of total and surface Notch3 levels by a mechanism involving endosomal sorting and suggest HDAC6 as potential novel therapeutic target to lower Notch signalling in T-ALL and other Notch3-addicted tumours.

Histone deacetylase 6 controls Notch3 trafficking and degradation in T-cell acute lymphoblastic leukemia cells

Several studies have revealed that endosomal sorting controls the steady-state levels of Notch at the cell surface in normal cells and prevents its inappropriate activation in the absence of ligands. However, whether this highly dynamic physiologic process can be exploited to counteract dysregulated Notch signaling in cancer cells remains unknown. T-ALL is a malignancy characterized by aberrant Notch signaling, sustained by activating mutations in Notch1 as well as overexpression of Notch3, a Notch paralog physiologically subjected to lysosome-dependent degradation in human cancer cells. Here we show that treatment with the pan-HDAC inhibitor Trichostatin A (TSA) strongly decreases Notch3 full-length protein levels in T-ALL cell lines and primary human T-ALL cells xenografted in mice without substantially reducing NOTCH3 mRNA levels. Moreover, TSA markedly reduced the levels of Notch target genes, including pTα, CR2, and DTX-1, and induced apoptosis of T-ALL cells. We further observed that Notch3 was post-translationally regulated following TSA treatment, with reduced Notch3 surface levels and increased accumulation of Notch3 protein in the lysosomal compartment. Surface Notch3 levels were rescued by inhibition of dynein with ciliobrevin D. Pharmacologic studies with HDAC1, 6, and 8-specific inhibitors disclosed that these effects were largely due to inhibition of HDAC6 in T-ALL cells. HDAC6 silencing by specific shRNA was followed by reduced Notch3 expression and increased apoptosis of T-ALL cells. Finally, HDAC6 silencing impaired leukemia outgrowth in mice, associated with reduction of Notch3 full-length protein in vivo. These results connect HDAC6 activity to regulation of total and surface Notch3 levels and suggest HDAC6 as a potential novel therapeutic target to lower Notch signaling in T-ALL and other Notch3-addicted tumors.

Thyroid Hormone Induces PGC-1α during Dendritic Outgrowth in Mouse Cerebellar Purkinje Cells.

Thyroid hormone 3,3′,5-Triiodo-L-thyronine (T3) is essential for proper brain development. Perinatal loss of T3 causes severe growth defects in neurons and glia, including strong inhibition of dendrite formation in Purkinje cells in the cerebellar cortex. Here we show that T3 promotes dendritic outgrowth of Purkinje cells through induction of peroxisome proliferator-activated receptor gamma (PPARγ) co-activator 1α (PGC-1α), a master regulator of mitochondrial biogenesis. PGC-1α expression in Purkinje cells is upregulated during dendritic outgrowth in normal mice, while it is significantly retarded in hypothyroid mice or in cultures depleted of T3. In cultured Purkinje cells, PGC-1α knockdown or molecular perturbation of PGC-1α signaling inhibits enhanced dendritic outgrowth and mitochondrial generation and activation caused by T3 treatment. In contrast, PGC-1α overexpression promotes dendrite extension even in the absence of T3. PGC-1α knockdown also downregulates dendrite formation in Purkinje cells in vivo. Our findings suggest that the growth-promoting activity of T3 is partly mediated by PGC-1α signaling in developing Purkinje cells.

Deptor Enhances Triple-Negative Breast Cancer Metastasis and Chemoresistance through Coupling to Survivin Expression

Transforming growth factor–β (TGF-β) functions to suppress tumorigenesis in normal mammary tissues and early-stage breast cancers and, paradoxically, acts to promote the metastasis and chemoresistance in late-stage breast cancers, particularly triple-negative breast cancers (TNBCs). Precisely how TGF-β acquires oncogenic characteristics in late-stage breast cancers remains unknown, as does the role of the endogenous mammalian target of rapamycin (mTOR) inhibitor, Dep domain–containing mTOR-interacting protein (Deptor), in coupling TGF-β to TNBC development and metastatic progression. Here we demonstrate that Deptor expression was downregulated in basal-like/TNBCs relative to their luminal counterparts. Additionally, Deptor expression was 1) inversely correlated with the metastatic ability of human (MCF10A) and mouse (4T1) TNBC progression series and 2) robustly repressed by several inducers of epithelial-mesenchymal transition programs. Functional disruption of Deptor expression in 4T07 cells significantly inhibited their proliferation and organoid growth in vitro, as well as prevented their colonization and tumor formation in the lungs of mice. In stark contrast, elevated Deptor expression was significantly associated with poorer overall survival of patients harboring estrogen receptor α–negative breast cancers. Accordingly, enforced Deptor expression in MDA-MB-231 cells dramatically enhanced their 1) organoid growth in vitro, 2) pulmonary outgrowth in mice, and 3) resistance to chemotherapies, an event dependent on the coupling of Deptor to survivin expression. Collectively, our findings highlight the dichotomous functions of Deptor in modulating the proliferation and survival of TNBCs during metastasis; they also implicate Deptor and its stimulation of survivin as essential components of TNBC resistance to chemotherapies and apoptotic stimuli.

Absence of SARM1 rescues development and survival of NMNAT2-deficient axons.

SummarySARM1 function and nicotinamide mononucleotide adenylyltransferase 2 (NMNAT2) loss both promote axon degeneration, but their relative relationship in the process is unknown. Here, we show that NMNAT2 loss and resultant changes to NMNAT metabolites occur in injured SARM1-deficient axons despite their delayed degeneration and that axon degeneration specifically induced by NMNAT2 depletion requires SARM1. Strikingly, SARM1 deficiency also corrects axon outgrowth in mice lacking NMNAT2, independently of NMNAT metabolites, preventing perinatal lethality. Furthermore, NAMPT inhibition partially restores outgrowth of NMNAT2-deficient axons, suggesting that the NMNAT substrate, NMN, contributes to this phenotype. NMNAT2-depletion-dependent degeneration of established axons and restricted extension of developing axons are thus both SARM1 dependent, and SARM1 acts either downstream of NMNAT2 loss and NMN accumulation in a linear pathway or in a parallel branch of a convergent pathway. Understanding the pathway will help establish relationships with other modulators of axon survival and facilitate the development of effective therapies for axonopathies.

CD4+ and CD8+ T Cells Can Act Separately in Tumour Rejection after Immunization with Murine Pneumotropic Virus Chimeric Her2/neu Virus-Like Particles

BackgroundImmunization with murine pneumotropic virus virus-like particles carrying Her2/neu (Her2MPtVLPs) prevents tumour outgrowth in mice when given prophylactically, and therapeutically if combined with the adjuvant CpG. We investigated which components of the immune system are involved in tumour rejection, and whether long-term immunological memory can be obtained.Methodology and ResultsDuring the effector phase in BALB/c mice, only depletion of CD4+ and CD8+ in combination, with or without NK cells, completely abrogated tumour protection. Depletion of single CD4+, CD8+ or NK cell populations only had minor effects. During the immunization/induction phase, combined depletion of CD4+ and CD8+ cells abolished protection, while depletion of each individual subset had no or negligible effect. When tumour rejection was studied in knock-out mice with a C57Bl/6 background, protection was lost in CD4−/−CD8−/− and CD4−/−, but not in CD8−/− mice. In contrast, when normal C57Bl/6 mice were depleted of different cell types, protection was lost irrespective of whether only CD4+, only CD8+, or CD4+ and CD8+ cells in combination were eradicated. No anti-Her2/neu antibodies were detected but a Her2/neu-specific IFNγ response was seen. Studies of long-term memory showed that BALB/c mice could be protected against tumour development when immunized together with CpG as long as ten weeks before challenge.ConclusionHer2MPtVLP immunization is efficient in stimulating several compartments of the immune system, and induces an efficient immune response including long-term memory. In addition, when depleting mice of isolated cellular compartments, tumour protection is not as efficiently abolished as when depleting several immune compartments together.

554 Triple negative breast cancer. Retrospective analysis of clinico-pathological features from a single institution (2005–2008)

Transforming growth factor–β (TGF-β) functions to suppress tumorigenesis in normal mammary tissues and early-stage breast cancers and, paradoxically, acts to promote the metastasis and chemoresistance in late-stage breast cancers, particularly triple-negative breast cancers (TNBCs). Precisely how TGF-β acquires oncogenic characteristics in late-stage breast cancers remains unknown, as does the role of the endogenous mammalian target of rapamycin (mTOR) inhibitor, Dep domain–containing mTOR-interacting protein (Deptor), in coupling TGF-β to TNBC development and metastatic progression. Here we demonstrate that Deptor expression was downregulated in basal-like/TNBCs relative to their luminal counterparts. Additionally, Deptor expression was 1) inversely correlated with the metastatic ability of human (MCF10A) and mouse (4T1) TNBC progression series and 2) robustly repressed by several inducers of epithelial-mesenchymal transition programs. Functional disruption of Deptor expression in 4T07 cells significantly inhibited their proliferation and organoid growth in vitro, as well as prevented their colonization and tumor formation in the lungs of mice. In stark contrast, elevated Deptor expression was significantly associated with poorer overall survival of patients harboring estrogen receptor α–negative breast cancers. Accordingly, enforced Deptor expression in MDA-MB-231 cells dramatically enhanced their 1) organoid growth in vitro, 2) pulmonary outgrowth in mice, and 3) resistance to chemotherapies, an event dependent on the coupling of Deptor to survivin expression. Collectively, our findings highlight the dichotomous functions of Deptor in modulating the proliferation and survival of TNBCs during metastasis; they also implicate Deptor and its stimulation of survivin as essential components of TNBC resistance to chemotherapies and apoptotic stimuli.

Target Deletion of the Cytoskeleton-Associated Protein Palladin Does Not Impair Neurite Outgrowth in Mice

Palladin is an actin cytoskeleton–associated protein which is crucial for cell morphogenesis and motility. Previous studies have shown that palladin is localized to the axonal growth cone in neurons and may play an important role in axonal extension. Previously, we have generated palladin knockout mice which display cranial neural tube closure defect and embryonic lethality before embryonic day 15.5 (E15.5). To further study the role of palladin in the developing nervous system, we examined the innervation of palladin-deficient mouse embryos since the 200 kd, 140 kd, 90–92 kd and 50 kd palladin isoforms were undetectable in the mutant mouse embryo brain. Contrary to the results of previous studies, we found no inhibition of the axonal extension in palladin-deficient mouse embryos. The cortical neurons derived from palladin-deficient mice also showed no significant difference in neurite outgrowth as compared with those from wild-type mice. Moreover, no difference was found in neurite outgrowth of neural stem cell derived-neurons between palladin-deficient mice and wild-type mice. In conclusion, these results suggest that palladin is dispensable for normal neurite outgrowth in mice.

Antitumor Activity of an EpCAM/CD3-bispecific BiTE Antibody During Long-term Treatment of Mice in the Absence of T-cell Anergy and Sustained Cytokine Release

muS110 is a BiTE antibody bispecific for murine EpCAM (CD326) and murine CD3. MT110, its human-specific analog, is in a clinical phase 1 trial for treatment of patients with adenocarcinoma of the lung or gastrointestinal tract. Recent studies have shown a therapeutic window for muS110, have explored single-dose toxicity of muS110, and have found that a 1-week low-dose treatment dramatically increased the tolerability of mice to very high doses of muS110 (Cancer Immunol. Immunother. 2009;58:95-109). Here we analyzed the impact of long-term, high-dose treatment of mice with muS110 on antitumor activity and functionality of T cells. After an initial self-limiting cytokine release, the 1-week adaptation period effectively blunted further cytokine production in response to a subsequent high-dose treatment with muS110. The much-increased tolerability of mice adapted to muS110 was not because of anergy of T cells. T cells isolated from chronically muS110-treated mice fully retained their cytotoxic potential, proliferative capacity, and responsiveness to stimulation by either muS110 or anti-CD3/anti-CD28/interleukin-2 when compared with T cells from control mice. Unimpaired T-cell performance was also evident from the effective prevention of orthotopic 4T1 breast tumor outgrowth in mice treated long term with escalating doses of muS110. Finally, we show that muS110 and MT110 recognize orthologous epitopes on mouse and human EpCAM proteins, suggesting that the target-related safety profile of muS110 in mice may be predictive for MT110 in humans.

The IGF System in Mammary Development and Breast Cancer

The IGFs, receptors and binding proteins have emerged as critical mediators in growth of mammary/breast epithelium both in normal developmental processes and in tumorigenesis. The function of this system in mammary development and breast cancer was first reviewed in this journal in 2000. At that time, an essential role for IGF-I in pubertal mammary growth and for the IGF-IR in TEB proliferation and ductal outgrowth in mice had just been established [1–3]. It was also recently clear that the IGF ligands were expressed locally in the gland in both epithelial and stromal locations [4, 5] raising the question of how circulating and local IGFs mediate epithelial growth. Local overexpression of the ligands primarily demonstrated that the IGFs were potent survival factors for mammary epithelial cells and could inhibit apoptosis during involution [6, 7]. A role for the IGF binding proteins, IGFBP-5 in particular, also was proposed in the apoptotic events of involution in part by inhibiting IGF actions as well as by IGFindependent functions [8, 9]. In breast cancer, the IGFs, IGFBPs and IGF-IR were an intense focus due to their proposed link with breast cancer susceptibility, role in breast tumor cell biology and interaction with estrogen receptor (ER) signaling. While these data were intriguing, there were no therapeutic strategies designed to test the function of IGF signaling in human subjects. With the clear demonstration that targeting growth factor receptor signaling in cancer has value (epidermal growth factor receptor, vascular endothelial growth factor receptor, etc.), the therapeutic field has changed dramatically. The reviews on IGF biology in mammary development and neoplasia in the 2000 reviews proposed hypotheses and raised a number of critical unanswered questions. Eight years later, we now have the opportunity in the current issue to revisit the IGF system in mammary development and breast cancer and to determine whether investigations during the intervening period answered any of these questions and led to revised hypotheses. Two major issues that were raised in the prior issue concerning IGF and IGFBP function in mammary/breast tissue were how these molecules are involved in hormone-mediated growth of epithelial cells and their role as endocrine versus autocrine/ paracrine growth factors. It is of note that a number of the updated reviews in this issue address these same questions both from the perspective of normal growth and developmental processes and of susceptibility and growth of breast cancers. New data presented here have led to a greater understanding of these questions but have not yet provided complete answers. While it was previously established that IGF-I is a local mediator of GH actions in mammary development, Kleinberg and Ruan review the increasingly complex roles for IGFs in both estrogen and progesterone events in mammary epithelial growth in rodents. Rowzee et al review new studies suggesting distinct roles for IGF-I expressed in epithelial and stromal compartments in the context of IGF-I as an endocrine or autocrine/paracrine mediator of growth. They also discuss the increasing evidence that IGF-I and IGF-II are independently regulated and have unique functions during postnatal mammary development. Endocrine IGF function is also the focus of the Lann and LeRoith review in the context of breast cancer. J Mammary Gland Biol Neoplasia (2008) 13:351–352 DOI 10.1007/s10911-008-9096-2