Orthotopic tumor models are critical to the development of new cancer therapies, as they have the greatest potential to mimic human cancer progression. However, such models are often underutilized due to practical challenges. These challenges include the need for specialized surgeries and the presence of complicating symptoms, such as ascites fluid accumulation in ovarian cancer and liver disease models, as well as ulceration of breast tumors growing in the mammary fat pad. An adaptable, quantitative animal health assessment system that employs simple, noninvasive visual measurements can enable researchers to realize the full potential of these models while safeguarding humane endpoints. The goal of this work was to establish health-scoring systems for translationally relevant ovarian (ID8-Defb29/Vegf-A) and breast cancer (triple-negative 4T1) models. We developed a tractable health assessment system by monitoring a combination of activity and responsiveness, hair coat condition, respiratory function, changes in body weight, posture, gait, tumor size (where measurable), and extent of ulceration, and scoring each parameter on a simple numerical scale. The scores at which euthanasia or other interventions were required were estimated and then adjusted based on clinical assessment by a team of researchers and veterinarians. These semiquantitative health-scoring systems were applied to survival experiments, in which mice were euthanized at a predetermined score or upon meeting a superseding health criterion, to confirm their applicability and effectiveness in maintaining humane endpoints. The scoring systems presented are intended to build on previously established generic tumor model scoring systems that do not account for the particular phenotypes present in orthotopic breast and ovarian cancer models.

Immunotherapy has revolutionized cancer treatment; however, only limited aspects of the tumor-immune interaction have been exploited successfully. While it is well-known that trafficking of T cells is often impaired during cancer, there are no available therapies that address this aspect of antitumor immunity. Clinically, expression levels of the sphingosine-1-phosphate producing enzyme sphingosine kinase 1 negatively correlates with survival of melanoma patients after immune checkpoint inhibitor (ICI) therapy. We therefore hypothesized that aberrant S1P signaling in tumors causes immune exclusion by impeding lymphocyte egress from the tumor draining lymph node (TDLN). We tested the efficacy of an engineered S1P-degrading enzyme (S1PL) on established murine tumors and performed immune profiling of TDLN and intratumoral T cell populations. S1PL increased both trafficking of lymphocytes out of TDLNs and T cell infiltration of tumors. Mice treated with S1PL evinced a higher proportion of S1P1+ circulating T cells relative to controls, suggesting that liberation of T cells from the TDLN occurred through the S1P/S1P1 axis. Increased lymphocyte trafficking correlated with strong inhibition of tumor growth and enhanced efficacy of ICI therapy. Administration of S1PL may be an attractive strategy for improving therapies that rely on antitumor adaptive immunity. This approach may thus be potent in settings wherein tumor-mediated immune exclusion is a dominant method of immune escape. Texas Biologics & CPRIT RP240454 Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)

Patients with tumors that have a “cold, or “immune desert” phenotype typically have poor outcomes on immune checkpoint therapy (ICT). Aberrant sphingosine-1-phosphate (S1P) signaling in tumors and in their draining lymph nodes (TDLN) appears to causes a common form of immune desertification by disabling or stalling lymphocyte egress from the TDLN, ultimately restricting immune trafficking to tumors. Clinically, tumor expression levels of the S1P producing enzyme sphingosine kinase 1 (SPHK1) strongly and negatively correlate with survival of patients after immune checkpoint inhibitor (ICI) therapy) and pre-clinically correlate with reduced tumor infiltrating lymphocytes (TILs). While there are five known S1P receptors (S1PR1-5), the expression of S1PR1 is required for lymphocyte egress from lymphatic organs. Abnormal S1P signaling or S1PR1 agonists functionally downregulate S1PR1 levels, trapping lymphocytes in the lymph node and is the mechanism of action of certain immunosuppressants (e.g., fingolimod) used to treat autoimmune diseases such as multiple sclerosis. In preliminary studies we have demonstrated in murine cancer models treated with pharmacologically optimized S1P degrading enzymes (SGPL1), that depleting extracellular S1P dramatically increases the trafficking of lymphocytes out of TDLNs, increases tumor T cell infiltration and strongly or completely inhibits tumor growth when administered as a single agent. Data from our lab suggests that deeply lowering extracellular S1P without eliminating internal stores of S1P needed for lymphatic egress is a powerful modality for increasing the level of S1PR1 on lymphocytes, releasing tumor specific T cells from TDLNs, and ultimately increasing tumor immune infiltration. Essentially the exact opposite mechanism as observed with immunosuppressants such as fingolimod. Current small molecule inhibitors of S1P production can only lower extracellular S1P levels by ∼ 50% via decreasing intracellular pools and only show limited anti-cancer activity. A cold tumor microenvironment is a common phenomenon that limits ICT efficacy and therapeutics that could enhance lymphocyte trafficking to the tumor would represent a significant advance for improving patient outcomes. Alessandra Araujo, Max Rodnick-Smith, Ranya Al-Khaledy, Mark Badeaux, Everett Stone. Reversing immune desertification for effective cancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 4884.

Abstract Introduction: Immunotherapy has revolutionized the landscape of cancer treatment; however, to date, only limited aspects of the tumor-immune interaction have been exploited successfully, e.g., relief of immune checkpoints, increase of tumor-associated antigen presentation. Although it is well-known that trafficking and entry of T cells from the circulation into tumors is often impaired, leading to an immune “cold”, checkpoint inhibitor-refractory state, there are no available therapies that address this important aspect of antitumor immunity. Sphingosine-1-phosphate (S1P) is a pleiotropic, bioactive lipid that has been shown to regulate lymphocyte egress from lymph nodes through its interaction with one of its several receptors, S1P1. Clinically, tumor expression levels of the sphingosine-1-phosphate-producing enzyme sphingosine kinase 1 (SPHK1) strongly and negatively correlates with survival of melanoma patients after immune checkpoint inhibitor (ICI) therapy, and pre-clinically correlates with reduced tumor infiltrating lymphocytes (TILs). We therefore hypothesize that aberrant S1P signaling in tumors and in their draining lymph nodes (TDLN) causes immune exclusion by impeding lymphocyte egress from the TDLN, ultimately restricting immune cell infiltration of tumors. Methods: We tested the therapeutic efficacy of an engineered S1P-degrading enzyme, S1P lyase (S1PL), on established (~100mm^3) syngeneic B16, CT26, and MC38 murine tumors. Using flow cytometry, we performed immune profiling of systemic, TDLN, and intratumoral T cell populations, assessing both activation status and expression of the S1P1 receptor. Results: Treatment of tumor-bearing animals with S1PL depleted systemic S1P, and dramatically increased both trafficking of lymphocytes out of TDLNs and T cell infiltration of tumors relative to control-treated animals. Mice treated with S1PL evinced a higher proportion of S1P1+ circulating and intratumoral T cells relative to control-treated mice, suggesting that the increase in liberation of T cells from the TDLN was effected through the S1P/S1P1 axis. This increased lymphocyte trafficking correlated with strong inhibition of tumor growth when S1PL was administered as a single agent. Conclusions: Therapeutic administration of S1PL may represent an attractive strategy for modulating lymphocyte trafficking to improve the efficacy of therapies that rely on the adaptive immune system to exert antitumor activity. This approach may be particularly potent in settings wherein tumor-mediated immune exclusion is a dominant method of immune escape. Citation Format: Alessandra Araujo, Max Rodnick-Smith, Ranya Al-Khaledy, George Georgiou, Mark Badeaux, Everett Stone. Therapeutic administration of an engineered sphingosine-1-phosphate lyase improves T cell trafficking to tumors and enhances antitumor response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2659.

<p>PDF file - 226KB, Figure S1. Schematic diagram of the genomic locus of miR-128 (Homo sapiens-miR-128). Figure S2. miR-128 mRNA was down-regulated in human PCa cell lines.Figure S3. Validation of transfection efficiencies and tumor suppression by miR-128. Figure S4. Characterizations of the miR-128 sensor. Figure S5. miR-128 'decoy' promoted clonal expansion and sphere-forming capacity in PC3 and Du145 cells. Figure S6. BMI-1 effects on proliferation of PCa cells as measured by MTT assays. Supplementary Table S1. Primers used in qPCR analysis miR-128 target quantification, BMI-1 and NANOG 3'-UTR cloning, and sequencing.</p>

Immune checkpoint blockers (ICBs) have failed in all phase III glioblastoma (GBM) trials. Here, we show that regulatory T (Treg) cells play a key role in GBM resistance to ICBs in experimental gliomas. Targeting glucocorticoid-induced TNFR-related receptor (GITR) in Treg cells using an agonistic antibody (αGITR) promotes CD4 Treg cell differentiation into CD4 effector T cells, alleviates Treg cell-mediated suppression of anti-tumor immune response, and induces potent anti-tumor effector cells in GBM. The reprogrammed GBM-infiltrating Treg cells express genes associated with a Th1 response signature, produce IFNγ, and acquire cytotoxic activity against GBM tumor cells while losing their suppressive function. αGITR and αPD1 antibodies increase survival benefit in three experimental GBM models, with a fraction of cohorts exhibiting complete tumor eradication and immune memory upon tumor re-challenge. Moreover, αGITR and αPD1 synergize with the standard of care treatment for newly-diagnosed GBM, enhancing the cure rates in these GBM models.

The effective treatment of cerebral metastases from HER2-positive breast cancer remains an unmet need. Recent studies indicate that activated astrocytes and brain endothelial cells exert chemoprotective effects on cancer cells through direct physical interaction. Here we report that the endothelin axis mediates protection of HER2-amplified brain metastatic breast cancers to the anti-HER2 antibody-drug conjugate ado-trastuzumab emtansine (T-DM1). Macitentan, a dual inhibitor of endothelin receptors A and B, improves the efficacy of T-DM1 against breast cancers grown in the brain. We show that direct contact of brain stroma with cancer cells is required for protection to T-DM1. Our data suggest that targeting the endothelin axis may be beneficial when anti-signaling agent and cytotoxic agent are combined. These findings may contribute to the development of therapeutic approaches with enhanced efficacy in the brain microenvironment.

Abstract Antigen presentation plays a major role in tumor cell recognition and targeting by immune cells, and is critical to the success of many cancer immunotherapies. How the abnormal tumor microenvironment affects tumor cell antigen presentation is unclear. Hypoxia is a prevalent feature of the tumor microenvironment. Here, we showed that the expression of major histocompatibility complex class I (MHCI) is associated with regions of hypoxia in human breast tumors. The association between hypoxia and MHCI is independent of the breast tumor hormone receptor and HER2 expression status. In vitro studies revealed that hypoxia directly regulates the expression levels of MHCI along with other components of the antigen presentation machinery. Multiple kinase regulators of MHCI expression are responsive to hypoxia. These results suggest that hypoxia effects on cancer cell antigen presentation may be a potential mechanism of tumor immune evasion and treatment resistance. Citation Format: Mei Rosa Ng, Francesco Sabbatino, Mark Duquette, Kamila Naxerova, Mark Badeaux, Gino B. Ferraro, Shan M. Chin, Divya Bezwada, Elena F. Brachtel, Soldano Ferrone, Rakesh K. Jain. Hypoxia regulation of antigen presentation machinery expression in breast cancer [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 LB-057.

Although targeted therapies are often effective systemically, they fail to adequately control brain metastases. In preclinical models of breast cancer that faithfully recapitulate the disparate clinical responses in these microenvironments, we observed that brain metastases evade phosphatidylinositide 3-kinase (PI3K) inhibition despite drug accumulation in the brain lesions. In comparison to extracranial disease, we observed increased HER3 expression and phosphorylation in brain lesions. HER3 blockade overcame the resistance of HER2-amplified and/or PIK3CA-mutant breast cancer brain metastases to PI3K inhibitors, resulting in marked tumor growth delay and improvement in mouse survival. These data provide a mechanistic basis for therapeutic resistance in the brain microenvironment and identify translatable treatment strategies for HER2-amplified and/or PIK3CAmutant breast cancer brain metastases.

This project was undertaken to address a critical cancer biology question: Is overexpression of the pluripotency molecule Nanog sufficient to initiate tumor development in a somatic tissue? Nanog1 is critical for the self-renewal and pluripotency of ES cells, and its retrotransposed homolog, NanogP8 is preferentially expressed in somatic cancer cells. Our work has shown that shRNA-mediated knockdown of NanogP8 in prostate, breast, and colon cancer cells inhibits tumor regeneration whereas inducible overexpression of NanogP8 promotes cancer stem cell phenotypes and properties. To address the key unanswered question whether tissue-specific overexpression of NanogP8 is sufficient to promote tumor development in vivo, we generated a NanogP8 transgenic mouse model, in which the ARR2PB promoter was used to drive NanogP8 cDNA. Surprisingly, the ARR2PB-NanogP8 transgenic mice were viable, developed normally, and did not form spontaneous tumors in >2 years. Also, both wild type and ARR2PB-NanogP8 transgenic mice responded similarly to castration and regeneration and castrated ARR2PB-NanogP8 transgenic mice also did not develop tumors. By crossing the ARR2PB-NanogP8 transgenic mice with ARR2PB-Myc (i.e., Hi-Myc) mice, we found that the double transgenic (i.e., ARR2PB-NanogP8; Hi-Myc) mice showed similar tumor incidence and histology to the Hi-Myc mice. Interestingly, however, we observed white dots in the ventral lobes of the double transgenic prostates, which were characterized as overgrown ductules/buds featured by crowded atypical Nanog-expressing luminal cells. Taken together, our present work demonstrates that transgenic overexpression of NanogP8 in the mouse prostate is insufficient to initiate tumorigenesis but weakly promotes tumor development in the Hi-Myc mouse model.

Breast tumors contain tumorigenic cancer cells, termed "tumor-initiating cells" (TICs), which are capable of both replenishing themselves and giving rise to populations of nontumorigenic breast cancer cells (non-TICs). However, the molecular mechanisms responsible for breast tumor initiation remain poorly understood. Here we describe a chemical screening strategy to identify small molecules that enhance the effect of chemotherapeutic agents on TIC-enriched breast cancer cells. We identified proteins that interact with the lead compound C108, including the stress granule-associated protein, GTPase-activating protein (SH3 domain)-binding protein 2, G3BP2. G3BP2 regulates breast tumor initiation through the stabilization of Squamous cell carcinoma antigen recognized by T cells 3 (SART3) mRNA, which leads to increased expression of the pluripotency transcription factors Octamer-binding protein 4 (Oct-4) and Nanog Homeobox (Nanog). Our findings suggest that G3BP2 is important for the process of breast cancer initiation. Furthermore, these data suggest a possible connection between stress granule formation and tumor initiation in breast cancer cells.

The cerebellum is a prominent part of the vertebrate hindbrain that is critically involved in the regulation of important body functions such as movement coordination, maintenance of balance and posture, and motor control. Here, we describe a cerebellar window that provides access to the mouse cerebellum for intravital imaging, thereby allowing for a detailed characterization of the dynamic processes in this region of the brain. First, the skull overlying the cerebellum is removed, and then the window is applied to the region of interest. Windows may be exchanged depending on the desired imaging modality. This technique has a variety of applications. In the setting of medulloblastoma, spontaneous or orthotopically implanted lesions can be imaged, and tumor morphology and size can be monitored using ultrasonography. Multiphoton laser-scanning microscopy (MPLSM) or optical frequency-domain imaging (OFDI) can be applied for in vivo visualization and analysis of cellular and vascular structures in a variety of disease states, including malignancies and ataxia telangiectasia. This protocol describes a novel and rapid method for cerebellar window construction that can be set up in under an hour.

Abstract Background: Brain metastases represent a major problem in the treatment of HER2-positive breast cancer (1). The antibody-drug conjugate ado-trastuzumab emtansine (T-DM1) has shown efficacy in trastuzumab-resistant systemic breast cancer. Here, we tested the hypothesis that T-DM1 could overcome trastuzumab resistance in murine models of brain metastases. Methods: We used previously established animal models of HER2-positive breast cancer brain metastases and organotypic brain slice cultures that recapitulate clinical scenarios (2). We treated mice bearing HER2-positive breast cancer brain metastases with trastuzumab or T-DM1 at equivalent or equipotent doses. Using intravital imaging, molecular techniques and histological analysis we determined tumor growth, mouse survival, cancer cell apoptosis and proliferation, tumor drug distribution, gene expression, and HER2 downstream signaling. Results: T-DM1 significantly delayed the growth of HER2-positive breast cancer brain metastases compared to trastuzumab. These findings were consistent between HER2-driven and PI3K-driven breast tumors. The activity of T-DM1 resulted in a striking survival benefit compared to trastuzumab (median survival for BT474 tumors: 28d for trastuzumab vs 112d for T-DM1, HR=6.2, P<0.001). A comparison of T-DM1 with trastuzumab revealed no difference in their tumor distribution, HER2 downstream signaling inhibition or immune cell enrichment. T-DM1, however, led to a significant increase in tumor cell apoptosis. Electron microscopy studies revealed increased numbers of abnormal mitotic figures in brain tumors treated with T-DM1. Whole-transcriptome microarray analysis of BT474 brain tumors treated with trastuzumab or T-DM1 showed an enrichment of genes that are associated with mitotic catastrophe in the group treated with the antibody-drug conjugate. These mechanistic studies support the hypothesis that the efficacy of ado-trastuzumab emtansine in the brain microenvironment is mediated through the cytotoxic chemotherapeutic effect of the DM1 component. Conclusions: Our findings suggest that T-DM1 can overcome resistance to HER2-targeted therapies in the CNS, and warrants clinical investigation for the effective treatment of HER2-positive breast cancer brain metastases. References: 1. Kodack DP, Askoxylakis V, Ferraro GB, et al. Emerging strategies for treating brain metastases from breast cancer. Cancer Cell 2015, 27(2):163-175. 2. Kodack DP, Chung E, Yamashita H, et al. Combined targeting of HER2 and VEGFR2 for effective treatment of HER2-amplified breast cancer brain metastases. Proc Natl Acad Sci U S A 2012, 109(45):E3119-3127. Citation Format: Askoxylakis V, Ferraro G, Kodack D, Badeaux M, Jain R. Ado-trastuzumab emtansine (T-DM1) is effective against established HER2-positive breast cancer brain metastases in mice. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P6-17-02.

Abstract Brain metastases represent a major problem in the treatment of HER2-positive breast cancer due to the poor efficacy of HER2-targeted therapies in the brain microenvironment. The antibody drug conjugate ado-trastuzumab emtansine (T-DM1) has shown efficacy in trastuzumab-resistant systemic breast cancer. We tested the hypothesis that T-DM1 could overcome trastuzumab resistance in murine models of brain metastases. Methods: We optimized established animal models of HER2-positive breast cancer brain metastases. We treated mice bearing BT474 (intracranial and intracarotid injections) or MDA-MB-361 (intracranial injection) tumors in the CNS with trastuzumab or T-DM1 at equivalent or equipotent doses. Using intravital imaging, molecular techniques and histological analysis we determined tumor growth, mouse survival, cancer cell apoptosis and proliferation, tumor drug distribution, and HER2 signaling. Results: Treatment with T-DM1 delayed tumor growth in comparison to trastuzumab and control IgG and improved survival. These findings were consistent between HER2-driven and PI3K-driven breast tumors. In BT474 tumors, median survival was 112 days for T-DM1 and 28 days for trastuzumab (p<0.05). Mechanistic studies revealed no difference in HER2 downstream signaling, drug distribution or immune cell enrichment between T-DM1 and trastuzumab treated mice. A significantly increased apoptotic rate was measured for brain metastases treated with the antibody-drug conjugate. Conclusions: T-DM1 can overcome resistance to trastuzumab therapy in HER2-driven or PI3K-driven breast cancer brain lesions due to the cytotoxicity of the DM1 component. The results of our studies indicate that T-DM1 is effective in the brain microenvironment and will directly inform clinical trials in patients with HER2+ breast cancer brain metastases. Citation Format: Gino B. Ferraro, Vasileios Askoxylakis, David P. Kodack, Mark Badeaux, Dai Fukumura, Jeffrey A. Engelman, Rakesh K. Jain. Ado-trastuzumab emtansine (T-DM1) controls tumor progression of established HER2-positive breast cancer brain metastases in mice. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Metastasis; 2015 Nov 30-Dec 3; Austin, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(7 Suppl):Abstract nr A48.

Background: Brain metastases represent a major problem in the treatment of HER2-positive breast cancer (1). The antibody-drug conjugate ado-trastuzumab emtansine (T-DM1) has shown efficacy in trastuzumab-resistant systemic breast cancer. Here, we tested the hypothesis that T-DM1 could overcome trastuzumab resistance in murine models of brain metastases. Methods: We used previously established animal models of HER2-positive breast cancer brain metastases and organotypic brain slice cultures that recapitulate clinical scenarios (2). We treated mice bearing HER2-positive breast cancer brain metastases with trastuzumab or T-DM1 at equivalent or equipotent doses. Using intravital imaging, molecular techniques and histological analysis we determined tumor growth, mouse survival, cancer cell apoptosis and proliferation, tumor drug distribution, gene expression, and HER2 downstream signaling. Results: T-DM1 significantly delayed the growth of HER2-positive breast cancer brain metastases compared to trastuzumab. These findings were consistent between HER2-driven and PI3K-driven breast tumors. The activity of T-DM1 resulted in a striking survival benefit compared to trastuzumab (median survival for BT474 tumors: 28d for trastuzumab vs 112d for T-DM1, HR=6.2, P Conclusions: Our findings suggest that T-DM1 can overcome resistance to HER2-targeted therapies in the CNS, and warrants clinical investigation for the effective treatment of HER2-positive breast cancer brain metastases. References: 1. Kodack DP, Askoxylakis V, Ferraro GB, et al. Emerging strategies for treating brain metastases from breast cancer. Cancer Cell 2015, 27(2):163-175. 2. Kodack DP, Chung E, Yamashita H, et al. Combined targeting of HER2 and VEGFR2 for effective treatment of HER2-amplified breast cancer brain metastases. Proc Natl Acad Sci U S A 2012, 109(45):E3119-3127. Citation Format: Askoxylakis V, Ferraro G, Kodack D, Badeaux M, Jain R. Ado-trastuzumab emtansine (T-DM1) is effective against established HER2-positive breast cancer brain metastases in mice. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P6-17-02.

Background: Central nervous system (CNS) metastases represent a major problem in the treatment of human epidermal growth factor receptor 2 (HER2)-positive breast cancer because of the disappointing efficacy of HER2-targeted therapies against brain lesions. The antibody-drug conjugate ado-trastuzumab emtansine (T-DM1) has shown efficacy in trastuzumab-resistant systemic breast cancer. Here, we tested the hypothesis that T-DM1 could overcome trastuzumab resistance in murine models of brain metastases.Methods: We treated female nude mice bearing BT474 or MDA-MB-361 brain metastases (n = 9-11 per group) or cancer cells grown in organotypic brain slice cultures with trastuzumab or T-DM1 at equivalent or equipotent doses. Using intravital imaging, molecular techniques and histological analysis we determined tumor growth, mouse survival, cancer cell apoptosis and proliferation, tumor drug distribution, and HER2 signaling. Data were analyzed with one-way analysis of variance (ANOVA), Kaplan-Meier analysis, and Coefficient of Determination. All statistical tests were two-sided.Results: T-DM1 delayed the growth of HER2-positive breast cancer brain metastases compared with trastuzumab. These findings were consistent between HER2-driven and PI3K-driven tumors. The activity of T-DM1 resulted in a survival benefit (median survival for BT474 tumors: 28 days for trastuzumab vs 112 days for T-DM1, hazard ratio = 6.2, 95% confidence interval = 6.1 to 85.84, P < .001). No difference in drug distribution or HER2-signaling was revealed between the two groups. However, T-DM1 led to a statistically significant increase in tumor cell apoptosis (one-way ANOVA for ApopTag, P < .001), which was associated with mitotic catastrophe.Conclusions: T-DM1 can overcome resistance to trastuzumab therapy in HER2-driven or PI3K-driven breast cancer brain lesions due to the cytotoxicity of the DM1 component. Clinical investigation of T-DM1 for patients with CNS metastases from HER2-positive breast cancer is warranted.

15-Lipoxygenase-2 (15-LOX2) is a human-specific lipid-peroxidizing enzyme most prominently expressed in epithelial cells of normal human prostate but downregulated or completely lost in>70% of prostate cancer (PCa) cases. Transgenic expression of 15-LOX2 in the mouse prostate surprisingly causes hyperplasia. Here we first provide evidence that 15-LOX2-induced prostatic hyperplasia does not progress to PCa even in p53(+/-) or p53(-/-) background. More important, by generating 15-LOX2; Hi-Myc double transgenic (dTg) mice, we show that 15-LOX2 expression inhibits Myc-induced PCa development, such that in the 3-month- and 6-month-old dTg mice, there is a significant reduction in prostate intraneoplasia (PIN) and PCa prevalent in age-matched Hi-Myc prostates. The dTg prostates show increased cell senescence and expression of several senescence-associated molecules, including p27, phosphorylated Rb, and Rb1cc1. We further show that in HPCa, 15-LOX2 and c-Myc manifest reciprocal protein expression patterns. Moreover, RB1CC1 accumulates in senescing normal human prostate (NHP) cells, and in both NHP and RWPE-1 cells, the 15-LOX2 metabolic products 15(S)-HPETE and 15(S)-HETE induce RB1CC1. We finally show that unlike 15-LOX2, RB1CC1 is not lost but rather frequently overexpressed in PCa samples. RB1CC1 knockdown in PC3 cells enhances clonal growth in vitro and tumor growth in vivo. Together, our present studies provide evidence for tumor-suppressive functions for both 15-LOX2 and RB1CC1.

Human Nanog1 is a 305-amino acid (aa) homeodomain-containing transcription factor critical for the pluripotency of embryonic stem (ES) and embryonal carcinoma (EC) cells. Somatic cancer cells predominantly express a retrogene homolog of Nanog1 called NanogP8, which is ~99% similar to Nanog at the aa level. Although the predicted M.W of Nanog1/NanogP8 is ∼35 kD, both have been reported to migrate, on Western blotting (WB), at apparent molecular masses of 29-80 kD. Whether all these reported protein bands represent authentic Nanog proteins is unclear. Furthermore, detailed biochemical studies on Nanog1/NanogpP8 have been lacking. By combining WB using 8 anti-Nanog1 antibodies, immunoprecipitation, mass spectrometry, and studies using recombinant proteins, here we provide direct evidence that the Nanog1 protein in NTERA-2 EC cells exists as multiple M.W species from ~22 kD to 100 kD with a major 42 kD band detectable on WB. We then demonstrate that recombinant NanogP8 (rNanogP8) proteins made in bacteria using cDNAs from multiple cancer cells also migrate, on denaturing SDS-PAGE, at ~28 kD to 180 kD. Interestingly, different anti-Nanog1 antibodies exhibit differential reactivity towards rNanogP8 proteins, which can spontaneously form high M.W protein species. Finally, we show that most long-term cultured cancer cell lines seem to express very low levels of or different endogenous NanogP8 protein that cannot be readily detected by immunoprecipitation. Altogether, the current study reveals unique biochemical properties of Nanog1 in EC cells and NanogP8 in somatic cancer cells.

MicroRNA-128 suppresses prostate cancer by inhibiting BMI-1 to inhibit tumor-initiating cells Min Jin*, Tao Zhang*, Can Liu, Mark A. Badeaux, Bigang Liu, Ruifang Liu, Collene Jeter, Xin Chen, Alexander V. Vlassov, and Dean G. Tang Department of Molecular Carcinogenesis, The University of Texas M.D Anderson Cancer Center, Science Park, Smithville, TX 78957, USA Cancer Center, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology (HUST), Wuhan, Hubei, 430023, China Research Center for Translational Medicine, Shanghai East Hospital, Tongji University School of Medicine, Shanghai 200120, China ThermoFisher Scientific, Austin, TX 78744, USA