Breast Cancer Cell Line Xenografts Research Articles

Abstract P3-04-15: The PI3K-inhibitor, copanlisib, has selective activity in luminal breast cancer cell lines and shows robust combined activity with hormonal blockade and CDK-4/6 inhibition in ER+ breast cancer cell line xenografts

Abstract Background: Genetic and epigenetic alterations in the PI3K/mTOR and cyclin D:CDK-4/6:Rb signaling axes occur frequently in breast cancer and have been attributed to resistance to both ER- and HER2-directed therapeutics. Pharmacologically targeting CDK-4/6 in combination with hormonal blockade provides clinical benefit in patients with advanced ER+ breast cancer. In this study, we evaluated the activity of the pan-class I PI3K inhibitor, copanlisib (BAY-80-6946), with potent alpha and delta activity as a single agent or in combination with CDK-4/6 inhibition and hormonal blockade in a panel of breast cancer cell lines. Methods: The growth inhibitory activity of copanlisib was evaluated against a large panel of 48 breast cancer cell lines molecularly characterized by genomic, transcriptomic and proteomic profiling. IC50 values were determined from direct cell counts using a Z1-particle counter. The activity of copanlisib in combination with hormone blockade and CDK-4/6 inhibition, by palbociclib, was assessed in two cell line xenograft models of ER+ breast cancer; MCF7(PIK3CA-E545K) and ZR751(PIK3CA WT). For xenograft studies, tumor bearing mice were treated once weekly (BID) by intravenous injection with clinically achievable doses of copanlisib (10 mg/kg) as single agent or in combination with tamoxifen or fulvestrant with or without 75 mg/kg daily palbociclib for 21 days. Results: A broad range of IC50 values (0.491-895 nM), with a high degree of separation between sensitive and resistant histologically defined subgroups were determined for copanlisib, indicating the potential for a wide therapeutic window. Luminal subtype, the presence of activating mutations in PIK3CA, high levels of ER, HER2, HER3 and EGFR protein enriched for sensitivity to copanlisib. Activating mutations of KRAS and BRAF were associated with resistance to copanlisib. Single agent copanlisib induced significant tumor growth inhibition (TGI) relative to vehicle control in each of the xenograft models. Modest increases in anti-tumor activity were achieved when copanlisib was combined with hormonal blockade by either tamoxifen or fulvestrant. However, robust tumor regressions were observed with the triple combinations of copanlisib-palbociclib-tamoxifen and copanlisib-palbociclib-fulvestrant. Furthermore, these triple combinations achieved a statistically significant improvement in anti-tumor activity over the standard of care combination of palbociclib plus fulvestrant. Each of the single agent and treatment combinations tested were well tolerated in animals. Discussion: These preclinical data illustrate the potent and selective activity of the pan class I PI3K inhibitor copanlisib in luminal breast cancers and support the clinical investigation of copanlisib in combination with CDK-4/6 inhibition and hormonal blockade in ER+ breast cancer. Citation Format: O'Brien NA, Conklin D, Luo T, Ayala R, Issakhanian S, Kalous O, Von Euw E, Politz O, Wilhelm S, Childs BH, Hurvitz SA, Slamon DJ. The PI3K-inhibitor, copanlisib, has selective activity in luminal breast cancer cell lines and shows robust combined activity with hormonal blockade and CDK-4/6 inhibition in ER+ breast cancer cell line xenografts [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P3-04-15.

Expression and function of MutT homolog 1 in distinct subtypes of breast cancer

Human MutT homolog 1 (MTH1) detoxifies the oxidized DNA precursor 8-oxo-2′-deoxyguanosine-5′-triphosphate and serves a tumor suppressive role in distinct types of cancer. In the present study, the expression of MTH1 was examined in various subtypes of breast cancer, and the effect of its suppression on breast cancer growth was characterized in vitro and in vivo. MTH1 mRNA and protein levels were assessed using the reverse transcription-quantitative polymerase chain reaction and immunohistochemistry. The effect of MTH1 expression on the proliferation of breast cancer cells was investigated in vitro using Cell Counting Kit-8 and colony formation assays, and in vivo using breast cancer cell line xenografts in mice. The toxicity of the MTH1 inhibitor TH588 was investigated in nude mice. A marked increase in MTH1 protein and mRNA levels was demonstrated in breast cancer tissues compared with the non-cancerous control. However, no apparent differences in MTH1 expression were observed between distinct molecular subtypes of breast cancer. MTH1 overexpression was demonstrated to be independent of patient age, tumor size and lymph node metastasis. Inhibition of MTH1 decreased cancer cell viability and the clonogenic potential of cancer cells in a dose-dependent manner. These results were confirmed by decreased in vivo proliferation of MCF7, MDA-MB-231 and MDA-MB-453 cancer cell lines, representing distinct subtypes of breast cancer. Although inhibition of MTH1 activity decreased xenograft growth in mice, no major adverse effects of TH588 were detected on the basis of blood biochemistry, and liver and kidney function. The results of the present study suggested that MTH1 is overexpressed in the majority of breast cancers, independent of the molecular identity and clinicopathological features of the tumor, including patient age, tumor size and lymph node metastasis. Inhibition of MTH1 activity suppressed the growth of three subtypes of breast cancer, including luminal, basal-like and human epidermal growth factor receptor 2-positive, in vitro and in vivo. Treatment with the MTH1 inhibitor appears to be safe; however, further studies are required prior to the clinical use of MTH1 inhibitors.

Abstract 2828: Preclinical activity of abemaciclib as a single agent or in combination with anti-mitotic or targeted therapies for breast cancer

Abstract The cyclin D:CDK-4/6:Rb axis is dysregulated in a number of different cancers and is implicated in resistance to hormonal therapy in breast cancer. Pharmacologically targeting cyclin dependent kinase 4 and 6 (CDK4 and 6) has proven to be a successful therapeutic approach in ER+ breast cancer (BC). This study aimed to identify the molecular subtypes of BC that are sensitive to the novel CDK4 and 6 inhibitor, abemaciclib, and identify the best combination strategies for the clinical development. Growth inhibition activity of abemaciclib was assessed in a panel of 46 BC cell lines molecularly characterized by genomic, transcriptomic and proteomic profiling. IC50 values were determined from direct cell counts using a Z1-particle counter. In vivo activity of abemaciclib was assessed in cell line xenograft models of ER+ and HER2+/ER+ BCs. For ER+ BC, mice were treated daily with clinically achievable doses of abemaciclib (50-75 mg/kg) as a single agent or in combination with tamoxifen or fulvestrant. Combinations with trastuzumab, docetaxel and tamoxifen were assessed in the HER2+/ER+ xenografts. Sensitivity to abemaciclib was observed predominately in multiple luminal BC cell lines and a small subset of triple negative cell lines that had intact Rb signaling. Activating mutations in PIK3CA also marked for abemaciclib sensitivity. Abemaciclib potentiated the anti-proliferative effects of cytotoxic/anti-mitotic agents when given simultaneously or 48 hours prior to treatment in vitro. Significant tumor growth inhibition (TGI) was observed with single agent abemaciclib in the ER+ BC cell line xenografts. In ZR751 xenografts, the addition of either tamoxifen or fulvestrant to abemaciclib induced complete inhibition of tumor growth for the 12 weeks of treatment. In the MCF7 model, treatment was withdrawn after five weeks, which triggered tumor regrowth in each of the single agent arms. However, complete responses were maintained in the combination arms for a further six weeks post drug withdrawal. In HER2 amplified xenografts, abemaciclib single agent treatment induced significant TGI in trastuzumab sensitive and resistant xenografts, and combination with trastuzumab further increased this anti-tumor effect. The addition of tamoxifen to this combination induced a further increment in TGI. Consistent with the in vitro findings, the combination of abemaciclib and the anti-mitotic agent docetaxel was not antagonistic in vivo, and the addition of docetaxel to the triple combination of abemaciclib, trastuzumab, and tamoxifen induced the most efficacy of any of the treatment arms tested. Combinations were well tolerated in animals. These data highlight the potential of abemaciclib to have single agent activity in addition to combined activity with anti-mitotic or targeted therapies for breast cancer. Citation Format: Neil A. O’Brien, Dylan Conklin, Tong Luo, Ondrej Kalous, Erika von Euw, Sara A. Hurvitz, Richard P. Beckmann, Colleen Mockbee, Dennis J. Slamon. Preclinical activity of abemaciclib as a single agent or in combination with anti-mitotic or targeted therapies for breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2828.

Construction of Nude Mouse Model of Triple Negative Breast Cancer and Value of Thermal Tomography

To explore the early detection of breast cancer by ultrasonic imaging and thermal tomography of luciferase or green fluorescent protein (GFP)-labeled MDA-MB-231 breast cancer cell line-xenografts in nude mice. Fluorescence-tagged lentiviral vectors were transfected into the triple-negative breast cancer cell line MDA-MB-231. These cells were implanted either subcutaneously under the right breast pad or intravenously into the tail vein of nude BALB/C mice. Thermal tomography and ultrasound imaging were used to detect tumor formation and to monitor tumor growth and metastasis in vivo. Triple negative breast cancer cell line-xenografts were used to successfully construct an orthotopic nude mice model of breast cancer metastasis in the peritoneum. Thermal tomography and ultrasound imaging were used together to detect small tumors. Thermal tomography imaging detected small tumors earlier than ultrasound imaging. Thermal tomography can be used to monitor changes in tumor growth and detect abnormal tissue. Therefore, it can serve as a convenient,rapid,sensitive, and reliable technique for early screening of human breast cancer.

Abstract C126: PRLR ADC: A novel antibody drug-conjugate for the treatment of PRLR positive breast cancer

Abstract Breast cancer is a heterogeneous disease comprised of various subtypes based on pathology and molecular profiling. Expression of hormone receptors (HR) and HER2 biomarkers are important determinants of therapy choice, due to the established role of these proteins as drivers of disease. Prolactin Receptor (PRLR) is a type 1 cytokine receptor that is expressed on a subset of breast cancers and may contribute to pathogenesis. Functionally, activation of PRLR by the hormone ligand Prolactin (PRL) induces PRLR dimerization and signaling resulting in cell proliferation and differentiation. While PRLR is expressed at low levels in some normal human tissues including the mammary gland, it is relatively overexpressed in ∼25% of human breast tumors and importantly, is rapidly internalized upon binding of anti-PRLR antibodies. We developed an anti-PRLR antibody-drug conjugate (ADC), PRLR ADC, to target PRLR positive breast cancer. PRLR ADC is comprised of a fully human high affinity function-blocking anti-PRLR IgG1 antibody conjugated via a non-cleavable SMCC linker to the cytotoxic maytansine derivative DM1. Both unconjugated anti-PRLR antibody and the PRLR ADC block PRL mediated activation in vitro and induce rapid internalization of the receptor into lysosomes. PRLR ADC induces potent cell cycle arrest and cytotoxicity in several PRLR-expressing cell lines. The in vivo efficacy of PRLR ADC was explored in breast cancer cell line xenograft models expressing both endogenous PRLR (MCF7, T47D) or transfected receptor (MCF7/PRLR). Treatment of tumor bearing SCID (T47D) or NCr Nude (MCF7) animals was initiated approximately 15 days post implantation of cells where tumor volumes averaged 150-200 mm3. In both T47D and MCF7/PRLR xenograft models, where PRLR is expressed highly, single or multiple (once weekly x 3) doses of 2.5-15 mg/kg resulted in significant inhibition of tumor xenograft growth. In the MCF7 model that expresses low levels of PRLR, inhibition and regression of tumors was observed at 10 and 15 mg/kg dose levels. In all models, higher doses resulted in greater and more prolonged repression of tumor growth. Conjugation of DM1 to anti-PRLR antibody was required for efficacy, as unconjugated antibody had no effect on tumor growth. Anti-tumor efficacy of PRLR ADC was also assessed in NSG mice bearing breast cancer Patient Derived Xenograft (PDXs) tumors with moderate and heterogeneous expression of PRLR. Treatment was initiated 21 days after implantation of the PDX tumors where the average tumor volume was ∼500mm3. Anti-tumor efficacy was observed following 10 or 20 mg/kg PRLR ADC dosed once weekly x 4. These studies demonstrate the promising anti-tumor activity of the PRLR ADC against PRLR positive breast cancers and support the continued development of this agent. Citation Format: Marcus P. Kelly, Sandra Coetzee, Carlos Hickey, Sosina Makonnen, Frank Delfino, Julian Andreev, Arthur Kunz, Christopher D'Souza, Jason Giurleo, Thomas Nittoli, Pamela A. Trail, Nicholas Papadopoulos, Gavin Thurston, Jessica R. Kirshner. PRLR ADC: A novel antibody drug-conjugate for the treatment of PRLR positive breast cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C126.

Abstract P5-05-04: Acquired resistance to everolimus occurs independently of mTORC1 inhibition in preclinical in vivo models of ER+ breast cancer

Abstract Background: The addition of the selective mTORC1 inhibitor everolimus (RAD001/Afinitor®), to exemestane significantly prolongs progression free survival in patients with advanced hormone receptor (ER+) positive breast cancer. However, despite these improved outcomes, the majority of patients that achieve an initial clinical benefit will eventually go on to develop progressive disease. In order to prevent or delay the onset of this acquired resistance, it is essential to identify the mechanisms by which continued proliferation is being driven in these resistant tumors. In this study, we used reverse phase protein array (RPPA) technology to compare cell signaling in ER+ breast cancer cell line xenografts that were responsive to everolimus versus those that had acquired resistance to everolimus. Materials and Methods: Xenograft models were established from three ER+ breast cancer cell lines containing different molecular alterations that confer activated PI3K/mTOR signaling; MCF7 and KPL-1 (both PIK3CA mutant) and ZR75-1 (PTEN-null). Mice were treated daily with either vehicle or 10 mg/kg everolimus until progression. Snap frozen tissue samples (n = 3) were collected from vehicle control tumors, from everolimus resistant tumors and from everolimus responsive tumors. Whole cell lysates were prepared from tumor tissues and processed for RPPA analysis. A customized panel of 27 known cancer/PI3K pathway associated signaling proteins were selected for the RPPA analysis. Results: In the vehicle controls, significantly higher levels of pAKT, pPRAS40 and p4EBP1 were detected in the PTEN-null ZR75-1 tumors compared to the PIK3CA mutant tumors, indicating potential variations in intensity of PI3K/AKT pathway signaling depending on the initiating molecular alteration. Complete inhibition of tumor proliferation was observed in response to everolimus in all 3 xenograft models for up to 7-weeks of treatment, until tumors showed signs of progression. Significantly lower S6 phosphorylation was observed in tumors responding to everolimus, which was accompanied by feedback activation of pAKT. Significant inhibition of S6 phosphorylation was also observed in each of the ZR75-1 and MCF7 tumors that were progressing on everolimus, indicating that although the tumor cells have acquired resistance to the anti-proliferative activity of everolimus, mTORC1 signaling is still being successfully blocked. Feedback activation of pAKT was observed in the ZR75-1 resistant model, however no feedback activation of AKT occurred in the MCF7 resistant model. The KPL-1 model showed the expected loss of S6 pathway inhibition upon resistance. No significant signaling changes were observed in any of the other signaling proteins measured, including ER, HER2, EGFR, MEK and ERK. Ongoing studies to measure the mRNA expression changes in these tumors using mRNA microarrays may identify signaling pathways that are driving the proliferation of these resistant tumors. Discussion: These preclinical data show that multiple pathways of resistance can develop in response to long-term everolimus treatment and resistance can occur despite continued inhibition of PI3K/mTOR signaling. Data from mRNA microarray experiments will be presented at the time of the meeting. Citation Format: Neil A O'Brien, Dylan Conklin, Michaela Ching, Luo Tong, Raul Ayala, Shawnt Issakhanian, Sara A Hurvitz, Emmanuelle di Tomaso, Ronald Linnartz, Richard S Finn, Samit Hirawat, Dennis J Slamon. Acquired resistance to everolimus occurs independently of mTORC1 inhibition in preclinical in vivo models of ER+ breast cancer [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P5-05-04.

Abstract P5-05-05: Low molecular weight cyclin E regulates response to aromatase inhibitors in post-menopausal breast cancer patients

Abstract Almost seventy percent of all breast cancer patients have estrogen receptor (ER) positive tumors requiring hormonal therapy. Aromatase inhibitors (AIs) are considered as the first line hormonal therapy for ER+ post-menopausal patients. However, resistance to these drugs remains a major challenge in clinic and the biology of such resistance is not clear. Previous studies have shown that cyclin E pathway, a key regulator in the G1 to S transition of cell cycle, is deregulated in breast cancer. Full-length cyclin E is abnormally cleaved into low molecular weight isoforms (LMW-E) that renders patients to poor survival. Here we hypothesize that cyclin E deregulation can confer resistance to AIs. To address this, we engineered aromatase overexpressing MCF7 cells to overexpress LMW-E under doxycycline inducible promoter. Full-length cyclin E, GFP and empty vector transfected cells were also generated and used as controls. Our results indicated that AIs inhibited proliferation by arresting the cells at G1 phase of the cell cycle. However, LMW-E expression significantly enhanced proliferation of the cells when treated with AIs. In addition, LMW-E bypassed G1 arrest following AI treatment. At the molecular level, AIs decreased CDK2, pCDK2, and Rb levels and attenuated Rb phosphorylation. However, these effects were completely rescued only when LMW-E was expressed. Moreover, using an in vitro kinase assay we indicated that AIs decreased CDK2 kinase activity while LMW-E expression reversed this effect by increasing CDK2 enzymatic activity. Taken together, these results suggest that LMW-E inactivates Rb protein as a tumor suppressor and renders the cells to bypass G1 checkpoint following AI treatment. In addition, this study provides early evidence that CDK2 inhibitors could be beneficial in combination with AIs for LMW-E expressing tumors. Currently we are investigating whether LMW-E can bypass the activity of AIs using inducible breast cancer cell line xenograft. We are also examining the correlation between cyclin E status and response to treatment in a cohort of patients who received AIs in the neo-adjuvant setting. Citation Format: Iman Doostan, Stacy L Moulder, Kelly K Hunt, Khandan Keyomarsi. Low molecular weight cyclin E regulates response to aromatase inhibitors in post-menopausal breast cancer patients [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P5-05-05.

Anti-metastasis activity of black rice anthocyanins against breast cancer: analyses using an ErbB2 positive breast cancer cell line and tumoral xenograft model.

Increasing evidence from animal, epidemiological and clinical investigations suggest that dietary anthocyanins have potential to prevent chronic diseases, including cancers. It is also noteworthy that human epidermal growth factor receptor 2 (ErbB2) protein overexpression or ErbB2 gene amplification has been included as an indicator for metastasis and higher risk of recurrence for breast cancer. The present experiments investigated the anti-metastasis effects of black rice anthocyanins (BRACs) on ErbB2 positive breast cancer cells in vivo and in vitro. Oral administration of BRACs (150 mg/kg/day) reduced transplanted tumor growth, inhibited pulmonary metastasis, and decreased lung tumor nodules in BALB/c nude mice bearing ErbB2 positive breast cancer cell MDA-MB-453 xenografts. The capacity for migration, adhesion, motility and invasion was also inhibited by BRACs in MDA-MB-453 cells in a concentration dependent manner, accompanied by decreased activity of a transfer promoting factor, urokinase-type plasminogen activator (u-PA). Together, our results indicated that BRACs possess anti-metastasis potential against ErbB2 positive human breast cancer cells in vivo and in vitro through inhibition of metastasis promoting molecules.

Drug-repositioning screening identified piperlongumine as a direct STAT3 inhibitor with potent activity against breast cancer.

Signal transducer and activator of transcription (STAT) 3 regulates many cardinal features of cancer including cancer cell growth, apoptosis resistance, DNA damage response, metastasis, immune escape, tumor angiogenesis, the Warburg effect, and oncogene addiction and has been validated as a drug target for cancer therapy. Several strategies have been employed to identify agents that target Stat3 in breast cancer but none has yet entered into clinical use. We used a high-throughput fluorescence microscopy search strategy to identify compounds in a drug-repositioning library (Prestwick library) that block ligand-induced nuclear translocation of Stat3 and identified piperlongumine (PL), a natural product isolated from the fruit of the pepper Piper longum. Piperlongumine inhibited Stat3 nuclear translocation, inhibited ligand-induced and constitutive Stat3 phosphorylation, and modulated expression of multiple Stat3-regulated genes. Surface plasmon resonance assay revealed that piperlongumine directly inhibited binding of Stat3 to its phosphotyrosyl (pY) peptide ligand. Phosphoprotein antibody array analysis revealed that PL does not modulate kinases known to activate Stat3 such as JAKs, Src kinase family members, or RTKs. PL inhibited anchorage-independent and anchorage-dependent growth of multiple breast cancer cell lines having increased pStat3 or total Stat3, and induced apoptosis. PL also inhibited mammosphere formation by tumor cells from patient derived xenografts (PDX). PL’s anti-tumorigenic function was causally linked to its Stat3-inhibitory effect. PL was non-toxic in mice up to a dose of 30 mg/Kg/day for 14 days and caused regression of breast cancer cell line xenografts in nude mice. Thus, PL represents a promising new agent for rapid entry into the clinic for use in treating breast cancer, as well as other cancers in which Stat3 plays a role.

Abstract 3726: Combinations of HDAC inhibitor, chemotherapeutic agent and retinoic acid induce growth arrest, differentiation and tumor regression in preclinical models of breast cancer.

Abstract The histone deacetylase inhibitor (HDACi), entinostat, is being actively explored as a new-generation epigenetic drug which can lead to the change in the expression status of genes/pathways, but has low efficacy in cancer monotherapy. All-trans retinoic acid (ATRA) induces the differentiation of various types of stem cells. Data from cell culture and xenograft models from our lab showed that a combination of entinostat (MS-275), doxorubicin and ATRA effectively decreased tumor size in three breast cancer cell line xenograft models. Here, we sought to further investigate the mechanism of action of the triple drug combination in cancer cells; in particular, its effect on the breast cancer stem cell population. We performed a comprehensive genome wide analysis of gene expression of MDA-MB-231 breast cancer cells treated with ATRA, MS-275 and doxorubicin as monotherapies and as combination therapies. We saw that the drug-response gene profile of ATRA is very similar to DMSO (vehicle)-treated cells. Accordingly, the addition of ATRA (A) to MS-275 (MA), Dox (AD) and MS-275/Dox (MAD) displayed minimal changes in the gene expression profile of each of the other treatments. Addition of Dox to MS-275 (MD), on the other hand, potentiated the “reprograming” effect of MS-275 and affected the expression of many antitumor genes known to be related to cell cycle and growth arrest. It also altered expression of genes involved in development and inflammation. The most differentially expressed genes, validated by qPCR, were novel genes from the cancer/testis antigens and tripartite motif (TRIM) family of proteins. Interestingly, in MDA-MB-231and SUM149 cells, even the addition of low doses of doxorubicin (12.5 nM) to MS-275 increased 2 and 2.6 fold the G2 cell cycle arrest in comparison to Dox and MS-275, respectively. Despite the gene expression pattern similarity between MS-275/Dox (MD) and MS-275/Dox/ATRA (MAD) groups, we saw that MAD was more effective in inducing cell death and apoptosis in vitro and in vivo. The epithelium specific ETS transcription factor-1 (ESE-1) was differentially regulated between MAD and MD and is, in fact, part of the MS-275/ATRA (MA) signature. Using limiting dilution transplantation assays in mammary fat pads of immunodeficient mice we observed that MAD treatment in vivo most effectively targeted breast cancer stem cells (BCSC) compared to any other combination of drugs. The cancer stem cell frequency of the cells isolated from MAD treated mice was 1 in 236,570. The second most effective treatment for BCSC was MA (1 in 150,721), followed by ATRA> MS-275>MD>Dox>DMSO>AD. In conclusion, the reprogramming events initiated by HDACi and retinoid sensitize the cells to low doses of doxorubicin. The combination therapy may have a significant effect in decreasing breast tumor growth and recurrence. Citation Format: Vanessa F. Merino, Nguyen Nguyen, Helen Sadik, Sean Cho, Xian Chong Zhou, Qian Chen, Duojia Pan, Saraswati Sukumar. Combinations of HDAC inhibitor, chemotherapeutic agent and retinoic acid induce growth arrest, differentiation and tumor regression in preclinical models of breast cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3726. doi:10.1158/1538-7445.AM2013-3726

Abstract 376: Limits of [18F]-FLT PET as a predictive imaging biomarker of proliferation in oncology

Abstract Background: Surrogate, non-invasive imaging biomarkers of cellular proliferation hold great promise for quantifying response to personalized medicine in oncology. An emerging imaging approach to assess tumor proliferation utilizes the positron emission tomography (PET) tracer 3′-deoxy-3′[18F]-fluorothymidine, [18F]-FLT. Though several studies have associated serial changes in [18F]-FLT-PET with elements of therapeutic response, the degree to which [18F]-FLT uptake quantitatively reflects proliferative index has been continuously debated for more that a decade. The goal of this preclinical study was to elucidate quantitative relationships between [18F]-FLT-PET and cellular, proteomic, and genomic metrics proliferation in treatment naive tumors. Methods and Findings: [18F]-FLT-PET imaging was conducted in mice bearing human colorectal or breast cancer cell line xenografts. Quantitative relationships between PET imaging, thymidine kinase 1 (TK1) protein and RNA levels, and immunostaining for proliferation markers (Ki67, TK1) were evaluated using imaging-matched tumor specimens. Overall, we found that [18F]-FLT-PET reflects TK1 protein levels in tumors. However, the cell cycle specificity of TK1 expression and the variable degree to which tumors utilize thymidine salvage for DNA synthesis tend to decouple [18F]-FLT-PET data from estimates of proliferation by standard, clinically employed means. Conclusions: Our findings illustrate that [18F]-FLT-PET reflects tumor proliferation as a function of thymidine salvage pathway utilization. Therefore, unlike more generalizable proliferation markers, such as Ki67 immunostaining, tumor uptake of [18F]-FLT reflects proliferative indices to variable and potentially unreliable extents. [18F]-FLT-PET can not discriminate moderately proliferative, thymidine salvage driven disease from highly proliferative tumors that rely primarily upon de novo thymidine synthesis. For those reasons, the magnitude of [18F]-FLT uptake in treatment naive tumors should not be considered a surrogate of proliferative index. These data provide rationale for the diversity of correlative results reported, both positive and discordant, comparing [18F]-FLT-PET and tumor proliferation across numerous single-center clinical studies and provide a basis for future clinical best-practices when this imaging biomarker is employed in oncology. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 376. doi:1538-7445.AM2012-376

FGFR Signaling Promotes the Growth of Triple-Negative and Basal-Like Breast Cancer Cell Lines Both In Vitro and In Vivo

The oncogenic drivers of triple-negative (TN) and basal-like breast cancers are largely unknown. Substantial evidence now links aberrant signaling by the fibroblast growth factor receptors (FGFR) to the development of multiple cancer types. Here, we examined the role of FGFR signaling in TN breast cancer. We examined the sensitivity of a panel of 31 breast cancer cell lines to the selective FGFR inhibitor PD173074 and investigated the potential mechanisms underlying sensitivity. TN breast cancer cell lines were more sensitive to PD173074 than comparator cell lines (P = 0.011), with 47% (7/15) of TN cell lines showing significantly reduced growth. The majority of TN cell lines showed only modest sensitivity to FGFR inhibition in two-dimensional growth but were highly sensitive in anchorage-independent conditions. PD173074 inhibited downstream mitogen-activated protein kinase and PI3K-AKT signaling and induced cell-cycle arrest and apoptosis. Basal-like breast cancer cell lines were found to express FGF2 ligand (11/21 positive) and, similarly, 62% of basal-like breast cancers expressed FGF2, as assessed by immunohistochemistry compared with 5% of nonbasal breast cancers (P < 0.0001). RNA interference targeting of FGF2 in basal-like cell lines significantly reduced growth in vitro and reduced down stream signaling, suggesting an autocrine FGF2 signaling loop. Treatment with PD173074 significantly reduced the growth of CAL51 basal-like breast cancer cell line xenografts in vivo. Basal-like breast cancer cell lines, and breast cancers, express autocrine FGF2 and show sensitivity to FGFR inhibitors, identifying a potential novel therapeutic approach for these cancers.

Abstract P5-02-01: FGFR Signalling Drives the Growth of Triple Negative and Basal-Like Breast Cancer Cell Lines Both In Vitro and In Vivo

Abstract Background: Substantial evidence now links aberrant signalling by the fibroblast growth factor receptors (FGFRs) to the development of multiple different cancer types. The oncogenic drivers of triple negative and basal-like breast cancers are largely unknown and we examined for a potential role of FGFR signalling in the oncogenesis of triple negative breast cancer. Methods: We examined the sensitivity of a panel of 40 breast cancer cell lines to the selective fibroblast growth factor receptor (FGFR) inhibitor PD173074. Results: Triple negative (TN) breast cancer cell lines were more sensitive to PD173074 than comparator cell lines (p=0.0028), with half (8/16) of TN cell lines demonstrating significantly reduced growth. The majority of TN cell lines demonstrated only modest sensitivity to FGFR inhibition in two-dimensional growth, but were highly sensitive in anchorage independent conditions. PD173074 inhibited down stream MAPK signalling and induced cell cycle arrest and apoptosis. Basal-like breast cancer cell lines were found to express autocrine FGF2 ligand, and RNA interference targeting of FGF2 significantly reduced anchorage independent growth. Finally, PD173074 significantly reduced the growth of basal-like breast cancer cell line xenografts. Conclusion: Basal-like breast cancer cell lines express autocrine FGF2 and demonstrate substantial sensitivity to FGFR inhibitors, identifying a potential novel therapeutic approach for these cancers. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P5-02-01.

In Vivo and In Vitro Evidence of Antitumor Activity of UCM-Gi028 [Novel FASN Inhibitor] Against Trastuzumab-Resistant Breast Cancer.

Abstract Introduction. The vast majority of Her-2 positive advanced breast cancer patients develop resistance to Trastuzumab based therapies within the first year of treatment. We have previously shown that inhibition of Fatty Acid Synthase (FASN) activity, an enzyme overexpressed and hiperactivated in breast carcinoma leads to apoptosis in breast cancer cells and its effect is tightly linked to Her-2 signalling pathways. Here we show the anticancer effect of UCM-Gi028, the lead-compound of the first generation of a novel family of synthetic FASN inhibitors, both in vitro and in an animal model of Her-2 amplified breast cancer.Material and Methods. We analysed the cellular and molecular effects of UCM-Gi028 alone or in combination with lapatinib, erlotinib, trastuzumab and gefitinib in a Her-2 dependent breast cancer cell line (AU565) using epigallocatechin-3-gallate (EGCG), a natural inhibitor of FASN, as a comparator. We also generated a long-term trastuzumab resistant pool from AU565 cells (AU565TTZR pool) to evaluate the effect of UCM-Gi028 in this setting. We further characterize the toxicity profile, anti-cancer activity and pharmacodynamic markers in an athymic mice model of Her-2 positive breast cancer cell line xenograft tumors.Results. UCM-Gi028 shows marked synergic antitumoral activity combined with lapatinib, erlotinib, trastuzumab and gefitinib (p &amp;lt;0.05). Response to co-exposure of UCM-Gi028 plus growth-signal inhibitors was significantly correlated with induction of apoptosis and with decrease in the activation forms of HER-2, MAPK and AKT, as markers of drug activity. Importantly, AU565TTZR pool displayed increased sensitivity to UCM-Gi028 compared to previously known FASN inhibitors (EGCG and C75). In the Her-2 positive breast cancer animal model study no weight loss or other relevant toxicity were observed after long term (7 weeks) exposure to UCM-Gi-28. A group of UCM-Gi028-treated mice showed tumour regression compared to control and, importantly, responsive tumours showed inhibition of FASN activity, increased apoptosis and decreased HER-2, AKT and MAPK activity.Discussion. As compared to its related natural compound (EGCG), UCM-Gi-28 shows increased antitumor activity with a favourable toxicity profile, in contrast with other, highly toxic, synthetic FASN inhibitor (C75). UCM-Gi028 antitumour activity remains unchanged in trastuzumab resistant cells. UCM-Gi-28 warrants further pre-clinical and clinical development alone or in combination with other anticancer agents in Her-2 positive breast cancer. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 5079.

In vitro and in vivo MMP gene expression localisation by In Situ-RT-PCR in cell culture and paraffin embedded human breast cancer cell line xenografts

BackgroundMembers of the matrix metalloproteinase (MMP) family of proteases are required for the degradation of the basement membrane and extracellular matrix in both normal and pathological conditions. In vitro, MT1-MMP (MMP-14, membrane type-1-MMP) expression is higher in more invasive human breast cancer (HBC) cell lines, whilst in vivo its expression has been associated with the stroma surrounding breast tumours. MMP-1 (interstitial collagenase) has been associated with MDA-MB-231 invasion in vitro, while MMP-3 (stromelysin-1) has been localised around invasive cells of breast tumours in vivo. As MMPs are not stored intracellularly, the ability to localise their expression to their cells of origin is difficult.MethodsWe utilised the unique in situ-reverse transcription-polymerase chain reaction (IS-RT-PCR) methodology to localise the in vitro and in vivo gene expression of MT1-MMP, MMP-1 and MMP-3 in human breast cancer. In vitro, MMP induction was examined in the MDA-MB-231 and MCF-7 HBC cell lines following exposure to Concanavalin A (Con A). In vivo, we examined their expression in archival paraffin embedded xenografts derived from a range of HBC cell lines of varied invasive and metastatic potential. Mouse xenografts are heterogenous, containing neoplastic human parenchyma with mouse stroma and vasculature and provide a reproducible in vivo model system correlated to the human disease state.ResultsIn vitro, exposure to Con A increased MT1-MMP gene expression in MDA-MB-231 cells and decreased MT1-MMP gene expression in MCF-7 cells. MMP-1 and MMP-3 gene expression remained unchanged in both cell lines. In vivo, stromal cells recruited into each xenograft demonstrated differences in localised levels of MMP gene expression. Specifically, MDA-MB-231, MDA-MB-435 and Hs578T HBC cell lines are able to influence MMP gene expression in the surrounding stroma.ConclusionWe have demonstrated the applicability and sensitivity of IS-RT-PCR for the examination of MMP gene expression both in vitro and in vivo. Induction of MMP gene expression in both the epithelial tumour cells and surrounding stromal cells is associated with increased metastatic potential. Our data demonstrate the contribution of the stroma to epithelial MMP gene expression, and highlight the complexity of the role of MMPs in the stromal-epithelial interactions within breast carcinoma.

Adenoviral vectors with E1A regulated by tumor-specific promoters are selectively cytolytic for breast cancer and melanoma.

We have previously demonstrated that a truncated form of the L-plastin promoter can confer tumor-specific patterns of expression on replication-incompetent adenoviral vector reporter and therapeutic transcription units. In this report, a 2.5-kb truncated version of the L-plastin promoter was placed 5' to the E1A gene of a wild-type adenovirus. The vector generated (Ad-Lp-E1A) was directly cytotoxic to established breast and ovarian cancer cell lines and to primary explant cultures derived from ovarian cancer, but was not cytotoxic to explant cultures of normal mammary epithelial cells. This vector was not cytotoxic to cell lines in which the L-plastin E1A transcription unit was not expressed, whereas the same cell lines were sensitive to the cytotoxic effect of a replication-competent adenoviral vector in which the cytomegalovirus (CMV) promoter drove E1A expression. When the tyrosinase promoter/enhancer was placed 5' to the E1A gene in the adenoviral backbone, the resulting vector (Ad-Tyr-E1A) was selectively toxic to melanoma cells and one percent as toxic to explants of ovarian cancer cells as the Ad-Lp-E1A vector. Injection of these vectors (Ad-Lp-E1A and Ad-Tyr-E1A) into nodules derived from the MCF-7 and MDA-MB-468 human breast cancer cell lines and the TF-2 human melanoma cell line, respectively, which were growing subcutaneously in severe combined immunodeficiency (SCID) mice, induced regression of these tumors. Such vectors may therefore be useful in cancer treatment.

Human breast cancer cell line xenografts as models of breast cancer. The immunobiologies of recipient mice and the characteristics of several tumorigenic cell lines.

The ability to maintain and study human tissues in an in vivo environment has proved to be a valuable tool in breast cancer research for several decades. The most widely studied tissues have been xenografts of established human breast cancer cell lines into athymic nude mice. Human breast tumor xenografts provide the opportunity to study various important interactions between the tumor and host tissues, including endocrinologic, immunologic, and tumor-stroma interactions. The nude mouse is not the only immune-deficient recipient system in which to study xenografts. Additional single and combined mutant strains have been used successfully, including mice homozygous for the severe combined immune deficiency mutation (scid), both the beige (bg) and nude (nu) mutations in combination (bg/nu), and mice bearing the combined bg/nu/xid mutations. The differing immunobiologies are discussed, with particular reference to the immunobiology of breast cancer, as are the characteristics of several of the more frequently utilized breast cancer xenografts and cell lines. The ability of several endocrine treatments to modulate effectors of cell mediated immunity, e.g., estrogens and antiestrogens, and the effect of site of inoculation on tumor take and metastasis, also are described.

Animal models of breast cancer: Their diversity and role in biomedical research

Animal models of breast cancer have been widely used to study various aspects of breast cancer biology, and are remarkably diverse, encompassing chemically and virally induced tumors, human tumor xenografts, and transgenic mouse models. Several novel models have become available during the past few years, including tumors induced by polyomavirus and adenovirus, and a series of human cell line variants. The several following articles describe, in some detail, the characteristics of these models and their relevance to the human disease. Descriptions of each of the major models, e.g., 7,12-dimethylbenz(a)anthracene-induced, MMTV-associated, and human breast cancer cell line xenografts, also are included. The limitations and advantages of several of these models, and some issues relating to the choice of models, are briefly discussed in this overview.

BRCA1 overexpression sensitizes cancer cells to lovastatin via regulation of cyclin D1-CDK4-p21WAF1/CIP1 pathway: analyses using a breast cancer cell line and tumoral xenograft model

It is well established that statins display potent anticancer activity in several types of proliferating tumor cells. However, how to promote the sensitivity of statins to mammary cancer is yet to be completely deciphered. The purpose of this study was to investigate whether breast cancer susceptibility gene 1 (BRCA1) overexpression sensitizes mammary cancer cells to statins. MCF-7 cells, which have only one wild-type BRCA1 allele, were transfected with pcDNA3-beta-HA-hsBRCA1 plasmids via liposomes to reconstitute BRCA1 overexpression human breast cancer cell line, and tumoral xenografts with BRCA1 overexpression were subsequently established in BALB/c nude mice. Then, the inhibitory activity of lovastatin on cancer cells and tumoral xenografts, and the underlying mechanism involving in cell-cycle regulatory proteins were analyzed. The proliferative ability of MCF-7 cells treated with lovastatin was reduced compared to normal, and further decreased in the presence of excess BRCA1, detected by methyl thiazolyl tetrazolium and flow cytometry techniques in vitro or by 5-bromodeoxyuridine incorporation in vivo. Additionally, the mRNA and protein expression of cyclin D1, cyclin-dependent kinase 4 (CDK4) and retinoblastoma protein (pRb), was further down-regulated under exposure to lovastatin in condition of BRCA1 overexpression, but the expression of p21WAF1/CIP1, a cyclin-dependent kinase inhibitor (CDKI), was further up-regulated, both in vitro and in vivo detected with quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot analysis. Moreover, we found the further reduced volume of tumoral xenografts treated with lovastatin in the presence of BRCA1 overexpression. Our results suggest that BRCA1 overexpression sensitizes cancer cells to lovastatin via regulation of cyclin D1-CDK4-p21WAF1/CIP1 pathway, which will provide an innovative experimental framework to study control of breast cancer cell proliferation.