Liver X receptors induce antiproliferative effects in basal-like breast cancer.

Liver X receptors (LXRs) are nuclear transcription factors important in the regulation of cholesterol transport, and glucose and fatty acid metabolism. The antiproliferative role of LXRs has been studied in a variety of malignancies and may represent a therapeutic opportunity in cancers lacking targeted therapies, such as triple-negative breast cancer. In this study, we investigated the impact of LXR agonists alone and in combination with carboplatin in preclinical models of breast cancer. In vitro experiments revealed a dose-dependent decrease in tumor cell proliferation in estrogen receptor-positive breast cancer cells, whereas LXR activation in vivo resulted in an increased growth inhibitory effect in a basal-like breast cancer model (in combination with carboplatin). Functional proteomic analysis identified differences in protein expression between responding and nonresponding models related to Akt activity, cell-cycle progression, and DNA repair. Furthermore, pathway analysis suggested that the LXR agonist in combination with carboplatin inhibits the activity of targets of E2F transcription factors and affects cholesterol homeostasis in basal-like breast cancer.

Breast cancer patient-derived explant cultures recapitulate in vivo drug responses.

Assessment of drug sensitivity in tumor tissue ex vivo may significantly contribute to functional diagnostics to guide personalized treatment of cancer. Tumor organoid- and explant-cultures have become attractive tools towards this goal, although culturing conditions for breast cancer (BC) tissue have been among the most challenging to develop. Validation of possibilities to detect concordant responses in individual tumors and their respective cultures ex vivo is still needed. Here we employed BC patient-derived xenografts (PDXs) with distinct drug sensitivity, to evaluate different conditions for tissue dissociation, culturing and monitoring of treatment efficacy ex vivo, aiming to recapitulate the in vivo drug responses. The common challenge of discriminating between tumor and normal cells in the cultured tissue was also addressed. Following conventional enzymatic dissociation of BC tissue, the tumor cells stayed within the non-disrupted tissue fragments, while the single cells represented mostly normal host cells. By culturing such fragments as explants, viable tumor tissue could be maintained and treated ex vivo, providing representative indications on efficacy of the tested treatment. Thus, drug sensitivity profiles, including acquired chemoresistance seen in the PDXs, were recapitulated in the respective explants. To detect the concordant responses, however, the effect monitoring had to be harmonized with the characteristics of the cultured tissue. In conclusion, we present the feasibility of BC explants ex vivo to capture differences in drug sensitivity of individual tumors. The established protocols will aid in setting up an analogous platform for BC patient biopsies with the aim to facilitate functional precision medicine.

Novel human melanoma brain metastasis models in athymic nude fox1nu mice: Site-specific metastasis patterns reflecting their clinical origin.

BACKGROUND: Malignant melanomas frequently metastasize to the brain, but metastases in the cerebellum are underrepresented compared with metastases in the cerebrum. METHODS: We established animal models by injecting intracardially in athymic nude fox1nu mice two human melanoma cell lines, originating from a cerebral metastasis (HM19) and a cerebellar metastasis (HM86). RESULTS: Using magnetic resonance imaging (MRI), metastases were first detected after a mean of 34.5 days. Mean survival time was 59.6 days for the mice in the HM86 group and significantly shorter (43.7 days) for HM19-injected animals (p < 0.001). In the HM86 group, the first detectable metastasis was located in the cerebellum in 15/55 (29%) mice compared with none in the HM19 group (p < 0.001). At sacrifice, cerebellar metastases were found in 34/55 (63%) HM86-injected mice compared with 1/53 (2%) in the HM19-injected (p < 0.001) mice. At that time, all mice in both groups had detectable metastases in the cerebrum. Comparing macroscopic and histologic appearances of the brain metastases with their clinical counterparts, the cell line-based tumors had kept their original morphologic characteristics. CONCLUSIONS: The present work demonstrates that human brain-metastatic melanoma cells injected intracardially in mice had retained inherent characteristics also in reproducing interaction with subtle microenvironmental brain tissue compartment-specific features. The models offer new possibilities for investigating tumor- and host-associated factors involved in determining tissue specificity of brain metastasis.

Detection of disseminated tumor cells in lymph nodes from patients with early stage non-small cell lung cancer.

BACKGROUND: The regional lymph node involvement is a major prognostic factor in patients with non-small cell lung cancer (NSCLC) undergoing surgical resection. Disease relapse is common, suggesting that early disseminated disease is already present in the regional lymph nodes at the time of surgery, and that the current nodal staging classification might be suboptimal. Early detection of disseminated tumor cells (DTCs) in lymph nodes could potentially enable identification of subcategories of patients with high risk of disease relapse. METHOD: Lymph node samples were collected from 128 NSCLC patients at the time of surgery and the presence of DTCs determined by immunomagnetic selection (IMS) using the MOC31 antibody recognizing EpCAM. Results obtained with IMS were compared to the pathological staging obtained by histopathology. Associations between the presence of DTCs and clinicopathological variables and patient outcome were investigated. RESULTS: DTCs were detected in 40 % of the lymph node samples by IMS. Their presence was significantly associated with pN status as assessed by histopathology, and samples from 83 % of the patients with lymph node metastases (pN1-2) had detectable DTCs. In the group of patients who were negative for lymph node metastases by standard histopathology (pN0) DTCs were detected in 32 %. The presence of DTCs was not associated with any other clinicopathological variables. Patients with IMS-positive samples showed decreased relapse free survival compared to patients with IMS-negative samples, but the difference was not statistically significant. The pN status was significantly associated with both relapse free and overall survival, but the presence of DTCs had no prognostic impact in the subcategory of patients with pN0 status. CONCLUSION: Our findings do not support further development of lymph node DTC detection for clinical use in early stage NSCLC.

Inhibition of in vivo tumour growth by the blocking of host alpha(v)beta3 and alphaII(b)beta3 integrins.

BACKGROUND: The alpha(v)beta3 integrin in the endothelial cell membrane is important for the growth and migration of capillaries into tumour tissue and is also a survival factor for these cells. The alphaII(b)beta3 (GPIIb/IIIa) integrin is responsible for platelet activation and, with concomitant release of different stored proangiogenic factors, and tumour cell-platelet interactions. MATERIALS AND METHODS: An immunodeficient nude rat model was used to study tumour growth in tibial bone, with tumour cells negative for the target alpha(v)beta3 and alphaII(b)beta3 integrins. RESULTS: Daily intraperitoneal injections of m7E3 F(ab')2 antibody fragment, blocking human and rat alpha(v)beta3 and alphaII(b)beta3 integrins, reduced the measured size of the tumours growing in the tibial bone by 35% (p = 0.012), and also the microvessel density in these tumours. The concentration of the important proangiogenic factor bFGF was significantly reduced by 41% in the treated tumours. The treatment slightly increased the time to the appearance of the tumour from 22.2 to 24.9 days, indicating a small but significant effect on the early stages of tumour growth and invasion through the bone tissue. CONCLUSION: Integrin-targeted treatment reduced tumour growth, solely targeting the host angiogenesis. This treatment strategy should be further exploited for use in combination with conventional treatment strategies, or the combined targeting of alternative antiangiogenic pathways.

AMP-activated protein kinase protects against anti-epidermal growth factor receptor-Pseudomonas exotoxin A immunotoxin-induced MA11 breast cancer cell death.

We have shown previously that our 425.3PE immunotoxin inhibits protein synthesis and induces apoptosis in human breast cancer cells. In attempts to further elucidate the intracellular pathways implicated in its cellular effects, we found that the immunotoxin induced an initial stress response, which rapidly caused an imbalance in the cellular energy status with an increase in reactive oxygen species. The AMP-activated protein kinase (AMPK), a sensor of increased cellular AMP/ATP ratio, was activated by 425.3PE. An immunotoxin-induced activation of c-Jun NH2-terminal kinase (JNK) preceded and overlapped caspase-mediated cleavage of the alpha-subunit of AMPK in a time- and dose-dependent manner. The JNK activation occurred already at a dose level too low to induce any detectable changes in the apoptotic machinery or protein synthesis. In contrast, cycloheximide, even at a concentration causing a 90% inhibition of protein synthesis, did neither affect the ATP level nor activate JNK and AMPK. Pretreatment of the cells with the specific AMPK inhibitor compound C and JNK inhibitor SP600125 blocked activation of AMPK and JNK, respectively, and subsequently sensitized the cells to 425.3PE-induced cell death. Whereas the antioxidant N-acetyl-l-cysteine blocked the generation of reactive oxygen species and activation of JNK and AMPK, it did not block immunotoxin-induced apoptosis. Together, the results show that 425.3PE induces several parallel signaling events, observed initially as an early activation of survival pathways, protecting the cells against the toxic effects of the immunotoxin, followed by subsequent apoptosis induction and protein synthesis inhibition. Conceivably, therapeutic manipulation of the signaling intermediates AMPK and JNK might provide a means to maximize the anticancer effects of the 425.3 immunotoxin.

Downregulation of the antiapoptotic MCL-1 protein and apoptosis in MA-11 breast cancer cells induced by an anti-epidermal growth factor receptor-Pseudomonas exotoxin a immunotoxin.

Pseudomonas exotoxin (PE)-containing immunotoxins (ITs) act by arresting protein synthesis and promoting apoptosis, but the mechanisms of the induced apoptosis and the relationship to protein synthesis inhibition is not well elucidated. We studied these effects in MA-11 human breast cancer cells treated with 425.3PE, an unmodified PE covalently linked to the 425.3 antibody, which targets the EGF receptor. This IT induced efficient inhibition of protein synthesis with simultaneous induction of apoptosis. Thus, treatment of cells with 10 ng/ml of IT for 5 hr caused 85% inhibition of protein synthesis in parallel with caspase-3, -8 and -9 activation and PARP inactivation. Even after 72 hr of IT treatment, preincubation with the broad-spectrum caspase inhibitor z-VAD-FMK caused a significant increase in cell survival without affecting IT-induced protein synthesis inhibition. Interestingly, a combination of z-VAD-FMK and the cathepsin B/L inhibitor z-FA-FMK prevented completely IT-induced cell death in MA-11 cells after 24 hr, indicating that cathepsin activation may be important for optimal induction of IT-induced cell death. IT treatment caused after 2.5 hr a significant decrease in the level of the antiapoptotic protein Mcl-1 but not of Bcl-2 and Bcl-XL. Furthermore, Mcl-1 expression was not sensitive to caspase inhibitors but was totally prevented by the lactacystin proteasome inhibitor, suggesting that IT-induced apoptosis may be triggered by a reduction in the Mcl-1 level. Mitochondrial membrane potential (DeltaPsi mito) decreased concurrently with caspase activation, showing the involvement of DeltaPsi mito as a regulator of IT-induced apoptosis. Our results demonstrate that 425.3PE-mediated cell death involves simultaneous induction of apoptosis and protein synthesis inhibition in MA-11 cells, thus contributing to an understanding of the mechanisms involved in IT-induced apoptosis.

Immunotoxin treatment targeted to the high-molecular-weight melanoma-associated antigen prolonging the survival of immunodeficient rats with invasive intracranial human glioblastoma multiforme.

OBJECT: The aim of this study was to target immunotoxin treatment to the high-molecular-weight melanoma-associated antigen (HMW-MAA) and thereby examine any changes in the survival of immunodeficient rats with human glioblastoma multiforme (GBM). METHODS: To target treatment specifically to human glioma cells, Pseudomonas exotoxin A (PE) was conjugated to the 9.2.27 antibody, which recognizes the HMW-MAA. Treatment of the antigen-positive glioma cell line U87MG with the resulting 9.2.27-PE caused cytotoxicity with a median inhibitory concentration of 1 ng/ml. Intratumoral 9.2.27-PE treatment of intracranial U87MG tumors in nude rats prolonged the survival of these animals by 43% compared with controls. In additional studies on the use of this targeted treatment, the authors precultured freshly dissected glioblastoma multiforme (GBM) biopsy tissue for 1 to 2 weeks. Inoculation of this tissue into the rat brain resulted in diffuse infiltrative gliomas. The markers glial fibrillary acidic protein and S100 protein were found to be expressed in the original biopsy specimens, as well as in the glioma xenografts in nude rat brains. Intratumoral immunotoxin treatment of such established tumors with 9.2.27-PE was effective and prolonged survival time from 30% to as high as 90% in animals with tumors originating from four different GBM specimens. CONCLUSIONS: Targeted treatment of highly invasive GBMs proved effective, and these results emphasize the clinical relevance of this antigen as a target molecule for immunotoxin treatment of human GBMs.

Plasminogen activator inhibitor-1 increases the expression of VEGF in human glioma cells.

The level of plasminogen activator inhibitor-1 (PAI-1) in tumor tissue has been shown to be an independent negative prognostic factor in different cancers. There are several proposed reasons for this, among these, the influence of PAI-1 on tumor neovascularization and cell migration. We report that PAI-1 stimulates expression and release of vascular endothelial growth factor (VEGF) in the human glioma cell line D54Mg, and thereby stimulates the proliferation of human umbilical vein endothelial cells (HUVEC) in vitro. To search for possible molecular effects of PAI-1 on malignant cells, cDNA array hybridization analysis of D54Mg glioma cells transfected with an adenoviral PAI-1 expression vector was performed. This revealed that the VEGF response was accompanied with the simultaneous upregulation of GADD153, Rho GTPase activating protein 4 (p115), Collagen type VI alpha 1 and cell division cycle 42 (CDC42) transcripts. Exogenous treatment of D54Mg cells with a constitutively active recombinant PAI-1 protein confirmed an upregulation of VEGF expression in a time- and dose-dependent manner, and supernatants from such cultures stimulated the proliferation of human umbilical vein endothelial cells in vitro. In 44 human glioma biopsies, patients, the protein levels of PAI-1 correlated strongly with the levels of VEGF in the tumor tissues. Whereas VEGF expression correlated inversely with survival, there was no statistically significant prediction of survival by PAI-1 in this group of patients. These clinical data support and strengthen the hypothesis that PAI-1 is one of the factors regulating and inducing the VEGF expression in human gliomas. The induction of VEGF expression and thus endothelial cell proliferation may represent an as yet undiscovered mechanism whereby PAI-1 contributes to tumor neoangiogenesis.

Intratumoral immunotoxin treatment of human malignant brain tumors in immunodeficient animals.

Treatment of malignant brain tumors remains a clinical challenge. New treatment modalities are under investigation and among these are intratumoral infusion of immunotoxins that bind to specific cell surface molecules on the malignant cells. We have compared the efficacy of the 425.3-PE immunotoxin (which targets the epidermal growth factor [EGF] receptor) with the well-known immunotoxin Tfn-CRM107 (which targets the transferrin receptor), for the treatment of subcutaneous and intracranial human gliomas in nude animals. Bolus intratumoral administration of 1 microg Tfn-CRM107 or 425.3-PE into sc U87Mg tumors in nude mice reduced the tumor volume to 29 and 79%, respectively, of that in the control group 18 days after start of treatment. Higher doses of Tfn-CRM107 were toxic to the animals, whereas 425.3-PE was tolerated, with a dose-response relationship of up to 8 microg, a dose that reduced the tumor volume to 2% of control. In nude rats, treatment of intracerebral U87Mg tumors with Tfn-CRM107 proved ineffective and doses above 10 ng/animal were toxic to tumor-bearing rats. In contrast, intratumoral administration of 4 microg 425.3-PE increased symptom-free survival from 23 days to 40 days, with 2/9 surviving more than 90 days. We have recently shown that immunodeficient rats inoculated intracerebrally with precultured glioblastoma biopsy specimens develop highly infiltrative brain tumors. Direct interstitial infusion of immunotoxins into such tumors reduced the number of animals with detectable tumors at autopsy after 3 months, from 8/9 in the control animals to 4/6 and 2/6 in animals treated with Tfn-CRM107 and 425.3-PE, respectively. In conclusion, the anti-EGF receptor immunotoxin 425.3-PE exhibited promising efficacy, comparable to or better than that of Tfn-CRM107, an immunotoxin that in early clinical trials has been found to give responses in patients with brain tumors.