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Purpose: Chemotherapy-induced apoptosis of immature hematopoietic cells is a major cause of anemia and thrombocytopenia in cancer patients. Although hematopoietic growth factors such as erythropoietin and colony-stimulating factors cannot prevent the occurrence of drug-induced myelosuppression, stem cell factor (SCF) has been previously shown to protect immature erythroid and megakaryocytic cells in vitro from drug-induced apoptosis. However, the effect of SCF in vivo as a single myeloprotective agent has never been elucidated.Experimental Design: The ability of SCF to prevent the occurrence of chemotherapy-induced anemia and thrombocytopenia was tested in a mouse model of cisplatin-induced myelosuppression. To highlight the importance of maintaining a continuous antiapoptotic signal in immature hematopoietic cells, we compared two treatment schedules: in the first schedule, SCF administration was interrupted during chemotherapy treatment and resumed thereafter, whereas in the second schedule, SCF was administered without interruption for 7 days, including the day of chemotherapy treatment.Results: The administration of SCF to cisplatin-treated mice could preserve bone marrow integrity, inhibit apoptosis of erythroid and megakaryocytic precursors, prevent chemotherapy-induced anemia, and rapidly restore normal platelet production. Treatment with SCF increased the frequency of Bcl-2/Bcl-XL–positive bone marrow erythroid cells and sustained Akt activation in megakaryocytes. Myeloprotection was observed only when SCF was administered concomitantly with cisplatin and kept constantly present during the days following chemotherapy treatment.Conclusions: SCF treatment can prevent the occurrence of chemotherapy-induced anemia and thrombocytopenia in mice, indicating a potential use of this cytokine in the supportive therapy of cancer patients. Clin Cancer Res; 17(19); 6185–91. ©2011 AACR.

Purpose: Current prostate cancer management calls for identifying novel and more effective therapies. Self-renewing tumor-initiating cells (TICs) hold intrinsic therapy resistance and account for tumor relapse and progression. As BMI-1 regulates stem cell self-renewal, impairing BMI-1 function for TIC-tailored therapies appears to be a promising approach.Experimental Design: We have previously developed a combined immunophenotypic and time-of-adherence assay to identify CD49bhiCD29hiCD44hi cells as human prostate TICs. We utilized this assay with patient-derived prostate cancer cells and xenograft models to characterize the effects of pharmacologic inhibitors of BMI-1.Results: We demonstrate that in cell lines and patient-derived TICs, BMI-1 expression is upregulated and associated with stem cell–like traits. From a screened library, we identified a number of post-transcriptional small molecules that target BMI-1 in prostate TICs. Pharmacologic inhibition of BMI-1 in patient-derived cells significantly decreased colony formation in vitro and attenuated tumor initiation in vivo, thereby functionally diminishing the frequency of TICs, particularly in cells resistant to proliferation- and androgen receptor–directed therapies, without toxic effects on normal tissues.Conclusions: Our data offer a paradigm for targeting TICs and support the development of BMI-1–targeting therapy for a more effective prostate cancer treatment. Clin Cancer Res; 22(24); 6176–91. ©2016 AACR.

Supplementary data Figure S1. Functional role(s) of BMI-1 in PCa. Figure S2. BMI-1 expression in subpopulations of DU145 cells. Figure S3. Pharmacological inhibition of BMI-1. Figure S4. Molecular and cellular effects of pharmacological targeting of BMI-1. Figure S5. Examining BMI-1 inhibitors in toxicological assays and effects on normal cells. Figure S6. Toxicity effects of C-209 and CHX on adult and embryo zebrafish. Figure S7. BMI-1 inhibition effect on the normal prostate and hematopoietic system. Figure S8. Representative images of BMI-1 expression in primary PCa samples. Figure S9. Xenografts of human primary PCa cells in embryonic zebrafish. Figure S10. Xenografts of primary PCa tissue in zebrafish embryos. Figure S11. Toxicology assays of chemotherapy and BMI-1 inhibitors in embryonic zebrafish. Figure S12. Anti-tumor activity of BMI-1 inhibitors. Figure S13. Representative H&E staining of mouse xenograft sections indicating the histological effects of treatments. Figure S14. In vivo pharmacological targeting of BMI-1 in androgen independent DU145 and androgen sensitive 22rv1 PCa mouse xenografts. Figure S15. Colony-formation frequency evaluated by dilution analysis of xenograft-derived cells from untreated mice and mice treated with C-209 60mg/kg/day. Figure S16. BMI-1 inhibition effects on androgen-dependent and -independent cells.

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Despite substantial effort and resources dedicated to drug discovery and development, new anticancer agents often fail in clinical trials. Among many reasons, the lack of reliable predictive preclinical cancer models is a fundamental one. For decades, immortalized cancer cell cultures have been used to lay the groundwork for cancer biology and the quest for therapeutic responses. However, cell lines do not usually recapitulate cancer heterogeneity or reveal therapeutic resistance cues. With the rapidly evolving exploration of cancer "omics," the scientific community is increasingly investigating whether the employment of short-term patient-derived tumor cell cultures (two- and three-dimensional) and/or patient-derived xenograft models might provide a more representative delineation of the cancer core and its therapeutic response. Patient-derived cancer models allow the integration of genomic with drug sensitivity data on a personalized basis and currently represent the ultimate approach for preclinical drug development and biomarker discovery. The proper use of these patient-derived cancer models might soon influence clinical outcomes and allow the implementation of tailored personalized therapy. When assessing drug efficacy for the treatment of glioblastoma multiforme (GBM), currently, the most reliable models are generated through direct injection of patient-derived cells or more frequently the isolation of glioblastoma cells endowed with stem-like features and orthotopically injecting these cells into the cerebrum of immunodeficient mice. Herein, we present the key strengths, weaknesses, and potential applications of cell- and animal-based models of GBM, highlighting our experience with the glioblastoma stem-like patient cell-derived xenograft model and its utility in drug discovery.

BACKGROUND: Hepatocellular carcinoma (HCC) represents one of the most lethal cancers worldwide due to therapy resistance and disease recurrence. Tumor relapse following treatment could be driven by the persistence of liver cancer stem-like cells (CSCs). The protein BMI1 is a member of the polycomb epigenetic factors governing cellular self-renewal, proliferation, and stemness maintenance. BMI1 expression also correlates with poor patient survival in various cancer types. OBJECTIVE: We aimed to elucidate the extent to which BMI1 can be used as a potential therapeutic target for CSC eradication in HCC. METHODS: We have recently participated in characterizing the first known pharmacological small molecule inhibitor of BMI1. Here, we synthesized a panel of novel BMI1 inhibitors and examined their ability to alter cellular growth and eliminate cancer progenitor/stem-like cells in HCC with different p53 backgrounds. RESULTS: Among various molecules examined, RU-A1 particularly downregulated BMI1 expression, impaired cell viability, reduced cell migration, and sensitized HCC cells to 5-fluorouracil (5-FU) in vitro. Notably, long-term analysis of HCC survival showed that, unlike chemotherapy, RU-A1 effectively reduced CSC content, even as monotherapy. BMI1 inhibition with RU-A1 diminished the number of stem-like cells in vitro more efficiently than the model compound C-209, as demonstrated by clonogenic assays and impairment of CSC marker expression. Furthermore, xenograft assays in zebrafish showed that RU-A1 abrogated tumor growth in vivo. CONCLUSIONS: This study demonstrates the ability to identify agents with the propensity for targeting CSCs in HCC that could be explored as novel treatments in the clinical setting.

Activation of the Hippo transducer TAZ is emerging as a novel oncogenic route in breast cancer and it has been associated with breast cancer stem cells. Additionally, TAZ expression has been linked with HER-2 positivity. We investigated the association between TAZ expression and pathological complete response in HER2-positive breast cancer patients treated with trastuzumab-based neoadjuvant therapy. TAZ was assessed in diagnostic core biopsies by immunohistochemistry. To categorize samples with low TAZ and samples with high TAZ we generated a score by combining staining intensity and cellular localization. The pathological complete response rate was 78.6% in patients with low TAZ tumors and 57.6% in patients with high TAZ tumors (p=0.082). In HER2-enriched tumors there was no significant association between TAZ and pathological complete response, whereas in the luminal B subtype the pathological complete response rate was 82.4% in tumors with low TAZ and 44.4% in tumors with high TAZ (p=0.035). This association remained statistically significant when restricting our analysis to triple-positive tumors with expression of both estrogen receptor and progesterone receptor ≥ 50% (p=0.035). Results from this exploratory study suggest that the TAZ score efficiently predicts pathological complete response in Luminal B, HER2-positive breast cancer patients who received neoadjuvant chemotherapy and trastuzumab.

Glioblastoma multiforme (GBM) are the most common and deadly primary brain tumors. GBMs are thought to derive from neuroglial stem or progenitor cells. Although surgery, radiotherapy and concomitant or alternating chemotherapy followed by anti-angiogenic therapy are still the mainstay of treatment, individually tailored strategies based on tumor-intrinsic dominant genomic, transcriptomic, epigenetic and antigenic tumor profiles may ultimately improve outcome. GBM recurrence is thought to be caused by a dynamically changing population of slow-cycling stem-like cells that are capable of evading current therapeutics which spare the radio/chemoresistant cells. Due to the extreme heterogeneity of these tumors, the likelihood of success of new therapeutic strategies for GBM relies on utilizing tailored combinations of targeted genetic-, epigenetic- and/or immune-modulating therapies. Targeting epigenetic polycomb repressor complex 1/2 (PRC1/2) and their key drivers BMI1 and EZH2 is highly attractive to target GBM stem-like and progenitor cells. Since PRC2 activity is dependent on PRC1 chromatin association, targeting BMI1 is a priority for GBM. BMI1 is strongly upregulated in most GBM subtypes, and we and others have demonstrated that reducing BMI1 levels results in a decrease in tumor cell self-renewal and eliminate the stem-cell like phenotypes in other tumor types. We have therefore developed novel small molecule BMI1 inhibitors, named RU-A compounds, for GBM therapy. First, we demonstrate that these RU-A compounds could eliminate BMI1 at low nanomolar conc. We further determined using modeling and luciferase assays that RU-A compounds exert effects on the BMI1 RNA, suggesting that RU-A compounds might bind to a pocket within the BMI1 untranslated region. Strikingly, RU-A compounds demonstrate complete ablation of self-renewal of GBM stem-like cells in serial clonogenic, sphere assays, and GBM organoids, and sensitize GBM cells to chemotherapy. We demonstrate the anti-GBM and reduction of tumor initiation activities of these inhibitors in a series of patient-derived xenografts and patient derived orthotopic mouse models of GBM. Due to the high potency of these compounds, when injected in mice, there were significant reductions in intratumor BMI1 associated with elimination of stem cell-like phenotypes, and no hematopoietic toxicity or neurotoxicity detected. These novel RU-A compounds by altering the stem-like populations of GBM cells offer effective targeted agents that could be utilized alone or combined with other targeted and immune modulatory agents in clinical trials for GBM patients. Citation Format: Michelle Chadwick, Eric Huselid, Monica Bartucci, Michele Patrizii, Kelly Jara, Monal Mehta, John Gilleran, David Augeri, Hatem Sabaawy. Development of novel BMI1 inhibitors targeting glioblastoma stem-like cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3073.

Prostate cancer (PCa) is the most common malignancy and the second most common cause of cancer death in Western men. Despite its prevalence, PCa has proven very difficult to propagate in vitro. PCa represents a complex organ-like multicellular structure maintained by the dynamic interaction of tumoral cells with parenchymal stroma, endothelial and immune cells, and components of the extracellular matrix (ECM). The lack of PCa models that recapitulate this intricate system has hampered progress toward understanding disease progression and lackluster therapeutic responses. Tissue slices, monolayer cultures and genetically engineered mouse models (GEMM) fail to mimic the complexities of the PCa microenvironment or reproduce the diverse mechanisms of therapy resistance. Moreover, patient derived xenografts (PDXs) are expensive, time consuming, difficult to establish for prostate cancer, lack immune cell-tumor regulation, and often tumors undergo selective engraftments. Here, we describe an interdisciplinary approach using primary PCa and tumor initiating cells (TICs), three-dimensional (3D) tissue engineering, genetic and morphometric profiling, and humanized mice to generate patient-derived organoids for examining personalized therapeutic responses in vitro and in mice co-engrafted with a human immune system (HIS), employing adaptive T-cell- and chimeric antigen receptor- (CAR) immunotherapy. The development of patient specific therapies targeting the vulnerabilities of cancer, when combined with antiproliferative and immunotherapy approaches could help to achieve the full transformative power of cancer precision medicine.

Purpose: Current prostate cancer management calls for identifying novel and more effective therapies. Self-renewing tumor-initiating cells (TICs) hold intrinsic therapy resistance and account for tumor relapse and progression. As BMI-1 regulates stem cell self-renewal, impairing BMI-1 function for TIC-tailored therapies appears to be a promising approach. Experimental Design: We have previously developed a combined immunophenotypic and time-of-adherence assay to identify CD49bhiCD29hiCD44hi cells as human prostate TICs. We utilized this assay with patient-derived prostate cancer cells and xenograft models to characterize the effects of pharmacologic inhibitors of BMI-1. Results: We demonstrate that in cell lines and patient-derived TICs, BMI-1 expression is upregulated and associated with stem cell–like traits. From a screened library, we identified a number of post-transcriptional small molecules that target BMI-1 in prostate TICs. Pharmacologic inhibition of BMI-1 in patient-derived cells significantly decreased colony formation in vitro and attenuated tumor initiation in vivo, thereby functionally diminishing the frequency of TICs, particularly in cells resistant to proliferation- and androgen receptor–directed therapies, without toxic effects on normal tissues. Conclusions: Our data offer a paradigm for targeting TICs and support the development of BMI-1–targeting therapy for a more effective prostate cancer treatment. Clin Cancer Res; 22(24); 6176–91. ©2016 AACR.

Cervical cancer cells commonly harbour a defective G1/S checkpoint owing to the interaction of viral oncoproteins with p53 and retinoblastoma protein. The activation of the G2/M checkpoint may thus become essential for protecting cancer cells from genotoxic insults, such as chemotherapy. In 52 cervical cancer patients treated with neoadjuvant chemotherapy, we investigated whether the levels of phosphorylated Wee1 (pWee1), a key G2/M checkpoint kinase, and γ-H2AX, a marker of DNA double-strand breaks, discriminated between patients with a pathological complete response (pCR) and those with residual disease. We also tested the association between pWee1 and phosphorylated Chk1 (pChk1), a kinase acting upstream Wee1 in the G2/M checkpoint pathway. pWee1, γ-H2AX and pChk1 were retrospectively assessed in diagnostic biopsies by immunohistochemistry. The degrees of pWee1 and pChk1 expression were defined using three different classification methods, i.e., staining intensity, Allred score, and a multiplicative score. γ-H2AX was analyzed both as continuous and categorical variable. Irrespective of the classification used, elevated levels of pWee1 and γ-H2AX were significantly associated with a lower rate of pCR. In univariate and multivariate analyses, pWee1 and γ-H2AX were both associated with reduced pCR. Internal validation conducted through a re-sampling without replacement procedure confirmed the robustness of the multivariate model. Finally, we found a significant association between pWee1 and pChk1. The message conveyed by the present analysis is that biomarkers of DNA damage and repair may predict the efficacy of neoadjuvant chemotherapy in cervical cancer. Further studies are warranted to prospectively validate these encouraging findings.

In response to pathophysiological stresses, cardiac myocytes undergo hypertrophic growth or apoptosis. Multiple signalling pathways have been implicated in these responses and among them, kinases such as mitogen-activated protein kinases (MAPKs) and Akt. However, the distinction between signalling pathways originally believed to be specific for either hypertrophy, apoptosis or cell survival is fading. The existing data, coming from different experimental systems, often are conflicting. In this study, we sought to compare aspects of intracellular signalling activated by diverse stimuli in a single experimental system, adult rat cardiac myocytes. Furthermore, we assessed the role of these stimuli in eliciting a particular cell phenotype, i.e. whether they promote hypertrophy, cell survival or apoptosis. The results demonstrate that the hypertrophic agonist phenylephrine is the most potent activator of MAPKs/mitogen and stress- activated kinase MSK1, although its effect on Akt phosphorylation is relatively minor. The pro-apoptotic concentration of H2O2 activates strongly both MAPKs and PI3K/Akt pathways. Insulin-like growth factor-1 has a minimal effect on phosphorylation of MAPKs/MSK1, but it is a potent activator of Akt. In conclusion, hypertrophic, pro-survival or apoptotic stimuli operate through the same signalling pathways with different time course and amplitude of kinase activation. Thus, to determine the effect of different stimuli on cell fate, it is important to assess signalling pathways as a network and not as a single pathway. Copyright © 2011 John Wiley & Sons, Ltd.

Thrombocytopenia is a common side effect of chemotherapy, responsible for increased risk of bleeding and delay of treatment schedules in cancer patients. It is currently unknown how chemotherapeutic agents affect platelet production and whether the platelet precursors megakaryocytes represent a direct target of cytotoxic drugs. We investigated the effects of chemotherapeutic agents on primary megakaryocytes by using a culture system that recapitulates in vitro human megakaryopoiesis and found that cytotoxic drugs predominantly destroyed megakaryocytic progenitors at early stages of differentiation. Immature megakaryocytes could be protected from chemotherapeutic agents by the cytokine stem cell factor (SCF), which binds the c-kit receptor expressed on hematopoietic stem and progenitor cells. In chemotherapy-treated megakaryocytes, SCF activated Akt, neutralized the mitochondrial apoptotic machinery, and inhibited caspase activity. Interfering with Akt activation abrogated the antiapoptotic effects of SCF, whereas exogenous expression of constitutively active Akt inhibited drug-induced apoptosis of primary megakaryocytes, indicating the Akt pathway as primarily responsible for SCF-mediated protection of megakaryocyte progenitors. These results indicate apoptosis of megakaryocyte progenitors as a major cause of chemotherapy-induced thrombocytopenia and suggest that SCF may be used to prevent platelet loss in cancer patients with c-kit–negative tumors. [Cancer Res 2007;67(10):4767–4773]

Metastatic growth in breast cancer (BC) has been proposed as an exclusive property of cancer stem cells (CSCs). However, formal proof of their identity as cells of origin of recurrences at distant sites and the molecular events that may contribute to tumor cell dissemination and metastasis development are yet to be elucidated. In this study, we analyzed a set of patient-derived breast cancer stem cell (BCSC) lines. We found that in vitro BCSCs exhibit a higher chemoresistance and migratory potential when compared with differentiated, nontumorigenic, breast cancer cells (dBCCs). By developing an in vivo metastatic model simulating the disease of patients with early BC, we observed that BCSCs is the only cell population endowed with metastatic potential. Gene-expression profile studies comparing metastagenic and non-metastagenic cells identified TAZ, a transducer of the Hippo pathway and biomechanical cues, as a central mediator of BCSCs metastatic ability involved in their chemoresistance and tumorigenic potential. Overexpression of TAZ in low-expressing dBCCs induced cell transformation and conferred tumorigenicity and migratory activity. Conversely, loss of TAZ in BCSCs severely impaired metastatic colonization and chemoresistance. In clinical data from 99 BC patients, high expression levels of TAZ were associated with shorter disease-free survival in multivariate analysis, thus indicating that TAZ may represent a novel independent negative prognostic factor. Overall, this study designates TAZ as a novel biomarker and a possible therapeutic target for BC.

In the last decade, many research studies suggested that solid tumors are hierarchically organized and sustained by a distinct pool of quiescent, therapy-resistant and highly aggressive cells identified as cancer stem cells (CSCs). In addition to being responsible for tumor initiation and growth, CSCs are expected to mediate cancer spread and relapse. While the scientific community is still debating on the legitimacy of the CSCs theory, an increasing amount of evidence indicates that their existence appears certainly valid in some cases. For instance, in breast cancer (BC) the relapse rate following therapy regimens can exceed 50% and may occur even 30 years after the initial diagnosis. Thus, one could speculate that conventional treatments might spare a small population of therapy-resistant cells that “quiescently” await until the right moment to re-initiate tumor growth; with these cells be, by definition, CSCs. Again in mammary tumors, metastatic growth has been proposed for many years as an exclusive property of CSCs, venturing that their biological plasticity may ease survival in secondary organs. Nevertheless, the proof of their identity as cells of origin of recurrences at distant sites was only recently given when, by employing fluorescent-labeled patient-derived cells and developing an in vivo metastatic model simulating BC clinical course, we show that, unlike the rest of the tumor bulk, only CSCs are capable to colonize distant organs [1]. Metastasis is an extraordinarily complex process responsible for nearly 90% of cancer-related deaths. High throughput technologies widened our comprehension on the genetic and the biochemical determinants associated with cancer development and progression [2]. Yet, the relevance of single pathways on metastatization is still unclear, especially when pondered within the pyramidal organization of tumors. Among others, the Hippo signaling is emerging as a fascinating oncogenic route mediating a variety of tumor-promoting functions such as epithelial-to-mesenchymal transition (EMT), therapeutic resistance and, accordingly, CSC generation [3, 4]. Thus, it was not totally surprising when a gene-expression profile comparison between metastagenic and non-metastagenic cells in BC linked one of the major Hippo signaling components, TAZ, to the metastatic ability of breast CSCs, along with their chemoresistance and tumorigenic potential. Genetic downregulation of TAZ expression level sensitized CSCs and relative tumor xenografts to chemotherapy, suggesting that the presence of this protein in CSCs contributes considerably to drug resistance in BC. More importantly, it led to the observation that metastases appearance was remarkably repressed in TAZ depleted CSCs, regardless the number of cells injected. These data were particularly significant because, although previous work suggested a relationship between TAZ expression and poor patient outcome in BC [5], a functional role of TAZ in tumor metastasis was not previously reported. TAZ is tightly linked to EMT, a complex cellular program well known for its association to epithelial plasticity and BC stemness [5, 6]. Accordingly, we observed that upon TAZ overexpression, non-tumorigenic BC cells developed a more spindle-like shape in vitro, suggesting an EMT transition, but also acquired tumorigenic capacity in vivo. To this regard, our and previous studies suggest a scenario in which, by inducing morphological changes, EMT determine TAZ stabilization and, in turn, acquisition of CSC, drug resistance and metastatic properties [1, 5]. Collectively, these data indicate that TAZ is instrumental in inciting metastasis in gain- and loss-of-function assays in mouse models, but does it correlate with overt metastatic lesions in human patients? The understanding of the biology of TAZ and the molecular outputs it elicited in our preclinical model, has encouraged a series of clinically focused analyses aimed at explore the prognostic/predictive significance of this protein. Initially we selected a number of primary BCs along with their paired metachronous metastases and observed that TAZ is overexpressed in breast secondary lesions. Then, to assess whether our findings had clinical relevance, we determined TAZ expression in a larger cohort of BCs patients with complete follow up data. We observed that high TAZ levels represent an independent negative prognostic factor together with the presence of metastatic lymph nodes and negativity for estrogen receptors [1]. Additionally, since some of our unpublished results revealed significant correlation between TAZ and HER2 positivity, in a follow up study we also investigated the association between TAZ expression and the pathological complete response in a subset of HER2-positive BCs. Results collected in this second study also suggest that the TAZ score efficiently predicts pathological complete response in Luminal B, HER2-positive BC patients previously treated with chemo- and targeted-therapy [7], designating a novel role for this protein in the clinical diagnostic. In conclusion, the understanding of the biological relevance of the Hippo pathway in cancer is very recent. The data collected so far from both preclinical and clinical studies, indicate that it deserves further and more thorough investigations, especially in BC, where TAZ holds the potential to implement the current pipeline of biomarkers and, possibly, therapeutic targets.

The advent of molecular targeted agents is changing the treatment of solid tumors. In non-small-cell lung cancer, compounds directed against oncogenic proteins offer novel therapeutic opportunities for a fraction of patients whose tumors harbor specific genetic defects. With the increased level of resolution achieved by high-throughput technologies, the taxonomy of lung cancer is rapidly changing. For instance, by cataloguing genetic abnormalities in squamous cell lung cancer the Cancer Genome Atlas Network revealed the existence of multiple molecular entities, each one characterized by specific molecular abnormalities, and by a different spectrum of activated/ inactivated molecular networks. Although this increased complexity could be perceived as a further drawback in effective anticancer therapy, on the other hand the combined interrogation of genomic and proteomic data is expected to provide the whole molecular map of each tumor, and to determine the information flow in the explored biological system. In particular, novel genetic and proteomic approaches are offering the opportunity for matching specific genetic defects and aberrant protein-protein interactions with active pathway-targeted inhibitors. Moreover, the isolation and characterization of a cellular pool endowed with stem-like traits, and able to recapitulate the parental disease in animals, is enabling investigators to recreate the individual patient tumor in the laboratory. In this article, we discuss how novel technologies and cellular and animal models, applied to lung cancer research, hold the potential to foster a new wave of biomarker-driven clinical trials.

Recombinant erythropoietin (EPO) analogs [erythropoiesis-stimulating agents (ESA)] are clinically used to treat anemia in patients with cancer receiving chemotherapy. After clinical trials reporting increased adverse events and/or reduced survival in ESA-treated patients, concerns have been raised about the potential role of ESAs in promoting tumor progression, possibly through tumor cell stimulation. However, evidence is lacking on the ability of EPO to directly affect cancer stem–like cells, which are thought to be responsible for tumor progression and relapse. We found that breast cancer stem–like cells (BCSC) isolated from patient tumors express the EPO receptor and respond to EPO treatment with increased proliferation and self-renewal. Importantly, EPO stimulation increased BCSC resistance to chemotherapeutic agents and activated cellular pathways responsible for survival and drug resistance. Specifically, the Akt and ERK pathways were activated in BCSC at early time points following EPO treatment, whereas Bcl-xL levels increased at later times. In vivo, EPO administration counteracted the effects of chemotherapeutic agents on BCSC-derived orthotopic tumor xenografts and promoted metastatic progression both in the presence and in the absence of chemotherapy treatment. Altogether, these results indicate that EPO acts directly on BCSC by activating specific survival pathways, resulting in BCSC protection from chemotherapy and enhanced tumor progression. Cancer Res; 73(21); 6393–400. ©2013 AACR.

The checkpoint kinase 1 (Chk1) is a key component of the DNA damage response, a molecular network deputed to maintain genome integrity. Nevertheless, cancer cells aberrantly exploit these circuits to overcome chemotherapy-induced cytotoxicity. Chk1 inhibitors have been developed as a chemopotentiating strategy and different molecular mechanisms underlying the synergism with chemotherapeutics have been uncovered. The monotherapy with Chk1 inhibitors seems to be endowed with antitumor activity against cancer cells characterized by specific defects in the DNA damage machinery or characterized by elevated levels of oncogene-induced replication stress. In this biological framework Chk1 neutralization represents a synthetic lethality-based therapeutic approach. Moreover, a dual targeting of the DNA damage machinery has been proposed envisioning the association of Chk1 abrogation with poly-ADP ribose polymerase inhibitors. The spectrum of antitumor properties of Chk1 antagonists is completed by the activity against cancer stem cells, the prominent tumorigenic population that is equipped to survive stressful conditions through multiple and interconnected mechanisms. Although the clinical development of the first generation of Chk1 antagonists was hindered by off-target effects and an unfavorable pharmacokinetic profile, a new wave of early clinical trials with more selective compounds are currently being carried out. To this end, the identification of predictive biomarkers and an in-depth characterization of molecular circuits governed by Chk1 are issues that need to be addressed for sharpening the therapeutic potential of Chk1 inhibitors.

252 Background: In spite of significant outcome gains in recurrent and de novo metastatic PC, resistance inevitably develops to androgen deprivation (ADT) and chemotherapy. Resistance is supported by clonal heterogeneity due to complex genomic and epigenetic alterations within PC foci in the primary and metastases (mets) and by the enrichment of residual tumor foci with PC SCs that survive therapy. PCSCs maintain self-renewal capacity, adapt over time to different microenvironments and retain tumor propagating activity leading to castration resistant (CR) PC progression. To study these processes, we generated patient SC-derived PC Org from primary and mets specimens. Methods: We isolated cells from normal epithelium and PC foci of 4 histopathologically mapped radical prostatectomy specimens and 2 CRPC mets. We developed a 3D culture system utilizing single tumor initiating cells expressing CD29hi/CD49bhi/CD44hiand co-cultured with epithelial/mesenchymal growth factors. Org growth peaked at 2 weeks, and Org could be passaged up to 6 months without significant deviation from early passages. We evaluated Org morphology, molecular markers and biologic response (proliferation, viability and secondary Org-forming potential) in response to ADT, docetaxel (Doc), abiraterone (Abi), enzalutamide (E), BEZ325,and a BMI-1 inhibitor. Results: The Org, ~6 per region, mirror original foci in morphology (well-organized glandular structure with central E-Cad+ cells of variable nuclear-cytoplasmic ratios, AR and PSA expression, peripheral Vimentin+cells, surrounding basement membrane) and in molecular alterations (TMRSS2-Ets fusions/Erg-overexpression, PTEN loss, RB, CHD1, BMI-1, AR-Vs). ADT decreased growth and Org forming potential. ADT plus Doc, Abi or E further reduced viability and Org-forming potential; ADT plus BEZ325 only reduced viability. The BMI-1 inhibitor selectively blocked self-renewal and restored Doc growth inhibition of a Doc-resistant CRPC-derived Org. Conclusions: We established a personalized SC-derived PC Org system to study in real time the potential efficacy of therapeutic alternatives, as well as mechanisms of resistance.

The discovery of tumor-initiating cells endowed with stem-like features has added a further level of complexity to the pathobiology of neoplastic diseases. In the attempt of dissecting the functional properties of this uncommon cellular subpopulation, investigators are taking full advantage of a body of knowledge about adult stem cells, as the “cancer stem cell model” implies that tissue-resident stem cells are the target of the oncogenic process. It is emerging that a plethora of molecular mechanisms protect cancer stem cells (CSC) against chemotherapy- and radiotherapy-induced death stimuli. The ability of CSCs to survive stressful conditions is correlated, among others, with a multifaceted protection of genome integrity by a prompt activation of the DNA damage sensor and repair machinery. Nevertheless, many molecular-targeted agents directed against DNA repair effectors are in late preclinical or clinical development while the identification of predictive biomarkers of response coupled with the validation of robust assays for assessing biomarkers is paving the way for biology-driven clinical trials. Mol Cancer Ther; 11(8); 1627–36. ©2012 AACR.

Cancer stem cell (SC) chemoresistance may be responsible for the poor clinical outcome of non-small-cell lung cancer (NSCLC) patients. In order to identify the molecular events that contribute to NSCLC chemoresistance, we investigated the DNA damage response in SCs derived from NSCLC patients. We found that after exposure to chemotherapeutic drugs NSCLC-SCs undergo cell cycle arrest, thus allowing DNA damage repair and subsequent cell survival. Activation of the DNA damage checkpoint protein kinase (Chk) 1 was the earliest and most significant event detected in NSCLC-SCs treated with chemotherapy, independently of their p53 status. In contrast, a weak Chk1 activation was found in differentiated NSCLC cells, corresponding to an increased sensitivity to chemotherapeutic drugs as compared with their undifferentiated counterparts. The use of Chk1 inhibitors in combination with chemotherapy dramatically reduced NSCLC-SC survival in vitro by inducing premature cell cycle progression and mitotic catastrophe. Consistently, the co-administration of the Chk1 inhibitor AZD7762 and chemotherapy abrogated tumor growth in vivo, whereas chemotherapy alone was scarcely effective. Such increased efficacy in the combined use of Chk1 inhibitors and chemotherapy was associated with a significant reduction of NSCLC-SCs in mouse xenografts. Taken together, these observations support the clinical evaluation of Chk1 inhibitors in combination with chemotherapy for a more effective treatment of NSCLC.

Clonal heterogeneity in primary prostate cancer (PC) is a well-established phenomenon from which rare subclones develop metastatic potential that seed distant sites. It is also well established that androgen deprivation increases clonal heterogeneity of PC foci, seeding between sites and progression to castration resistant (CR) disease that is poorly controlled by subsequent therapies. Genomic sequencing and expression profiling have revealed the complexity and variation of the molecular changes within foci and across patients. In spite of the heterogeneity, a number of gene/pathway alterations have been identified that discriminate molecular subtype and potential differences in response to therapies. To study the heterogeneity of locally advanced PC in high-risk patients, identify foci with metastatic potential and differences in biologic behavior, we generated patient-derived organoids. To this end, we isolated cells from PC foci and normal epithelial counterparts from histopathologically mapped radical prostatectomy specimens and developed a 3D culture system utilizing single cell-derived tumor initiating cells (TICs) and epithelial/mesenchymal growth factors. Organoid growth peaks at 2 weeks and serially passaged organoids can be maintained in culture for up to 6 months with no significant deviation from early passages. The organoids mirror the morphologic features (well-organized glandular structures with central E-Cad+ cells, peripheral Vimentin+ cells and a surrounding thick basement membrane), biomarker expression (AR and PSA) and molecular subtypes of the foci from which they were derived, including alterations in TMRSS2-Ets fusions, loss of PTEN, RB and CHD1, overexpression of Erg, BMI-1, AR and AR-Vs. To study the biology and responsiveness of these organoids to established therapies in the androgen sensitive and castration resistant stages of PC progression, we measured single-cell-derived organoid cell proliferation, viability and secondary organoid- forming potential in response to androgen deprivation, docetaxel, abiraterone, enzalutamide, BEZ325, BMS-345541 IKB/IKK inhibitor and a novel BMI-1 inhibitor. The results show that androgen deprivation decreases cellular growth compared to control untreated organoids, although they can be passaged for up to 4 additional weeks. Monotherapy with docetaxel, abiraterone or BEZ325 significantly reduced tumor cell viability in the absence of DHT but only abiraterone and docetaxel reduced the organoid-forming potential. The BMI-1 inhibitor selectively inhibited self-renewal, and combinations of the BMI-1 inhibitor with docetaxel rescued resistance to docetaxel in CRPC-derived cells. In conclusion, we developed a personalized and genetically defined patient-derived PC organoid model that can be utilized to identify clones with metastatic potential which may help planning systemic therapeutic interventions. Citation Format: Hatem E. Sabaawy, Monica Bartucci, Mark N. Stein, Isaac Yi Kim, Joseph R. Bertino, Robert S. DiPaola, Anna Ferrari. Patient-derived organoids from high-risk prostate cancer identify different biological subtypes and responsiveness to therapy. [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 C36.

Purpose: Chemotherapy-induced apoptosis of immature hematopoietic cells is a major cause of anemia and thrombocytopenia in cancer patients. Although hematopoietic growth factors such as erythropoietin and colony-stimulating factors cannot prevent the occurrence of drug-induced myelosuppression, stem cell factor (SCF) has been previously shown to protect immature erythroid and megakaryocytic cells in vitro from drug-induced apoptosis. However, the effect of SCF in vivo as a single myeloprotective agent has never been elucidated. Experimental Design: The ability of SCF to prevent the occurrence of chemotherapy-induced anemia and thrombocytopenia was tested in a mouse model of cisplatin-induced myelosuppression. To highlight the importance of maintaining a continuous antiapoptotic signal in immature hematopoietic cells, we compared two treatment schedules: in the first schedule, SCF administration was interrupted during chemotherapy treatment and resumed thereafter, whereas in the second schedule, SCF was administered without interruption for 7 days, including the day of chemotherapy treatment. Results: The administration of SCF to cisplatin-treated mice could preserve bone marrow integrity, inhibit apoptosis of erythroid and megakaryocytic precursors, prevent chemotherapy-induced anemia, and rapidly restore normal platelet production. Treatment with SCF increased the frequency of Bcl-2/Bcl-XL–positive bone marrow erythroid cells and sustained Akt activation in megakaryocytes. Myeloprotection was observed only when SCF was administered concomitantly with cisplatin and kept constantly present during the days following chemotherapy treatment. Conclusions: SCF treatment can prevent the occurrence of chemotherapy-induced anemia and thrombocytopenia in mice, indicating a potential use of this cytokine in the supportive therapy of cancer patients. Clin Cancer Res; 17(19); 6185–91. ©2011 AACR.

The incidence of colon cancer has increased in developed countries, possibly due to sedentary lifestyle and high caloric diet. Experimental and epidemiological evidence suggests a link between colon cancer development and adipose tissue-derived circulating hormones. Leptin, a pluripotent cytokine secreted by adipocytes, is a key regulator of appetite and energy balance acting in the brain. On the other hand, leptin also controls many physiological and pathological processes in peripheral organs. Recent studies in colon cancer cell lines and human tumors suggested that leptin and its receptor (ObR) are implicated in colon carcinogenesis, and may serve as new biomarkers and pharmacological targets. Here, we explored, for the first time, whether leptin can affect the biology of colorectal tumor stem cells (CTSCs). We found that our previously established and characterized CTSC clones express ObR and respond to leptin with cell proliferation, activation of the extracellular signal-related kinase (ERK)1/2 and AKT signaling pathways, enhanced growth in soft agar, and improved sphere formation associated with E-cadherin overexpression. Moreover, leptin counteracted cytotoxic effects of 5-fluorouracil, a common colon cancer therapeutic agent. These results suggest that obesity and increased leptin levels might promote colorectal cancer by increasing growth and survival of CTSCs.

MicroRNAs (miRNAs) are noncoding small RNAs that repress protein translation by targeting specific messenger RNAs. miR-15a and miR-16-1 act as putative tumor suppressors by targeting the oncogene BCL2. These miRNAs form a cluster at the chromosomal region 13q14, which is frequently deleted in cancer. Here, we report that the miR-15a and miR-16-1 cluster targets CCND1 (encoding cyclin D1) and WNT3A, which promotes several tumorigenic features such as survival, proliferation and invasion. In cancer cells of advanced prostate tumors, the miR-15a and miR-16 level is significantly decreased, whereas the expression of BCL2, CCND1 and WNT3A is inversely upregulated. Delivery of antagomirs specific for miR-15a and miR-16 to normal mouse prostate results in marked hyperplasia, and knockdown of miR-15a and miR-16 promotes survival, proliferation and invasiveness of untransformed prostate cells, which become tumorigenic in immunodeficient NOD-SCID mice. Conversely, reconstitution of miR-15a and miR-16-1 expression results in growth arrest, apoptosis and marked regression of prostate tumor xenografts. Altogether, we propose that miR-15a and miR-16 act as tumor suppressor genes in prostate cancer through the control of cell survival, proliferation and invasion. These findings have therapeutic implications and may be exploited for future treatment of prostate cancer.

Clinically effective next generation androgen deprivation therapy (ADT) and androgen receptor (AR)-directed therapies have been developed to treat castration-resistant prostate cancer (CRPC). However, disease progression inevitably occurs, and majority of relapsed tumors continue to overexpress AR. AR mutations and constitutively active AR splice variants (AR-Vs) are detected during ADT and may lead to drug resistance. Therefore, there is dire need to develop models that provide a better understanding of AR signaling and determine markers of ADT resistance in prostate cancer patient cells. Mouse models and existing human prostate cancer cell lines have inherent limitations for deciphering mechanisms of therapy resistance. Unlike the recently reported prostate tissue organoids that were derived at a low efficiency from 7/49 metastatic prostate cancer samples, we have developed a novel and exciting technology to generate normal, benign prostatic hyperplasia (BPH) and cancer “mini-prostates” from both early-stage and advanced prostate cancer patient samples. We have successfully rederived mini-prostates from 17 out of 19 primary prostatectomy tissues. We incorporated developmental epithelial and mesenchymal signals, stem-like cells and growth requirements in 3D cultures for maintaining primary normal, BPH and prostate cancer patient-derived cells as stem-like cell-derived organoids. Our preliminary data show the establishment of patient- single cell-derived normal, BPH and cancer mini-prostates comprised of organoids with glandular tissues that have a thick basement membrane, an outer mesenchymal and inner epithelial cells conveying AR signaling, expressing cytokeratins and secreting PSA. RNA sequencing, expression profiling and ADT revealed that mini-prostate cells maintain the genetic, chemoresistance, and growth rate features of the primary prostate. To model tumor heterogeneity, prostate cells from surgical tissues were isolated upon digital imaging and tissue mapping in mirror sections for AMACR, AR, AR-Vs, p63, Ki67, ERG expression and FISH analysis for TMPRSS2-ERG fusion. Living cells from mapped BPH and cancer clones are being utilized to derive single cells into mini-prostates. Our studies generated novel therapy sensitivity and drug discovery models that would allow for precision medicine and coclinical approaches to be enlisted to reveal chemo- or hormonal-resistance profiles in genetically defined mini-prostates, apply combinatory-targeted therapies and study therapy-induced clonal selection processes to ultimately guide future clinical trials. Citation Format: Monica Bartucci, Michele Patrizii, Eric Huselid, Shamila Yussuf, Nitu Bansal, Kathleen Flaherty, Denis Tolkunov, Hua Zhong, Mark N. Stein, Joseph Bertino, Robert DiPaola, Isaac Kim, Hatem E. Sabaawy. Generation of single cell-derived normal, benign and cancer mini-prostates from primary patient-derived tissues. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 223. doi:10.1158/1538-7445.AM2015-223

The INK4b-ARF-INK4a locus on human chromosome 9p21 is a hot spot genomic region that undergoes frequent inactivation or deletion in a wide spectrum of human cancers. The locus encodes three cell cycle inhibitory proteins: p15INK4b encoded by CDKN2b, p16INK4a encoded by CDKN2a and p14ARF (p19Arf in mice) encoded by an alternative reading frame (ARF) of CDKN2a. We have recently identified a homologous INK4 genomic locus in zebrafish that is surprisingly devoid of ARF sequences, and encodes a single zebrafish ink4ab gene that functions to activate stress-induced senescence. Therefore, evolution of the mammalian INK4 locus to include multiple tumor suppressors sharing regulatory features could attribute to stronger mechanisms of tumor suppression in these longer-lived vertebrates. Oxidative and/or oncogenic stress provokes cellular senescence involving the retinoblastoma (Rb) and ARF/p53 pathways, leading to silencing of growth-promoting genes by methylation of histone H3 lysine 9 (H3K9me) with the histone methyltransferase Suv39h1. We found that ink4ab deficient zebrafish had deregulated Rb signaling and senescence responses, and reduced suv39h1 expression levels that led to poor overall survival. Ink4ab deficient zebrafish had increased lymphocyte proliferation, splenomegaly, and developed multiple spontaneous tumors including metastatic melanoma, osteosarcoma, hepatocellular adenoma, leukemias and myelodysplastic disorders. Moreover, zebrafish heterozygous for both p53 and ink4ab mutations displayed significantly higher tumor incidence compared to p53 heterozygotes with wild-type ink4ab, indicating that ink4ab haploinsufficiency promotes tumorigenesis. To this end, the combined deficiency of ink4ab and p53 accelerates tumor latency, likely by reversing cellular senescence responses. To further examine ARF-independent senescence regulatory roles of INK4 products in a mammalian model, we have generated conditional mice deficient for all three INK4 open reading frames in the adult hematopoietic system. Within seven months, homozygous Ink4a/Ink4b/ARF-/- mice exhibited marked splenomegaly and developed spontaneous tumors, including leukemias and soft tissue sarcomas. Peripheral blood, splenic and bone marrow analyses of homozygous mutant mice showed deregulated lymphocyte development, uncontrolled proliferation, apoptosis and Suv39h1-dependent senescence when compared to heterozygous and wild type mice. These results identify ARF-independent and Suv39h1-dependent senescence regulatory and tumor suppressor mechanisms whose inactivation permits tumor formation in response to oncogenic and/or oxidative stress. Our models offer the opportunity to reveal novel paradigms for promoting cellular senescence as an alternative strategy for cancer prevention and therapy. Citation Format: Kathleen Flaherty, Daniel Jones, Shamila Yussuf, Stephani Davis, Eric Huselid, Wei Wang, Monica Bartucci, Hatem E. Sabaawy. Conditional mouse and zebrafish models of INK4-mediated tumor suppression reveal ARF-independent regulation of cellular senescence. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1264. doi:10.1158/1538-7445.AM2015-1264

Life expectancy of patients affected by glioblastoma multiforme is extremely low. The therapeutic use of tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) has been proposed to treat this disease based on its ability to kill glioma cell lines in vitro and in vivo. Here, we show that, differently from glioma cell lines, glioblastoma multiforme tumors were resistant to TRAIL stimulation because they expressed low levels of caspase-8 and high levels of the death receptor inhibitor PED/PEA-15. Inhibition of methyltransferases by decitabine resulted in considerable up-regulation of TRAIL receptor-1 and caspase-8, down-regulation of PED/PEA-15, inhibition of cell growth, and sensitization of primary glioblastoma cells to TRAIL-induced apoptosis. Exogenous caspase-8 expression was the main event able to restore TRAIL sensitivity in primary glioblastoma cells. The antitumor activity of decitabine and TRAIL was confirmed in vivo in a mouse model of glioblastoma multiforme. Evaluation of tumor size, apoptosis, and caspase activation in nude mouse glioblastoma multiforme xenografts showed dramatic synergy of decitabine and TRAIL in the treatment of glioblastoma, whereas the single agents were scarcely effective in terms of reduction of tumor mass, apoptosis induction, and caspase activation. Thus, the combination of TRAIL and demethylating agents may provide a key tool to overcome glioblastoma resistance to therapeutic treatments. (Cancer Res 2005; 65(24): 11469-77)

In breast cancer cells, 17-beta-estradiol (E2) upregulates the expression of insulin receptor substrate 1 (IRS-1), a molecule transmitting insulin-like growth factor-I (IGF-I) signals through the PI-3K/Akt survival pathways. The stimulation of IRS-1 by E2 has been documented on the transcriptional level. Here we studied whether the expression of estrogen receptor (ER)-alpha affects IRS molecules post-transcriptionally. We used ER-alpha-negative MDA-MB-231 breast cancer cells and MDA-MB-231 cells with re-expressed ER-alpha. In MDA-MB-231 cells cultured under serum-free conditions, IRS-1 and IRS-2 were degraded through the 26S proteasome and calpain pathways. Re-expression of ER-alpha in MDA-MB-231 cells correlated with enhanced stability of IRS molecules. This effect coincided with significantly reduced ubiquitination of IRS-1 and IRS-2, but did not involve increased IRS-1 and IRS-2 transcription. The interference of ER-alpha with IRS-1 and IRS-2 turnover could rely on the competition for common degradation pathways, as in MDA-MB-231/ER cells, ER-alpha processing was blocked by proteasome and calpain inhibitors. Notably, a fraction of the cytosolic ER-alpha colocalized and coprecipitated with IRS-1 and IRS-2, indicating a possible common destination for these proteins. The stabilization of IRS-1 in MDA-MB-231/ER cells was paralleled by the upregulation of the IRS-1/Akt/GSK-3 pathway and improved survival in the presence of IGF-I, whereas IRS-2 was not involved in IGF-I signaling.

The structural disintegration of normal epithelium is an early manifestation of cancer in mammary gland. Yet our knowledge on mechanisms controlling breast epithelial cell adhesion is still rudimentary. The increased content of the insulin-like growth factor I receptor (IGF-IR) and its close homologue the insulin receptor (IR) has been well documented in human breast cancer specimens. In this study we investigated how these hormone receptors modulate cell adhesion in breast carcinoma cells. Previously we developed MCF-7 human breast cancer cells overexpressing the IGF-IR. The major achievement of this work is the establishment of a new model consisting of MCF-7-derived cells overexpressing the IR that allowed the comparison of IR and IGF-IR functions. Our results strongly confirmed the specificity of the IGF-IR in promoting the large non-invasive tumor aggregates on biological matrix. For the first time we demonstrated that in breast cancer cells IGF-IR tyrosine kinase modulates the intercellular balance of Beta-catenin, the element of E-cadherin/catenin cell-cell adhesion complex. We continued to explore anti-tumor potentials of the antiestrogens and found that a synthetic steroid analog ICI 182,780 is a potent inhibitor of non-invasive breast tumor aggregates in three-dimensional culture.

Hyperactivation of the insulin-like growth factor I receptor (IGF-IR) contributes to primary breast cancer development, but the role of the IGF-IR in tumor metastasis is unclear. Here we studied the effects of the IGF-IR on intercellular connections mediated by the major epithelial adhesion protein, E-cadherin (E-cad). We found that IGF-IR overexpression markedly stimulated aggregation in E-cad-positive MCF-7 breast cancer cells, but not in E-cad-negative MDA-MB-231 cells. However, when the IGF-IR and E-cad were co-expressed in MDA-MB-231 cells, cell-cell adhesion was substantially increased. The IGF-IR-dependent cell-cell adhesion of MCF-7 cells was not related to altered expression of E-cad or alpha-, beta-, or gamma-catenins but coincided with the up-regulation of another element of the E-cad complex, zonula occludens-1 (ZO-1). ZO-1 expression (mRNA and protein) was induced by IGF-I and was blocked in MCF-7 cells with a tyrosine kinase-defective IGF-IR mutant. By co-immunoprecipitation, we found that ZO-1 associates with the E-cad complex and the IGF-IR. High levels of ZO-1 coincided with an increased IGF-IR/alpha-catenin/ZO-1-binding and improved ZO-1/actin association, whereas down-regulation of ZO-1 by the expression of an anti-ZO-1 RNA inhibited IGF-IR-dependent cell-cell adhesion. The results suggested that one of the mechanisms by which the activated IGF-IR regulates E-cad-mediated cell-cell adhesion is overexpression of ZO-1 and the resulting stronger connections between the E-cad complex and the actin cytoskeleton. We hypothesize that in E-cad-positive cells, the IGF-IR may produce antimetastatic effects.

The oncogenic SHC proteins are signaling substrates for most receptor and cytoplasmic tyrosine kinases (TKs) and have been implicated in cellular growth, transformation, and differentiation. In tumor cells overexpressing TKs, the levels of tyrosine phosphorylated SHC are chronically elevated. The significance of amplified SHC signaling in breast tumorigenesis and metastasis remains unknown. Here we demonstrate that seven- to ninefold overexpression of SHC significantly altered interactions of cells with fibronectin (FN). Specifically, in human breast cancer cells overexpressing SHC (MCF-7/SHC) the association of SHC with α5β1 integrin (FN receptor) was increased, spreading on FN was accelerated, and basal growth on FN was reduced. These effects coincided with an early decline of adhesion-dependent MAP kinase activity. Basal motility of MCF-7/SHC cells on FN was inhibited relative to that in several cell lines with normal SHC levels. However, when EGF or IGF-I was used as the chemoattractant, the locomotion of MCF-7/SHC cells was greatly (approx fivefold) stimulated, while it was only minimally altered in the control cells. These data suggest that SHC is a mediator of the dynamic regulation of cell adhesion and motility on FN in breast cancer cells.

We have previously shown that estrogens binding to estrogen receptor (ER) α increase proliferation of Leydig tumor cells. Estrogens can also bind to G protein-coupled ER (GPER) and activation of this receptor can either increase or decrease cell proliferation of several tumor types. The aim of this study was to investigate GPER expression in R2C rat tumor Leydig cells, evaluate effects of its activation on Leydig tumor cell proliferation and define the molecular mechanisms triggered in response to its activation. R2C cells express GPER and its activation, using the specific ligand G-1, is associated with decreased cell proliferation and initiation of apoptosis. Apoptosis after G-1 treatment was asserted by appearance of DNA condensation and fragmentation, decrease in Bcl-2 and increase in Bax expression, cytochrome c release, caspase and poly (ADP-ribose) polymerase-1 (PARP-1) activation. These effects were dependent on GPER activation because after silencing of the gene, using a specific small interfering RNA, cyt c release, PARP-1 activation and decrease in cell proliferation were abrogated. These events required a rapid, however, sustained extracellular regulated kinase 1/2 activation. G-1 was able to decrease the growth of R2C xenograft tumors in CD1 nude mice while increasing the number of apoptotic cells. In addition, in vivo administration of G-1 to male CD1 mice did not cause any alteration in testicular morphology, while cisplatin, the cytotoxic drug currently used for the therapy of Leydig tumors, severely damaged testicular structure, an event associated with infertility in cisplatin-treated patients. These observations indicate that GPER targeting for the therapy of Leydig cell tumor may represent a good alternative to cisplatin to preserve fertility in Leydig tumor patients.