Three-dimensional Laminin-rich Extracellular Matrix Research Articles

Aqueous extracts from Dioscorea sansibarensis Pax show cytotoxic and radiosensitizing potential in 3D growing HPV-negative and HPV-positive human head and neck squamous cell carcinoma models

Numerous natural substances have anti-cancer properties. Especially indigenous people use aqueous plant extracts for tea or ointments including Dioscorea sansibarensis Pax to treat various diseases. The aim of this study was to evaluate the cytotoxic and radiosensitizing potential of aqueous extracts from Dioscorea sansibarensis Pax collected from Kenya in a panel of HPV-negative and -positive head and neck squamous cell carcinoma (HNSCC) cells grown in three-dimensional laminin-rich extracellular matrix (3D lrECM). The results show cytotoxicity, radiosensitization and increased levels of residual double strand breaks (DBS) by Dioscorea sansibarensis Pax extracts in HPV-negative and -positive HNSCC models in a concentration- and cell model-dependent manner. Application of ROS scavengers indicated an association between ROS-induced DSB and radiosensitization through Dioscorea sansibarensis Pax pretreatment. High-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) based characterization of Dioscorea sansibarensis Pax identified the main components of the extract including camptothecin. Overall, Dioscorea sansibarensis Pax aqueous extracts alone and in combination with X-ray irradiation showed effective anticancer properties, which are worthy of further mechanistic investigation.

Transient external force induces phenotypic reversion of malignant epithelial structures via nitric oxide signaling.

Non-malignant breast epithelial cells cultured in three-dimensional laminin-rich extracellular matrix (lrECM) form well organized, growth-arrested acini, whereas malignant cells form continuously growing disorganized structures. While the mechanical properties of the microenvironment have been shown to contribute to formation of tissue-specific architecture, how transient external force influences this behavior remains largely unexplored. Here, we show that brief transient compression applied to single malignant breast cells in lrECM stimulated them to form acinar-like structures, a phenomenon we term 'mechanical reversion.' This is analogous to previously described phenotypic 'reversion' using biochemical inhibitors of oncogenic pathways. Compression stimulated nitric oxide production by malignant cells. Inhibition of nitric oxide production blocked mechanical reversion. Compression also restored coherent rotation in malignant cells, a behavior that is essential for acinus formation. We propose that external forces applied to single malignant cells restore cell-lrECM engagement and signaling lost in malignancy, allowing them to reestablish normal-like tissue architecture.

Abstract P3-06-19: Gene expression profiles accompanying phenotypic changes during non-malignant breast epithelial cells acini formation to explain MRI phenotypes

Abstract Magnetic resonance imaging (MRI) captures the three dimensional way in which tumors are organized in the breast, defined as imaging phenotypes in the I-SPY 1 trial. We developed a gene set based on the way in which breast epithelial cells aggregate and organize in three dimensional cultures. We investigated whether these organizational genes correspond to the imaging phenotypes. MRI phenotypes have been shown to correspond to the degree of response to neoadjuvant chemotherapy, and are used to predict the ability to achieve breast conservation treatment (Mukhtar et al, Ann Surg Oncol, 20: 3823–3830, 2013). We hypothesized that the molecular profile accompanying phenotypic changes occurring during the organization process of non-malignant acini may explain the molecular basis of MRI tumor phenotypes. We have developed prediction models for MRI phenotypes based on expression profiles identified during the organization process of non-malignant breast epithelial cells in three-dimensional laminin-rich extracellular matrix (Fournier et al. Cancer Res, 66, 7095-102, 2006). We analyzed a subset of 324 organizational genes in 147 samples from stage II-III breast cancer in the I-SPY 1 trial cohort with Agilent microarrays and MRI annotation (CALGB 150007/150012; ACRIN 6657). MRI phenotype of the index lesion was assessed by the site radiologists using the following radiographic criteria: A) well defined unicentric mass; B) well-defined multilobulated mass; C) area enhancement with nodularity; D) area enhancement without nodularity; E) Septal spreading. The distribution of phenotypes in I-SPY 1 was: A: 16%, B: 33%, C: 30%, D: 15%, E: 7%. We developed predictors for MRI phenotypes dichotomized as either "well-defined" (A and B) or "non-well-defined" (C, D and E). Using patent-pending algorithms we selected a subset of the organizational genes with the greatest predictive power to identify MRI "well-defined" phenotypes in the I-SPY 1 cohort. Then prediction models were developed using the 50 top ranking genes and logistic regression methods. We followed Monte-Carlo cross validation method to make sure that the performance of a model is not a result of over-fitting the model to the sample data using separate datasets to support the modeling. The samples were randomly partitioned 10,000 times in a 85% training, and 15% test ratio to test models performance. The resulting performance of predictive models in the test set had an average classification accuracy of greater than 80% (ROC statistics AUC>0.8) using gene expression measurements from between 16 to 20 genes. For random lists of 16-20 genes, the accuracy was approximately 50% (ROC AUC∼0.5). Amongst the MRI models were several genes that are known to regulate key cellular pathways such as cell division, metabolism, and migration using MetaCore pathway analysis. Taken together, the results suggest that the MRI phenotypes may be a manifestation of the organization genes that determine behavior in three dimensional culture. Future research includes confirming these results using an independent dataset, defining the potential drivers of MRI phenotypes, and determining if putative drivers provide a key contribution to tumor subtypes. Citation Format: Marcia V Fournier, Alan Derr, Alex Margulis, Kevin Reid, Sara Brumbaugh, Richard Anderson, W Fraser Symmans, Laura Esserman, Nola Hylton. Gene expression profiles accompanying phenotypic changes during non-malignant breast epithelial cells acini formation to explain MRI phenotypes [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 P3-06-19.

Simultaneous β1 integrin-EGFR targeting and radiosensitization of human head and neck cancer.

Signaling from integrins and receptor tyrosine kinases (RTKs) contributes substantially to therapy resistance of malignant tumors. We investigated simultaneous β1 integrin-epidermal growth factor receptor (EGFR) targeting plus radiotherapy in human head and neck squamous cell carcinomas (HNSCCs). Ten HNSCC cell lines were grown in three-dimensional laminin-rich extracellular matrix cell cultures and two of them as tumor xenografts in nude mice (n = 12-16 per group). Targeting of β1 integrin and EGFR with monoclonal inhibitory antibodies (AIIB2 and cetuximab, respectively) was combined with x-ray irradiation. Clonogenic survival, tumor growth, and tumor control (evaluated by Kaplan-Meier analysis), apoptosis, phosphoproteome (interactome, network betweeness centrality analysis), receptor expression (immunohistochemistry), and downstream signaling (western blotting) were assessed. Various mutants of the integrin signaling mediator focal adhesion kinase (FAK) were employed for mechanistic studies. All statistical tests were two-sided. Compared with β1 integrin or EGFR single inhibition, combined β1 integrin-EGFR targeting resulted in enhanced cytotoxicity and radiosensitization in eight out of 10 tested HNSCC cell lines, which responded with an FAK dephosphorylation after β1 integrin inhibition. In vivo, simultaneous anti-β1 integrin/anti-EGFR treatment and radiotherapy of UTSCC15 responder xenografts enabled better tumor control compared with anti-EGFR monotherapy and irradiation (hazard ratio [HR] = 6.9, 95% confidence interval [CI] = 1.6 to 30.9, P = .01), in contrast to the SAS nonresponder tumor model (HR = 0.9, 95% CI = 0.4 to 2.3, P = .83). Mechanistically, a protein complex consisting of FAK- and Erk1-mediated prosurvival signals for radiation resistance, which was effectively compromised by β1 integrin and EGFR blocking. Concomitant targeting of β1 integrin and EGFR seems a powerful and promising approach to overcome radioresistance of HNSCCs.

β1-integrin via NF-κB signaling is essential for acquisition of invasiveness in a model of radiation treated in situ breast cancer

IntroductionDuctal carcinoma in situ (DCIS) is characterized by non-invasive cancerous cell growth within the breast ducts. Although radiotherapy is commonly used in the treatment of DCIS, the effect and molecular mechanism of ionizing radiation (IR) on DCIS are not well understood, and invasive recurrence following radiotherapy remains a significant clinical problem. This study investigated the effects of IR on a clinically relevant model of Akt-driven DCIS and identified possible molecular mechanisms underlying invasive progression in surviving cells.MethodsWe measured the level of phosphorylated-Akt (p-Akt) in a cohort of human DCIS specimens by immunohistochemistry (IHC) and correlated it with recurrence risk. To model human DCIS, we used Akt overexpressing human mammary epithelial cells (MCF10A-Akt) which, in three-dimensional laminin-rich extracellular matrix (lrECM) and in vivo, form organotypic DCIS-like lesions with lumina expanded by pleiomorphic cells contained within an intact basement membrane. In a population of cells that survived significant IR doses in three-dimensional lrECM, a malignant phenotype emerged creating a model for invasive recurrence.ResultsP-Akt was up-regulated in clinical DCIS specimens and was associated with recurrent disease. MCF10A-Akt cells that formed DCIS-like structures in three-dimensional lrECM showed significant apoptosis after IR, preferentially in the luminal compartment. Strikingly, when cells that survived IR were repropagated in three-dimensional lrECM, a malignant phenotype emerged, characterized by invasive activity, up-regulation of fibronectin, α5β1-integrin, matrix metalloproteinase-9 (MMP-9) and loss of E-cadherin. In addition, IR induced nuclear translocation and binding of nuclear factor-kappa B (NF-κB) to the β1-integrin promoter region, associated with up-regulation of α5β1-integrins. Inhibition of NF-κB or β1-integrin signaling abrogated emergence of the invasive activity.ConclusionsP-Akt is up-regulated in some human DCIS lesions and is possibly associated with recurrence. MCF10A-Akt cells form organotypic DCIS-like lesions in three-dimensional lrECM and in vivo, and are a plausible model for some forms of human DCIS. A population of Akt-driven DCIS-like spheroids that survive IR progresses to an invasive phenotype in three-dimensional lrECM mediated by β1-integrin and NF-κB signaling.

Integrin α3β1–CD151 complex regulates dimerization of ErbB2 via RhoA

Integrin α3β1 regulates adhesive interactions of cells with laminins and have a critical role in adhesion-dependent cellular responses. Here, we examined the role of α3β1-integrin in ErbB2-dependent proliferation of breast cancer cells in three-dimensional laminin-rich extracellular matrix (3D lr-ECM). Depletion of α3β1 in ErbB2-overexpressing breast cancer cells suppressed growth and restore cell polarity in 3D lr-ECM. The phenotype of α3β1-depleted cells was reproduced upon depletion of tetraspanin CD151 and mirrored that of the cells treated with Herceptin, an established ErbB2 antagonist. Breast cancer cells expressing the α3β1-CD151 complex have higher steady-state phosphorylation of ErbB2 and show enhanced dimerization of the protein when compared with α3β1-/CD151-depleted cells. Furthermore, Herceptin-dependent dephosphorylation of ErbB2 was only observed in α3β1-CD151-expressing cells. Importantly, the inhibitory activity of Herceptin was more pronounced when cells expressed both α3β1 and CD151. We also found that the level of active RhoA was increased in α3β1- and CD151-depleted cells and that Rho controls dimerization of ErbB2. Expression of α3β1 alone did not have significant prognostic value in patients with invasive ductal carcinoma of the breast. However, expression of α3β1 in combination with CD151 represented a more stringent indicator of poor survival than CD151 alone. Taken together, these results demonstrate that the α3β1-CD151 complex has a critical regulatory role in ErbB2-dependent signalling and thereby may be involved in breast cancer progression.

NF-κB Regulates Radioresistance Mediated By β1-Integrin in Three-Dimensional Culture of Breast Cancer Cells

β1-integrin induction enhances breast cancer cell survival after exposure to ionizing radiation (IR), but the mechanisms of this effect remain unclear. Although NF-κB initiates prosurvival signaling pathways post-IR, the molecular function of NF-κB with other key elements in radioresistance, particularly with respect to extracellular matrix-induced signaling, is not known. We discovered a typical NF-κB-binding site in the β1-integrin promoter region, indicating a possible regulatory role for NF-κB. Using three-dimensional laminin-rich extracellular matrix (3D lrECM) culture, we show that NF-κB is required for β1-integrin transactivation in T4-2 breast cancer cells post-IR. Inhibition of NF-κB reduced clonogenic survival and induced apoptosis and cytostasis in formed tumor colonies. In addition, T4-2 tumors with inhibition of NF-κB activity exhibit decreased growth in athymic mice, which was further reduced by IR with downregulated β1-integrin expression. Direct interactions between β1-integrin and NF-κB p65 were induced in nonmalignant breast epithelial cells, but not in malignant cells, indicating context-specific regulation. As β1-integrin also activates NF-κB, our findings reveal a novel forward feedback pathway that could be targeted to enhance therapy.

Abstract A34: Microenvironment controls breast cancer cells sensitivity to doxorubicin by autophagy inhibition

Abstract Drug chemotherapy resistance is the leading cause of death for cancer patients. Most of the studies involving chemotherapy resistance are based on experiments making use of cancer cell lines maintained in two-dimensional (2D) cultures. In these conditions, the influence of extracellular matrix (ECM) and the three-dimensional (3D) microenvironment are not considered, despite clear evidence of their role in genesis and progression of cancer. In the present work, we evaluated the microenvironment control in the MCF-7 cellular response to doxorubicin using the three-dimensional laminin-rich ECM (3D lrECM) cell culture model. Based on cell plating on top of growth factor-reduced Basement Membrane (BD MatrigelTM), the 3D lrECM culture consists of a more physiologically applicable approach for ex vivo analysis of cell behavior. When maintained in a 3D context, these human breast cancer cells showed morphological differences, as viewed by phase contrast microscopy, forming round colonies. The MCF-7 growth rate showed also to be dependent of the cellular culture model used. Despite cancerous, these cells undergo a significant growth arrest on top of laminin-rich ECM. Using the cell viability XTT assays for doxorubicin sensitivity analysis, we observed a drug resistance event in 2D cells treated with low chemotherapy doses (250 and 625 nM of doxorubicin). Thus, while the cell growth on lrECM led to decrease on doxorubicin survival at 60%, the survival rates were almost unchanged (higher than 95% of survival) in monolayer cultures at same treatment conditions. Autophagy, a lysosome-dependent catabolic degradation process, has been implicated in anticancer drug resistance. Moreover, recent reports demonstrated that this cellular phenomenon plays a crucial role as a pro-survival factor in MCF-7 cells treated with epirubicin, a doxorubicin analog. Thus, we investigated whether autophagy is involved in doxorubicin response changes promoted by maintenance in 3D culture. By co-treatment with chloroquine, a pharmacological inhibitor of autophagy, we observed a synergistic effect with doxorubicin only in 2D, with 30% reduction of survival. We also evaluated the chloroquine effect in breast cancer cell response to doxorubicin using specific fluorescent dyes for living, apoptotic and necrotic cells. Inhibition of autophagy increased the cell death percentage in monolayer, by not in 3D cell culture, confirming that ECM abolishes autophagy pro-survival action. Subsequently, the autophagy levels were measured by LC3-II accumulation in both cell culture conditions. Our results suggested that autophagy is significantly inhibited by cellular growth in 3D lrECM. While a clear synergic LC3-II accumulation was observed in 2D cultured cells both treated with doxorubicin and chloroquine, very low levels of LC3-II expression were detected in 3D treated cells. Altogether, our results suggest that autophagy is essential for the tissue microenvironment control of breast cancer cells response to anticancer drugs. Therefore, presence of ECM compounds might prevent the autophagy protective effect from doxorubicin-induced cell death. Financial Support: FAPESP and CNPq Citation Format: Luciana R. Gomes, Alexandre T. Vessoni, Carlos FM Menck. Microenvironment controls breast cancer cells sensitivity to doxorubicin by autophagy inhibition. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr A34.

Abstract P4-06-05: NF-κB Upregulates β1-integrin via Increased Transcriptional Activity in Three-dimensional Culture: a Mechanism by which Malignant Breast Cells Acquire Radioresistance

Abstract The molecular mechanisms giving rise to tumor viability after therapeutic radiation remain elusive. We showed previously that β1-integrin is associated with enhanced cell survival of breast cancer cells after exposure to high dose ionizing radiation (IR). Recently, we discovered that a typical binding site for Nuclear factor-κB (NF-κB), a stress-sensitive heterodimeric transcription factor, was located in human β1-integrin promoter, which indicates that β1-integrin-mediated resistance to radiation may be functionally regulated by NF-κB. The aim of the present study was to investigate the relationship between NF-κB and β1-integrin pathways in radioprotection of malignant T4-2 breast cells in three-dimensional laminin-rich extracellular matrix (3D lrECM). We show that NF-κB is required for radiation-induced β1-integrin transactivation. Inhibition of NF-κB activity significantly reduced clonogenic survival with the inhibition of IR-induced total and phosphorylated (Thr 788/789) β1-integrins, correlated with increased DNA binding activity at the β1- integrin promoter. Treatment of T4-2 colonies with NF-κB activation inhibitor before IR resulted in a reduction of the size of colonies, associated with a decrease in proliferation and increase in apoptosis, indicating an increased susceptibility to IR. Further studies revealed that a direct interaction of β1-integrin with α5-integrin was highly induced, but not with NF-κB p65, in T4-2 cells surviving IR. These interactions were found to be oppositely regulated in radiosensitive S1 cells, suggesting that IR induces the formation of α5β1-integrin complexes leading to increased survival post-IR. Together these results provide the first evidence that NF-κB-induced β1-integrin transactivation is responsible for increased survival post IR in breast cancer. Since β1-integrin is shown to activate NF-κB, our data further suggest a loop-like β1-integrin–NF-κB–β1-integrin pathway in tumor radioresistance. This novel pathway may serve as an efficient drug target to re-sensitize radioresistant tumor cells. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P4-06-05.

P3-01-06: NF-kB Inhibition Promotes Radiosensitivity of Breast Cancer Cells in Three-Dimensional Culture through Abating b1-Integrin Expression.

Abstract Therapy-associated tumor resistance, giving rise to recurrence and mortality, is a critical issue in cancer therapy. The molecular mechanisms causing tumor resistnace to the therapeutic radiation remain elusive. Nuclear factor-KB (NF-KB), a stress-sensitive heterodimeric transcription factor in the regulation of the stress-responsive genes, has been shown to initiate prosurvival signaling pathways. The cooperative function of NF-KB with other key stress elements in radioresistance remains to be elucidated. We have previously reported that induction of α5β1-integrin is associated with the enhanced cell survival of breast cancer cells after exposure to high dose IR (ionizing radiation). Because a typical NF-KB binding site was located in human β1-integrin promoter region, β1-integrin-mediated resistance to radiation may be regulated by NF-KB. The aim of the present study was to reveal a connection between NF-KB and β1-integrin pathways in radioprotection of malignant T4-2 mammary epithelial cells in 3D ***lrECM (three-dimensional laminin-rich extracellular matrix). We show that the elevated NF-KB activity was correlated with enhanced clonogenic survival, and increased NF-KB heterodimer p50/p65 levels were associated with an increase in total and phosphorylated (Thr 788/789) β1-integrins. Inhibition of NF-KB activation significantly reduced clonogenic survival with the inhibition of β1-integrin. These results indicate that NF-KB-mediated induction of β1-integrin is associated with an increased radiation resistance. Treatment of T4-2 colonies, formed at day 4, with NF-KB activation inhibitor in 3D lrECM before exposure to IR (4-Gy X-ray) resulted in a reduction of the size of colonies. The surviving colonies were associated with a decrease in proliferation and increase in apoptosis, indicating a decrease of resistance to IR. Together, these results provide the first evidence that NF-KB-mediated β1-integrin expression is responsible for tumor radioresistance. The NF-KB/β1-integrin pathway may serve as an efficient drug target to re-sensitize radioresistant tumor cells. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P3-01-06.

Abstract C130: Inhibition of NF-κB activity promotes radiosensitivity of breast cancer cells in three-dimensional culture via a β1-integrin-mediated pathway.

Abstract Radioresistance markedly impairs the efficacy of tumor radiotherapy and involves prosurvival signal transduction pathways that prevent radiation-induced cell death. Nuclear factor-κB (NF-κB), a stress-sensitive heterodimeric transcription factor in the regulation of the stress-responsive genes, has been shown to initiate prosurvival signaling in response to ionizing radiation (IR). The cooperative function of NF-κB with other key stress elements in radioresistance has not been clearly defined. We have previously reported that induction of β1-integrin is associated with the enhanced cell survival of breast cancer cells after exposure to clinically relevant doses of IR. Because a typical NF-κB binding site was identified in human β1-integrin promoter region, β1-integrin-mediated resistance to radiation may be regulated by NF-κB. The aim of the present study was to reveal a connection between NF-κB and β1-integrin pathways in radioprotection of malignant T4-2 mammary epithelial cells in 3D lrECM (three-dimensional laminin-rich extracellular matrix). We show that the elevated NF-κB activity was correlated with enhanced clonogenic survival with an increase in total and phosphorylated (Thr 788/789) β1-integrins. Inhibition of NF-κB activation significantly reduced clonogenic survival with the inhibition of β1-integrin, suggesting that NF-κB-mediated induction of β1-integrin is associated with an increased radiation resistance. Treatment of malignant human breast cell colonies, T4-2 with NF-κB activation inhibitor at Day 4 in 3D lrECM before exposure to IR (4-Gy X-ray) resulted in a reduction of the size of colonies. The surviving colonies were associated with a decrease in proliferation and increase in apoptosis, indicating a decrease of resistance to IR. Using an ELISA-based DNA binding assay, we next determined that the binding of NF-κB to its target sequence on β1-integrin promoter region was significantly increased by IR. These data suggest that IR-induced increase in β1-integrin protein expression is mediated by NF-κB activation and subsequent binding of NF-κB to the β1-integrin promoter. Taken together, our results provide the first evidence that NF-κB-mediated β1-integrin expression is responsible for tumor radioresistance. The NF-κB/β1-integrin pathway could provide new insight into the development of new therapeutic modalities to re-sensitize radioresistant tumor cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C130.

Breast Cancer Cells in Three-dimensional Culture Display an Enhanced Radioresponse after Coordinate Targeting of Integrin α5β1 and Fibronectin

Tactics to selectively enhance cancer radioresponse are of great interest. Cancer cells actively elaborate and remodel their extracellular matrix (ECM) to aid in survival and progression. Previous work has shown that beta1-integrin inhibitory antibodies can enhance the growth-inhibitory and apoptotic responses of human breast cancer cell lines to ionizing radiation, either when cells are cultured in three-dimensional laminin-rich ECM (3D lrECM) or grown as xenografts in mice. Here, we show that a specific alpha heterodimer of beta1-integrin preferentially mediates a prosurvival signal in human breast cancer cells that can be specifically targeted for therapy. 3D lrECM culture conditions were used to compare alpha-integrin heterodimer expression in malignant and nonmalignant cell lines. Under these conditions, we found that expression of alpha5beta1-integrin was upregulated in malignant cells compared with nonmalignant breast cells. Similarly, we found that normal and oncofetal splice variants of fibronectin, the primary ECM ligand of alpha5beta1-integrin, were also strikingly upregulated in malignant cell lines compared with nonmalignant acini. Cell treatment with a peptide that disrupts the interactions of alpha5beta1-integrin with fibronectin promoted apoptosis in malignant cells and further heightened the apoptotic effects of radiation. In support of these results, an analysis of gene expression array data from breast cancer patients revealed an association of high levels of alpha5-integrin expression with decreased survival. Our findings offer preclinical validation of fibronectin and alpha5beta1-integrin as targets for breast cancer therapy.

Suppression of HER2/HER3-Mediated Growth of Breast Cancer Cells with Combinations of GDC-0941 PI3K Inhibitor, Trastuzumab, and Pertuzumab

Oncogenic activation of the phosphatidylinositol 3-kinase (PI3K) signaling pathway is prevalent in breast cancer and has been associated with resistance to HER2 inhibitors in the clinic. We therefore investigated the combinatorial activity of GDC-0941, a novel class I PI3K inhibitor, with standard-of-care therapies for HER2-amplified breast cancer. Three-dimensional laminin-rich extracellular matrix cultures of human breast cancer cells were utilized to provide a physiologically relevant approach to analyze the efficacy and molecular mechanism of combination therapies ex vivo. Combination studies were done using GDC-0941 with trastuzumab (Herceptin), pertuzumab, lapatinib (Tykerb), and docetaxel, the principal therapeutic agents that are either approved or being evaluated for treatment of early HER2-positive breast cancer. Significant GDC-0941 activity (EC(50) <1 micromol/L) was observed for >70% of breast cancer cell lines that were examined in three-dimensional laminin-rich extracellular matrix culture. Differential responsiveness to GDC-0941 as a single agent was observed for luminal breast cancer cells upon stimulation with the HER3 ligand, heregulin. Combined treatment of GDC-0941, trastuzumab, and pertuzumab resulted in growth inhibition, altered acinar morphology, and suppression of AKT mitogen-activated protein kinase (MAPK) / extracellular signed-regulated kinase (ERK) kinase and MEK effector signaling pathways for HER2-amplified cells in both normal and heregulin-supplemented media. The GDC-0941 and lapatinib combination further showed that inhibition of HER2 activity was essential for maximum combinatorial efficacy. PI3K inhibition also rendered HER2-amplified BT-474M1 cells and tumor xenografts more sensitive to docetaxel. GDC-0941 is efficacious in preclinical models of breast cancer. The addition of GDC-0941 to HER2-directed treatment could augment clinical benefit in breast cancer patients.

Interaction of E-cadherin and PTEN Regulates Morphogenesis and Growth Arrest in Human Mammary Epithelial Cells

Phosphatase and tensin homologue deleted on chromosome 10 (PTEN) is a dual-function phosphatase with tumor suppressor function compromised in a wide spectrum of cancers. Because tissue polarity and architecture are crucial modulators of normal and malignant behavior, we postulated that PTEN may play a role in maintenance of tissue integrity. We used two nonmalignant human mammary epithelial cell lines that form polarized, growth-arrested structures (acini) when cultured in three-dimensional laminin-rich extracellular matrix gels (lrECM). As acini begin to form, PTEN accumulates both in the cytoplasm and at cell-cell contacts where it colocalizes with the E-cadherin/beta-catenin complex. Reduction of PTEN levels by shRNA in lrECM prevents formation of organized breast acini and disrupts growth arrest. Importantly, disruption of acinar polarity and cell-cell contact by E-cadherin function-blocking antibodies reduces endogenous PTEN protein levels and inhibits its accumulation at cell-cell contacts. Conversely, in Skbr-3 breast cancer cells lacking endogenous E-cadherin expression, exogenous introduction of E-cadherin gene causes induction of PTEN expression and its accumulation at sites of cell interactions. These studies provide evidence that E-cadherin regulates both the PTEN protein levels and its recruitment to cell-cell junctions in three-dimensional lrECM, indicating a dynamic reciprocity between architectural integrity and the levels and localization of PTEN. This interaction thus seems to be a critical integrator of proliferative and morphogenetic signaling in breast epithelial cells.

β1 Integrin Inhibition Dramatically Enhances Radiotherapy Efficacy in Human Breast Cancer Xenografts

Beta(1) integrin signaling has been shown to mediate cellular resistance to apoptosis after exposure to ionizing radiation (IR). Other signaling molecules that increase resistance include Akt, which promotes cell survival downstream of beta(1) integrin signaling. We previously showed that beta(1) integrin inhibitory antibodies (e.g., AIIB2) enhance apoptosis and decrease growth in human breast cancer cells in three-dimensional laminin-rich extracellular matrix (lrECM) cultures and in vivo. Here, we asked whether AIIB2 could synergize with IR to modify Akt-mediated IR resistance. We used three-dimensional lrECM cultures to test the optimal combination of AIIB2 with IR treatment of two breast cancer cell lines, MCF-7 and HMT3522-T4-2, as well as T4-2 myr-Akt breast cancer colonies or HMT3522-S-1, which form normal organotypic structures in three-dimensional lrECM. Colonies were assayed for apoptosis and beta(1) integrin/Akt signaling pathways were evaluated using Western blot. In addition, mice bearing MCF-7 xenografts were used to validate the findings in three-dimensional lrECM. We report that AIIB2 increased apoptosis optimally post-IR by down-regulating Akt in breast cancer colonies in three-dimensional lrECM. In vivo, addition of AIIB2 after IR significantly enhanced tumor growth inhibition and apoptosis compared with either treatment alone. Remarkably, the degree of tumor growth inhibition using AIIB2 plus 2 Gy radiation was similar to that of 8 Gy alone. We previously showed that AIIB2 had no discernible toxicity in mice; here, its addition allowed for a significant reduction in the IR dose that was necessary to achieve comparable growth inhibition and apoptosis in breast cancer xenografts in vivo.

Integrin-linked kinase: Dispensable for radiation survival of three-dimensionally cultured fibroblasts

Purpose Cancer treatment by conventional radiotherapy is limited by normal tissue side-effects. Fibroblasts as “non-target” stromal cell type are considered as strong promoter of tumor growth and for developing a therapy resistant phenotype. Regarding application of novel molecular therapeutics combined with radiotherapy, evaluation of a specific targeted molecule in both tumor and normal cells is mandatory for efficacy and tolerability assessment. Previous work showed integrin-linked kinase (ILK), a mediator of β-integrin signals and putative phosphorylator of AKT, as potent anti-survival regulator in human cancer cell lines. Materials and methods To evaluate the role of ILK in normal fibroblast survival, ILK-wild-type (ILK fl/fl), ILK −/− and ILK N-terminal and ILK C-terminal domain expressing fibroblasts were irradiated with X-rays on different substrata or in three-dimensional laminin-rich extracellular matrix (lrECM). Results On control substrata, ILK-deficient and ILK-mutant fibroblasts showed significant increase in radiation survival relative to ILK-wild-type cells. This effect was compensated by growth on ECM proteins and in 3D lrECM. ILK regulated AKT activity in a phosphatidylinositol-3 kinase (PI3K)-dependent manner. Upon PI3K inhibition, only ILK-wild-type fibroblasts showed significant radiosensitization. Conclusions These findings obtained in 3D cell cultures suggest ILK to be dispensable for the radiation survival response of normal fibroblasts. However, targeting the PI3K/AKT signaling axis pharmacologically might be critical for survival of normal fibroblasts exposed to ionizing radiation.