Targeting the chromatin binding of exportin-1 disrupts NFAT and T cell activation.

Exportin-1 (XPO1/CRM1) plays a central role in the nuclear-to-cytoplasmic transport of hundreds of proteins and contributes to other cellular processes, such as centrosome duplication. Small molecules targeting XPO1 induce cytotoxicity, and selinexor was approved by the Food and Drug Administration in 2019 as a cancer chemotherapy for relapsed multiple myeloma. Here, we describe a cell-type-dependent chromatin-binding function for XPO1 that is essential for the chromatin occupancy of NFAT transcription factors and thus the appropriate activation of T cells. Additionally, we establish a class of XPO1-targeting small molecules capable of disrupting the chromatin binding of XPO1 without perturbing nuclear export or inducing cytotoxicity. This work defines a broad transcription regulatory role for XPO1 that is essential for T cell activation as well as a new class of XPO1 modulators to enable therapeutic targeting of XPO1 beyond oncology including in T cell-driven autoimmune disorders.

Neutrophil extracellular traps induced by chemotherapy inhibit tumor growth in murine models of colorectal cancer.

Neutrophil extracellular traps (NETs), a web-like structure of cytosolic and granule proteins assembled on decondensed chromatin, kill pathogens and cause tissue damage in diseases. Whether NETs can kill cancer cells is unexplored. Here, we report that a combination of glutaminase inhibitor CB-839 and 5-FU inhibited the growth of PIK3CA-mutant colorectal cancers (CRCs) in xenograft, syngeneic, and genetically engineered mouse models in part through NETs. Disruption of NETs by either DNase I treatment or depletion of neutrophils in CRCs attenuated the efficacy of the drug combination. Moreover, NETs were present in tumor biopsies from patients treated with the drug combination in a phase II clinical trial. Increased NET levels in tumors were associated with longer progression-free survival. Mechanistically, the drug combination induced the expression of IL-8 preferentially in PIK3CA-mutant CRCs to attract neutrophils into the tumors. Further, the drug combination increased the levels of ROS in neutrophils, thereby inducing NETs. Cathepsin G (CTSG), a serine protease localized in NETs, entered CRC cells through the RAGE cell surface protein. The internalized CTSG cleaved 14-3-3 proteins, released BAX, and triggered apoptosis in CRC cells. Thus, our studies illuminate a previously unrecognized mechanism by which chemotherapy-induced NETs kill cancer cells.

Piezo1 facilitates optimal T cell activation during tumor challenge.

Functional effector T cells in the tumor microenvironment (TME) are critical for successful anti-tumor responses. T cell anti-tumor function is dependent on their ability to differentiate from a naïve state, infiltrate into the tumor site, and exert cytotoxic functions. The factors dictating whether a particular T cell can successfully undergo these processes during tumor challenge are not yet completely understood. Piezo1 is a mechanosensitive cation channel with high expression on both CD4+ and CD8+ T cells. Previous studies have demonstrated that Piezo1 optimizes T cell activation and restrains the CD4+ regulatory T cell (Treg) pool in vitro and under inflammatory conditions in vivo. However, little is known about the role Piezo1 plays on CD4+ and CD8+ T cells in cancer. We hypothesized that disruption of Piezo1 on T cells impairs anti-tumor immunity in vivo by hindering inflammatory T cell responses. We challenged mice with T cell Piezo1 deletion (P1KO) with tumor models dependent on T cells for immune rejection. P1KO mice had the more aggressive tumors, higher tumor growth rates and were unresponsive to immune-mediated therapeutic interventions. We observed a decreased CD4:CD8 ratio in both the secondary lymphoid organs and TME of P1KO mice that correlated inversely with tumor size. Poor CD4+ helper T cell responses underpinned the immunodeficient phenotype of P1KO mice. Wild type CD8+ T cells are sub-optimally activated in vivo with P1KO CD4+ T cells, taking on a CD25loPD-1hi phenotype. Together, our results suggest that Piezo1 optimizes T cell activation in the context of a tumor response.

Functional tumor cell-intrinsic STING, not host STING, drives local and systemic antitumor immunity and therapy efficacy following cryoablation.

BACKGROUND: Despite its potential utility in delivering direct tumor killing and in situ whole-cell tumor vaccination, tumor cryoablation produces highly variable and unpredictable clinical response, limiting its clinical utility. The mechanism(s) driving cryoablation-induced local antitumor immunity and the associated abscopal effect is not well understood. METHODS: The aim of this study was to identify and explore a mechanism of action by which cryoablation enhances the therapeutic efficacy in metastatic tumor models. We used the subcutaneous mouse model of the rhabdomyosarcoma (RMS) cell lines RMS 76-9STINGwt or RMS 76-9STING-/-, along with other murine tumor models, in C57BL/6 or STING-/- (TMEM173-/- ) mice to evaluate local tumor changes, lung metastasis, abscopal effect on distant tumors, and immune cell dynamics in the tumor microenvironment (TME). RESULTS: The results show that cryoablation efficacy is dependent on both adaptive immunity and the STING signaling pathway. Contrary to current literature dictating an essential role of host-derived STING activation as a driver of antitumor immunity in vivo, we show that local tumor control, lung metastasis, and the abscopal effect on distant tumor are all critically dependent on a functioning tumor cell-intrinsic STING signaling pathway, which induces inflammatory chemokine and cytokine responses in the cryoablated TME. This reliance extends beyond cryoablation to include intratumoral STING agonist therapy. Additionally, surveys of gene expression databases and tissue microarrays of clinical tumor samples revealed a wide spectrum of expressions among STING-related signaling components. CONCLUSIONS: Tumor cell-intrinsic STING pathway is a critical component underlying the effectiveness of cryoablation and suggests that expression of STING-related signaling components may serve as a potential therapy response biomarker. Our data also highlight an urgent need to further characterize tumor cell-intrinsic STING pathways and the associated downstream inflammatory response evoked by cryoablation and other STING-dependent therapy approaches.

Oral TGF-ßR1 inhibitor Vactosertib promotes osteosarcoma regression by targeting tumor proliferation and enhancing anti-tumor immunity.

Osteosarcoma (OS) is an aggressive malignant bone cancer, with refractory and metastatic disease remaining a significant challenge. Transforming growth factor-ß1 (TGF-ß) is a potent immune suppressive cytokine in OS and the TGF-ß is increased in the sera of OS patients and this increase is associated with high-grade OS and lung metastases. Therefore, blocking TGF-ß1 signaling may be a novel therapy for OS treatment. Here we show that blocking TGF-ß1 signaling using TGF-ßR1 inhibitor, Vactosertib, significantly inhibited OS proliferation in vitro and in vivo. Notably, Vactosertib inhibits c-Myc expression in the OS cells. Vactosertib increased immune effectors (IFNγ+CD8+ cells and NK cells) and inhibited immune suppressors (M2-like TAM, MDSC) in the OS tumor microenvironment. Our results suggest that inhibition of TGF-ß1 signaling is an effective therapeutic strategy against OS through a multi-pronged approach that targets tumor intrinsic and extrinsic factors to achieve optimal immune-effector functions and maximal clinical response.

Preconditioning hippocampal slices with hypothermia promotes rapid tolerance to hypoxic depolarization and swelling: Mediation by erythropoietin.

We suggested previously that hippocampal slices were protected from hypoxic depolarization and swelling by preincubating them at room temperature (Kreisman et al., 2000). We postulated that hypothermic preconditioning induced tolerance in our slices, which protected against hypoxic depolarization and swelling. Control hippocampal slices were incubated at 34-35 °C for two hours and the response to 10 min of severe hypoxia was compared to slices which were preconditioned for two hours at room temperature (22-23 °C) prior to warming to 34-35 °C. Recordings of the extracellular DC potential provided an index of tissue depolarization and changes in tissue light transmittance provided an index of swelling. Hypothermic preconditioning significantly reduced hypoxia-induced swelling, particularly in CA3 and the dentate inner blade. Since erythropoietin (EPO) had been shown to mediate hypoxic preconditioning, we tested whether EPO also mediated hypothermic preconditioning in our slices. Recombinant rat EPO (1-10 micromolar) mitigated hypoxia-induced swelling and depolarization in dentate inner blade of unconditioned slices in a dose-dependent manner. We also blocked the protective effects of hypothermic preconditioning on hypoxic depolarization and swelling in the inner blade of the dentate gyrus by administering soluble EPO receptor in the bath and treating slices with wortmannin to block phosphorylation of PI3 kinase, a critical step in the activation of the downstream neuroprotectant, Akt. These results suggest that EPO mediates tolerance to hypoxic depolarization and swelling induced by hypothermic preconditioning. They also emphasize that various preincubation protocols used in experiments with hippocampal slices may differentially affect basal electrophysiological and metabolic properties of those slices.

Treatment response and outcome of children with T-cell acute lymphoblastic leukemia expressing the gamma-delta T-cell receptor.

T-cell malignancies expressing the γδ T-cell receptor (TCR) are often associated with poor prognosis. Here, we determined the clinical outcome of pediatric patients with T-cell acute lymphoblastic leukemia (T-ALL) expressing the γδ TCR. Of 100 newly diagnosed T-ALL patients, 93 had γδ TCR analysis performed at diagnosis. Repertoire was evaluated by paired sequencing of the rearranged TCR. All patients received intensified chemotherapy and those with minimal residual disease (MRD) ≥ 1% on day 42-46 became candidates for hematopoietic cell transplantation. Of the 93 T-ALL patients, 12 (13%) had γδ T-ALL and 11 (12%) had early T-cell precursor (ETP) ALL. Compared to the remaining 70 T-ALL patients, the γδ T-ALL patients were more likely to have MRD ≥ 1% on day 15-19 (67% vs. 33%, P = 0.03) and day 42-49 (33% vs. 7%; P = 0.007) of remission induction. The 10-year overall survival for γδ T-ALL patients (66.7% ± 22.2%) were lower than that of T-ALL patients (93.3% ± 7.3%, P = 0.001). TCR analysis demonstrated a conserved clonotype. In conclusion, the data suggest that children with γδ T-ALL may have a poor response to remission induction, based on MRD levels and decreased survival than the other T-ALL patients, despite receiving risk-directed therapy.

Rapid cloning, expression, and functional characterization of paired αß and γδ T-cell receptor chains from single-cell analysis.

Transgenic expression of antigen-specific T-cell receptor (TCR) genes is a promising approach for immunotherapy against infectious diseases and cancers. A key to the efficient application of this approach is the rapid and specific isolation and cloning of TCRs. Current methods are often labor-intensive, nonspecific, and/or relatively slow. Here, we describe an efficient system for antigen-specific αßTCR cloning and CDR3 substitution. We demonstrate the capability of cloning influenza-specific TCRs within 10 days using single-cell polymerase chain reaction (PCR) and Gibson Assembly techniques. This process can be accelerated to 5 days by generating receptor libraries, requiring only the exchange of the antigen-specific CDR3 region into an existing backbone. We describe the construction of this library for human γδ TCRs and report the cloning and expression of a TRGV9/TRDV2 receptor that is activated by zoledronic acid. The functional activity of these αß and γδ TCRs can be characterized in a novel reporter cell line (Nur77-GFP Jurkat 76 TCRα(-)ß(-)) for screening of TCR specificity and avidity. In summary, we provide a rapid method for the cloning, expression, and functional characterization of human and mouse TCRs that can assist in the development of TCR-mediated therapeutics.

Enhanced cytotoxic function of natural killer and CD3+CD56+ cells in cord blood after culture.

Rate of immune reconstitution directly correlates with the number of hematopoietic stem cells infused and is particularly delayed in patients undergoing cord blood (CB) transplantation (CBT). Methods to increase the number of CB natural killer (NK) cells have the potential to improve immune reconstitution after CBT. NK cells are the first lymphocyte population to recover after hematopoietic stem cells transplantation and are central to preventing early relapse and infection. CB NK cells are low in number and are known to be incomplete in maturation and require activation for effective function. Here, we report a clinically relevant ex vivo expansion method that increases the number of activated CB NK cells. We report a multilog increase in NK cell number when CB mononuclear cells are cocultured with IL-2 and IL-15. Furthermore, NK cells expressing activating receptors and adhesion molecules responsible for cytotoxicity increased throughout culture, whereas inhibitory receptor expression remained low. Additionally, cytotoxic function against various malignancies was significantly enhanced in cultured NK cells but not CD3(+)CD56(+) cells. These data suggest that ex vivo expansion and activation of CB NK cells is a clinically feasible and relevant approach to prevent early infection and relapse after CBT.

Mesenchymal stem cells as a novel vaccine platform.

Vaccines are the most efficient and cost-effective means of preventing infectious disease. However, traditional vaccine approaches have thus far failed to provide protection against human immunodeficiency virus (HIV), tuberculosis, malaria, and many other diseases. New approaches to vaccine development are needed to address some of these intractable problems. In this report, we review the literature identifying stimulatory effects of mesenchymal stem cells (MSC) on immune responses and explore the potential for MSC as a novel, universal vaccination platform. MSC are unique bone marrow-derived multipotent progenitor cells that are presently being exploited as gene therapy vectors for a variety of conditions, including cancer and autoimmune diseases. Although MSC are predominantly known for anti-inflammatory properties during allogeneic MSC transplant, there is evidence that MSC can actually promote adaptive immunity under certain settings. MSC have also demonstrated some success in anti-cancer therapeutic vaccines and anti-microbial prophylactic vaccines, as we report, for the first time, the ability of modified MSC to express and secrete a viral antigen that stimulates antigen-specific antibody production in vivo. We hypothesize that the unique properties of modified MSC may enable MSC to serve as an unconventional but innovative, vaccine platform. Such a platform would be capable of expressing hundreds of proteins, thereby generating a broad array of epitopes with correct post-translational processing, mimicking natural infection. By stimulating immunity to a combination of epitopes, it may be possible to develop prophylactic and even therapeutic vaccines to tackle major health problems including those of non-microbial and microbial origin, including cancer, or an infectious disease like HIV, where traditional vaccination approaches have failed.

Toll-like receptor 3 and suppressor of cytokine signaling proteins regulate CXCR4 and CXCR7 expression in bone marrow-derived human multipotent stromal cells.

BACKGROUND: The use of bone marrow-derived human multipotent stromal cells (hMSC) in cell-based therapies has dramatically increased in recent years, as researchers have exploited the ability of these cells to migrate to sites of tissue injury, inflammation, and tumors. Our group established that hMSC respond to "danger" signals--by-products of damaged, infected or inflamed tissues--via activation of Toll-like receptors (TLRs). However, little is known regarding downstream signaling mediated by TLRs in hMSC. METHODOLOGY/PRINCIPAL FINDINGS: We demonstrate that TLR3 stimulation activates a Janus kinase (JAK) 2/signal transducer and activator of transcription (STAT) 1 pathway, and increases expression of suppressor of cytokine signaling (SOCS) 1 and SOCS3 in hMSC. Our studies suggest that each of these SOCS plays a distinct role in negatively regulating TLR3 and JAK/STAT signaling. TLR3-mediated interferon regulatory factor 1 (IRF1) expression was inhibited by SOCS3 overexpression in hMSC while SOCS1 overexpression reduced STAT1 activation. Furthermore, our study is the first to demonstrate that when TLR3 is activated in hMSC, expression of CXCR4 and CXCR7 is downregulated. SOCS3 overexpression inhibited internalization of both CXCR4 and CXCR7 following TLR3 stimulation. In contrast, SOCS1 overexpression only inhibited CXCR7 internalization. CONCLUSION/SIGNIFICANCE: These results demonstrate that SOCS1 and SOCS3 each play a functionally distinct role in modulating TLR3, JAK/STAT, and CXCR4/CXCR7 signaling in hMSC and shed further light on the way hMSC respond to danger signals.

A new mesenchymal stem cell (MSC) paradigm: polarization into a pro-inflammatory MSC1 or an Immunosuppressive MSC2 phenotype.

BACKGROUND: Our laboratory and others reported that the stimulation of specific Toll-like receptors (TLRs) affects the immune modulating responses of human multipotent mesenchymal stromal cells (hMSCs). Toll-like receptors recognize "danger" signals, and their activation leads to profound cellular and systemic responses that mobilize innate and adaptive host immune cells. The danger signals that trigger TLRs are released following most tissue pathologies. Since danger signals recruit immune cells to sites of injury, we reasoned that hMSCs might be recruited in a similar way. Indeed, we found that hMSCs express several TLRs (e.g., TLR3 and TLR4), and that their migration, invasion, and secretion of immune modulating factors is drastically affected by specific TLR-agonist engagement. In particular, we noted diverse consequences on the hMSCs following stimulation of TLR3 when compared to TLR4 by our low-level, short-term TLR-priming protocol. PRINCIPAL FINDINGS: Here we extend our studies on the effect on immune modulation by specific TLR-priming of hMSCs, and based on our findings, propose a new paradigm for hMSCs that takes its cue from the monocyte literature. Specifically, that hMSCs can be polarized by downstream TLR signaling into two homogenously acting phenotypes we classify here as MSC1 and MSC2. This concept came from our observations that TLR4-primed hMSCs, or MSC1, mostly elaborate pro-inflammatory mediators, while TLR3-primed hMSCs, or MSC2, express mostly immunosuppressive ones. Additionally, allogeneic co-cultures of TLR-primed MSCs with peripheral blood mononuclear cells (PBMCs) predictably lead to suppressed T-lymphocyte activation following MSC2 co-culture, and permissive T-lymphocyte activation in co-culture with MSC1. SIGNIFICANCE: Our study provides an explanation to some of the conflicting reports on the net effect of TLR stimulation and its downstream consequences on the immune modulating properties of stem cells. We further suggest that MSC polarization provides a convenient way to render these heterogeneous preparations of cells more uniform while introducing a new facet to study, as well as provides an important aspect to consider for the improvement of current stem cell-based therapies.

Leucine leucine-37 uses formyl peptide receptor-like 1 to activate signal transduction pathways, stimulate oncogenic gene expression, and enhance the invasiveness of ovarian cancer cells.

Emerging evidence suggests that the antimicrobial peptide, leucine leucine-37 (LL-37), could play a role in the progression of solid tumors. LL-37 is expressed as the COOH terminus of human cationic antimicrobial protein-18 (hCAP-18) in ovarian, breast, and lung cancers. Previous studies have shown that the addition of LL-37 to various cancer cell lines in vitro stimulates proliferation, migration, and invasion. Similarly, overexpression of hCAP-18/LL-37 in vivo accelerates tumor growth. However, the receptor or receptors through which these processes are mediated have not been thoroughly examined. In the present study, expression of formyl peptide receptor-like 1 (FPRL1) was confirmed on ovarian cancer cells. Proliferation assays indicated that LL-37 does not signal through a G protein-coupled receptor, such as FPRL1, to promote cancer cell growth. By contrast, FPRL1 was required for LL-37-induced invasion through Matrigel. The peptide stimulated mitogen-activated protein kinase and Janus-activated kinase/signal transducers and activators of transcription signaling cascades and led to the significant activation of several transcription factors, through both FPRL1-dependent and FPRL1-independent pathways. Likewise, expression of some LL-37-stimulated genes was attenuated by the inhibition of FPRL1. Increased expression of CXCL10, EGF, and PDGF-BB as well as other soluble factors was confirmed from conditioned medium of LL-37-treated cells. Taken together, these data suggest that LL-37 potentiates a more aggressive behavior from ovarian cancer cells through its interaction with FPRL1.

The pro-inflammatory peptide LL-37 promotes ovarian tumor progression through recruitment of multipotent mesenchymal stromal cells.

Bone marrow-derived mesenchymal stem cells or multipotent mesenchymal stromal cells (MSCs) have been shown to engraft into the stroma of several tumor types, where they contribute to tumor progression and metastasis. However, the chemotactic signals mediating MSC migration to tumors remain poorly understood. Previous studies have shown that LL-37 (leucine, leucine-37), the C-terminal peptide of human cationic antimicrobial protein 18, stimulates the migration of various cell types and is overexpressed in ovarian, breast, and lung cancers. Although there is evidence to support a pro-tumorigenic role for LL-37, the function of the peptide in tumors remains unclear. Here, we demonstrate that neutralization of LL-37 in vivo significantly reduces the engraftment of MSCs into ovarian tumor xenografts, resulting in inhibition of tumor growth as well as disruption of the fibrovascular network. Migration and invasion experiments conducted in vitro indicated that the LL-37-mediated migration of MSCs to tumors likely occurs through formyl peptide receptor like-1. To assess the response of MSCs to the LL-37-rich tumor microenvironment, conditioned medium from LL-37-treated MSCs was assessed and found to contain increased levels of several cytokines and pro-angiogenic factors compared with controls, including IL-1 receptor antagonist, IL-6, IL-10, CCL5, VEGF, and matrix metalloproteinase-2. Similarly, Matrigel mixed with LL-37, MSCs, or the combination of the two resulted in a significant number of vascular channels in nude mice. These data indicate that LL-37 facilitates ovarian tumor progression through recruitment of progenitor cell populations to serve as pro-angiogenic factor-expressing tumor stromal cells.

Toll-like receptors on human mesenchymal stem cells drive their migration and immunomodulating responses.

Adult human bone marrow-derived mesenchymal stem cells (hMSCs) are under study as therapeutic delivery agents that assist in the repair of damaged tissues. To achieve the desired clinical outcomes for this strategy requires a better understanding of the mechanisms that drive the recruitment, migration, and engraftment of hMSCs to the targeted tissues. It is known that hMSCs are recruited to sites of stress or inflammation to fulfill their repair function. It is recognized that toll-like receptors (TLRs) mediate stress responses of other bone marrow-derived cells. This study explored the role of TLRs in mediating stress responses of hMSCs. Accordingly, the presence of TLRs in hMSCs was initially established by reverse transcription-polymerase chain reaction assays. Flow cytometry and fluorescence immunocytochemical analyses confirmed these findings. The stimulation of hMSCs with TLR agonists led to the activation of downstream signaling pathways, including nuclear factor kappaB, AKT, and MAPK. Consequently, activation of these pathways triggered the induction and secretion of cytokines, chemokines, and related TLR gene products as established from cDNA array, immunoassay, and cytokine antibody array analyses. Interestingly, the unique patterns of affected genes, cytokines, and chemokines measured identify these receptors as critical players in the clinically established immunomodulation observed for hMSCs. Lastly, hMSC migration was promoted by TLR ligand exposure as demonstrated by transwell migration assays. Conversely, disruption of TLRs by neutralizing TLR antibodies compromised hMSC migration. This study defines a novel TLR-driven stress and immune modulating response for hMSCs that is critical to consider in the design of stem cell-based therapies.

Ovarian cancers overexpress the antimicrobial protein hCAP-18 and its derivative LL-37 increases ovarian cancer cell proliferation and invasion.

The role of the pro-inflammatory peptide, LL-37, and its pro-form, human cationic antimicrobial protein 18 (hCAP-18), in cancer development and progression is poorly understood. In damaged and inflamed tissue, LL-37 functions as a chemoattractant, mitogen and pro-angiogenic factor suggesting that the peptide may potentiate tumor progression. The aim of this study was to characterize the distribution of hCAP-18/LL-37 in normal and cancerous ovarian tissue and to examine the effects of LL-37 on ovarian cancer cells. Expression of hCAP-18/LL-37 was localized to immune and granulosa cells of normal ovarian tissue. By contrast, ovarian tumors displayed significantly higher levels of hCAP-18/LL-37 where expression was observed in tumor and stromal cells. Protein expression was statistically compared to the degree of immune cell infiltration and microvessel density in epithelial-derived ovarian tumors and a significant correlation was observed for both. It was demonstrated that ovarian tumor tissue lysates and ovarian cancer cell lines express hCAP-18/LL-37. Treatment of ovarian cancer cell lines with recombinant LL-37 stimulated proliferation, chemotaxis, invasion and matrix metalloproteinase expression. These data demonstrate for the first time that hCAP-18/LL-37 is significantly overexpressed in ovarian tumors and suggest LL-37 may contribute to ovarian tumorigenesis through direct stimulation of tumor cells, initiation of angiogenesis and recruitment of immune cells. These data provide further evidence of the existing relationship between pro-inflammatory molecules and ovarian cancer progression.

AKT regulation of estrogen receptor beta transcriptional activity in breast cancer.

Growth factor activation of the phosphatidylinositol 3-kinase (PI3K)-AKT pathway has been shown to activate the estrogen receptor (ER) alpha and to mediate tamoxifen resistance in breast cancer. Here, we investigated the regulation of the transcriptional activity of the newer ER beta by PI3K-AKT signaling. Tissue arrays of breast cancer specimens showed a positive association between the expressions of AKT and ER beta in the clinical setting. Reporter gene assays using pharmacologic and molecular inhibitors of AKT and constitutively active AKT revealed for the first time the ability of AKT to (a) potentiate ER beta activity and (b) target predominantly the activation function-2 (AF2) domain of the receptor, with a requirement for residue K269. Given the importance of coactivators in ER transcriptional activity, we further investigated the possible involvement of steroid receptor coactivator 1 (SRC1) and glucocorticoid receptor-interacting protein 1 (GRIP1) in AKT regulation of ER beta. Mammalian two-hybrid assays revealed that AKT enhanced both SRC1 and GRIP1 recruitment to the ER beta-AF2 domain, and reporter gene analyses revealed that AKT and GRIP1 cooperatively potentiated ER beta-mediated transcription to a level much greater than either factor alone. Investigations into AKT regulation of GRIP with mammalian one-hybrid assays showed that AKT potentiated the activation domains of GRIP1 itself, and in vitro kinase assays revealed that AKT directly phosphorylated GRIP1. The cross-talk between the PI3K-AKT and ER beta pathways, as revealed by the ability of AKT to regulate several components of ER beta-mediated transcription, may represent an important aspect that may influence breast cancer response to endocrine therapy.