Combining Dual Checkpoint Immunotherapy with Ablative Radiation to All Sites of Oligometastatic Non-Small Cell Lung Cancer: Toxicity and Efficacy Results of a Phase 1b Trial.

PURPOSE: Ablative local treatment of all radiographically detected metastatic sites in patients with oligometastatic non-small cell lung cancer (NSCLC) increases progression-free survival (PFS) and overall survival (OS). Prior studies demonstrated the safety of combining stereotactic body radiation therapy (SBRT) with single-agent immunotherapy. We investigated the safety of combining SBRT to all metastatic tumor sites with dual checkpoint, anticytotoxic T-lymphocyte-associated protein 4 (anti-CTLA-4), and anti-programmed cell death ligand 1 (anti-PD-L1) immunotherapy for patients with oligometastatic NSCLC. METHODS AND MATERIALS: We conducted a phase 1b clinical trial in patients with oligometastatic NSCLC with up to 6 sites of extracranial metastatic disease. All sites of disease were treated with SBRT to a dose of 30 to 50 Gy in 5 fractions. Dual checkpoint immunotherapy was started 7 days after completion of radiation using anti-CTLA-4 (tremelimumab) and anti-PD-L1 (durvalumab) immunotherapy for a total of 4 cycles followed by durvalumab alone until progression or toxicity. RESULTS: Of the 17 patients enrolled in this study, 15 patients received at least 1 dose of combination immunotherapy per protocol. The study was closed early (17 of planned 21 patients) due to slow accrual during the COVID-19 pandemic. Grade 3+ treatment-related adverse events were observed in 6 patients (40%), of which only one was possibly related to the addition of SBRT to immunotherapy. Median PFS was 42 months and median OS has not yet been reached. CONCLUSIONS: Delivering ablative SBRT to all sites of metastatic disease in combination with dual checkpoint immunotherapy did not result in excessive rates of toxicity compared with historical studies of dual checkpoint immunotherapy alone. Although the study was not powered for treatment efficacy results, durable PFS and OS results suggest potential therapeutic benefit compared with immunotherapy or radiation alone in this patient population.

A Multicenter Retrospective Chart Review of Clinical Outcomes Among Patients With KRAS G12C Mutant Non-Small Cell Lung Cancer.

BACKGROUND: On May 28, 2021, the United States Food and Drug Administration (FDA) granted accelerated approval to sotorasib for second-line or later treatment of patients with locally advanced or metastatic KRAS G12C mutant non-small cell lung cancer (NSCLC). This was the first FDA-approved targeted therapy for this patient population. Due to a paucity of real world data describing clinical outcomes in patients with locally advanced or metastatic KRAS G12C mutated NSCLC in the second-line or later, we sought to compile a large, academic medical center-based historical dataset to clarify clinical outcomes in this patient population. MATERIALS AND METHODS: The clinical outcomes of 396 patients with stage IV (n = 268, 68%) or recurrent, metastatic (n = 128, 32%) KRAS G12C mutant NSCLC were evaluated in this multicenter retrospective chart review conducted through the Academic Thoracic Oncology Medical Investigator's Consortium (ATOMIC). Patients treated at 13 sites in the United States and Canada and diagnosed between 2006 and 2020 (30% 2006-2015, 70% 2016-2020) were included. Primary outcomes included real-world PFS (rwPFS) and overall survival (OS) from time of stage IV or metastatic diagnosis, with particular interest in patients treated with second-line docetaxel-containing regimens, as well as clinical outcomes in the known presence or absence of STK11 or KEAP1 comutations. RESULTS: Among all patients with stage IV or recurrent, metastatic KRAS G12C mutant NSCLC (n = 201 with KRAS G12C confirmed prior to first line systemic therapy), the median first-line rwPFS was 9.3 months (95% CI, 7.3-11.8 months) and median OS was 16.8 months (95% CI, 12.7-22.3 months). In this historical dataset, first line systemic therapy among these 201 patients included platinum doublet alone (44%), PD-(L)1 inhibitor monotherapy (30%), platinum doublet chemotherapy plus PD-(L)1 inhibitor (18%), and other regimens (8%). Among patients with documented second-line systemic therapy (n = 123), the second-line median rwPFS was 8.3 months (95% CI, 6.1-11.9 months), with median rwPFS 4.6 months (95% CI, 1.4-NA) among 10 docetaxel-treated patients (9 received docetaxel and 1 received docetaxel plus ramucirumab). Within the total study population, 49 patients (12%) had a co-occurring STK11 mutation and 3 (1%) had a co-occurring KEAP1 mutation. Among the 49 patients with a co-occurring KRAS G12C and STK11 mutation, median rwPFS on first-line systemic therapy (n = 23) was 6.0 months (95% CI, 4.7-NA), and median OS was 14.0 months (95% CI, 10.8-35.3 months). CONCLUSION: In this large, multicenter retrospective chart review of patients with KRAS G12C mutant NSCLC we observed a relatively short median rwPFS of 4.6 months among 10 patients with KRAS G12C mutant NSCLC treated with docetaxel with or without ramucirumab in the second-line setting, which aligns with the recently reported CodeBreak 200 dataset.

A clinical-grade liquid biomarker detects neuroendocrine differentiation in prostate cancer.

BackgroundNeuroendocrine prostate cancer (NEPC) is an aggressive subtype, the presence of which changes the prognosis and management of metastatic prostate cancer.MethodsWe performed analytical validation of a Circulating Tumor Cell (CTC) multiplex RNA qPCR assay to identify the limit of quantification (LOQ) in cell lines, synthetic cDNA, and patient samples. We next profiled 116 longitudinal samples from a prospectively collected institutional cohort of 17 patients with metastatic prostate cancer (7 NEPC, 10 adenocarcinoma) as well as 265 samples from 139 patients enrolled in 3 adenocarcinoma phase II trials of androgen receptor signaling inhibitors (ARSIs). We assessed a NEPC liquid biomarker via the presence of neuroendocrine markers and the absence of androgen receptor (AR) target genes.ResultsUsing the analytical validation LOQ, liquid biomarker NEPC detection in the longitudinal cohort had a per-sample sensitivity of 51.35% and a specificity of 91.14%. However, when we incorporated the serial information from multiple liquid biopsies per patient, a unique aspect of this study, the per-patient predictions were 100% accurate, with a receiver-operating-curve (ROC) AUC of 1. In the adenocarcinoma ARSI trials, the presence of neuroendocrine markers, even while AR target gene expression was retained, was a strong negative prognostic factor.ConclusionOur analytically validated CTC biomarker can detect NEPC with high diagnostic accuracy when leveraging serial samples that are only feasible using liquid biopsies. Patients with expression of NE genes while retaining AR-target gene expression may indicate the transition to neuroendocrine differentiation, with clinical characteristics consistent with this phenotype.FundingNIH (DP2 OD030734, 1UH2CA260389, R01CA247479, and P30 CA014520), Department of Defense (PC190039 and PC200334), and Prostate Cancer Foundation (Movember Foundation - PCF Challenge Award).

Integrated analysis of the tumor microenvironment using a reconfigurable microfluidic cell culture platform.

The tumor microenvironment (TME) is a complex network of non-malignant cells and stroma that perform a wide array of vital roles in tumor growth, immune evasion, metastasis, and therapeutic resistance. These highly diverse roles have been shown to be critically important to the progression of cancers and have already shown potential as therapeutic targets. Therefore, there has been a tremendous push to elucidate the pathways that underlie these roles and to develop new TME-directed therapies for cancer treatment. Unfortunately, TME-focused research has been limited by a lack of translational in vitro culture platforms that can model this highly complex niche and can support the integrated analysis of cell biology and function. In the current study, we investigate whether an independently developed reconfigurable microfluidic platform, known as Stacks, can address the critical need for translational multi-cellular tumor models and integrated analytics in TME research. We present data on multi-cellular culture of primary human cells in Stacks as well as the orthogonal analysis of cellular polarization, differentiation, migration, and cytotoxicity in this reconfigurable system. These expanded capabilities of Stacks are highly relevant to the cancer research community with the potential to enhance clinical translation of pre-clinical TME studies and to yield novel biological insight into TME crosstalk, metastasis, and responses to novel drug combinations or immune therapies.

Longitudinal Molecular Profiling of Circulating Tumor Cells in Metastatic Renal Cell Carcinoma.

PURPOSE: Liquid biopsies in metastatic renal cell carcinoma (mRCC) provide a unique approach to understand the molecular basis of treatment response and resistance. This is particularly important in the context of immunotherapies, which target key immune-tumor interactions. Unlike metastatic tissue biopsies, serial real-time profiling of mRCC is feasible with our noninvasive circulating tumor cell (CTC) approach. METHODS: We collected 457 longitudinal liquid biopsies from 104 patients with mRCC enrolled in one of two studies, either a prospective cohort or a phase II multicenter adaptive immunotherapy trial. Using a novel CTC capture and automated microscopy platform, we profiled CTC enumeration and expression of HLA I and programmed cell death-ligand 1 (PD-L1). Given their diametric immunological roles, we focused on the HLA I to PD-L1 ratio (HP ratio). RESULTS: Patients with radiographic responses showed significantly lower CTC abundances throughout treatment. Furthermore, patients whose CTC enumeration trajectory was in the highest quartile (> 0.12 CTCs/mL annually) had shorter overall survival (median 17.0 months v 21.1 months, P < .001). Throughout treatment, the HP ratio decreased in patients receiving immunotherapy but not in patients receiving tyrosine kinase inhibitors. Patients with an HP ratio trajectory in the highest quartile (≥ 0.0012 annually) displayed significantly shorter overall survival (median 18.4 months v 21.2 months, P = .003). CONCLUSION: In the first large longitudinal CTC study in mRCC to date to our knowledge, we identified the prognostic importance of CTC enumeration and the change over time of both CTC enumeration and the HP ratio. These insights into changes in both tumor burden and the molecular profile of tumor cells in response to different treatments provide potential biomarkers to predict and monitor response to immunotherapy in mRCC.

Analytical validation and initial clinical testing of quantitative microscopic evaluation for PD-L1 and HLA I expression on circulating tumor cells from patients with non-small cell lung cancer.

INTRODUCTION: PD-L1 expression in non-small cell lung cancer (NSCLC) predicts response to immune checkpoint blockade, however is an imperfect biomarker given tumor heterogeneity, and the antigen presentation pathway requiring other components including HLA I expression. HLA I downregulation may contribute to resistance, warranting its evaluation in attempts to guide patient selection. In addition, earlier detection of acquired resistance could prompt earlier change in treatment and prolong patient survival. Analysis of circulating tumor cells (CTCs) captures heterogeneity across multiple sites of metastases, enables detection of changes in tumor burden that precede radiographic response, and can be obtained in serial fashion. METHODS: To quantify the expression of both PD-L1 and HLA I on CTCs, we developed exclusion-based sample preparation technology, achieving high-yield with gentle magnetic movement of antibody-labeled cells through virtual barriers of surface tension. To achieve clinical-grade quantification of rare cells, we employ high quality fluorescence microscopy image acquisition and automated image analysis together termed quantitative microscopy. RESULTS: In preparation for clinical laboratory implementation, we demonstrate high precision and accuracy of these methodologies using a diverse set of control materials. Preliminary testing of CTCs isolated from patients with NSCLC demonstrate heterogeneity in PD-L1 and HLA I expression and promising clinical value in predicting PFS in response to PD-L1 targeted therapies. CONCLUSIONS: By confirming high performance, we ensure compatibility for clinical laboratory implementation and future application to better predict and detect resistance to PD-L1 targeted therapy in patients with NSCLC.

Advancing Treatment of Bone Metastases through Novel Translational Approaches Targeting the Bone Microenvironment.

Bone metastases represent a lethal condition that frequently occurs in solid tumors such as prostate, breast, lung, and renal cell carcinomas, and increase the risk of skeletal-related events (SREs) including pain, pathologic fractures, and spinal cord compression. This unique metastatic niche consists of a multicellular complex that cancer cells co-opt to engender bone remodeling, immune suppression, and stromal-mediated therapeutic resistance. This review comprehensively discusses clinical challenges of bone metastases, novel preclinical models of the bone and bone marrow microenviroment, and crucial signaling pathways active in bone homeostasis and metastatic niche. These studies establish the context to summarize the current state of investigational agents targeting BM, and approaches to improve BM-targeting therapies. Finally, we discuss opportunities to advance research in bone and bone marrow microenvironments by increasing complexity of humanized preclinical models and fostering interdisciplinary collaborations to translational research in this challenging metastatic niche.

Live cell molecular analysis of primary prostate cancer organoids identifies persistent androgen receptor signaling.

Prostate Cancer (PC) is a disease with remarkable tumor heterogeneity that often manifests in significant intra-patient variability with regards to clinical outcomes and treatment response. Commonly available PC cell lines do not accurately reflect the complexity of this disease and there is critical need for development of new models to recapitulate the intricate hierarchy of tumor pathogenesis. In current study, we established ex vivo primary patient-derived cancer organoid (PDCO) cultures from prostatectomy specimens of patients with locally advanced PC. We then performed a comprehensive multi-parameter characterization of the cellular composition utilizing a novel approach for live-cell staining and direct imaging in the integrated microfluidic Stacks device. Using orthogonal flow cytometry analysis, we demonstrate that primary PDCOs maintain distinct subsets of epithelial cells throughout culture and that these cells conserve expression of androgen receptor (AR)-related elements. Furthermore, to confirm the tumor-origin of the PDCOs we have analyzed the expression of PC-associated epigenetic biomarkers including promoter methylation of the GSTP1, RASSF1 and APC and RARb genes by employing a novel microfluidic rare-event screening protocol. These results demonstrate that this ex vivo PDCO model recapitulates the complexity of the epithelial tumor microenvironment of multifocal PC using orthogonal analyses. Furthermore, we propose to leverage the Stacks microfluidic device as a high-throughput, translational platform to interrogate phenotypic and molecular endpoints with the capacity to incorporate a complex tumor microenvironment.

Immune profiling of the bone marrow microenvironment in patients with high-risk localized prostate cancer.

Bone marrow (BM) is a primary metastatic site in prostate cancer (PC) and bone invasion is considered incurable. T cell-mediated immune surveillance is essential in controlling both tumorigenesis and initiation of metastases. Beside tropism, dissemination of PC cells to the BM may be facilitated by defects in BM immune homeostasis predisposing this niche to colonization. To evaluate the BM immune microenvironment in locally advanced, non-metastatic PC, we performed flow cytometry analysis of myeloid and lymphoid subsets in BM aspirates and peripheral blood collected during prostatectomy. Healthy BM aspirates served to establish a reference range for comparison. We found alterations in BM immune composition of PC patients, including an increased CD4/CD8 ratio, enrichment of CD4+ T cells, increased CD56+CD3+ NKT and CD56+CD3- NK yields compared to healthy controls. The lymphoid phenotype remained comparable regarding T cell activation and chemokine receptor-based polarization patterns. Additionally, we found increased B7H3 expression in the myeloid monocyte/macrophage subset and decreased DC infiltration in BM of PC patients. These findings suggest that alterations in the immune milieu may limit immune surveillance that compromise the ability of the BM microenvironment to prevent tumor dissemination, and predispose development of bone metastases in a subset of patients with localized PC.

Identification of PD1-mediated regulation of antitumor antigen response in patients with NSCLC using the trans vivo DTH assay.

OBJECTIVES: Emerging evidence has shown a role for tumor antigen-specific regulation in cancer. Identifying individuals with pre-existing regulatory responses may be key to understand those who are more likely to respond to Programmed Death-1 (PD-1) or PD-1 Ligand 1 (PD-L1) checkpoint blockade. We hypothesized that a functional assay could identify the role of PD-1/PD-L1 interactions on tumor-specific immune cells in the peripheral blood in patients with advanced non-small-cell lung cancer (NSCLC). METHODS: We performed the trans vivo delayed-type hypersensitivity assay to identify the role of PD-1/PD-L1-mediated tumor-specific immune regulation in ten patients with advanced NSCLC. RESULTS: The majority of patients had PD-1-mediated anergic immune responses towards their tumor antigens. Eight out of nine of these patients did not respond to their own tumor antigens but responded in the presence of anti-PD-1 antibody ('PD-1 anergy' phenotype). A minority (3/9) also had 'active' PD-1-mediated immune suppressive regulatory responses. Our results suggest that PD-1-anergy is a common feature of NSCLC immune responses, whereas PD-1-mediated immune suppression is present only in a minority of patients. The latter was associated with poor clinical outcomes in our sample. CONCLUSIONS: Overall, our results indicate that bystander suppression or the 'anergy-only' phenomenon may be novel biomarkers in NSCLC and suggest prediction value based on these phenotypes.

Spectrum of genomic alterations in FGFR3: current appraisal of the potential role of FGFR3 in advanced urothelial carcinoma.

Molecular analysis has identified subsets of urothelial carcinoma (UC) expressing distinct genetic signatures. Genomic alterations in the oncogenic fibroblast growth factor receptor 3 (FGFR3) pathway are among the most well described in UC and have led to extensive and ongoing investigation of FGFR3-targeted therapies in this disease, although no new drugs have yet been approved. Given the unmet need for effective treatments in advanced and metastatic UC, a better understanding of the known molecular alterations of FGFR3 and of the previous and ongoing clinical investigations of this promising target in UC deserves attention. The objective of the present review is to describe the landscape of alterations and biology of FGFR3 in UC, comprehensively summarize the current state of UC clinical trials of FGFR3 inhibitors, and discuss future therapeutic applications. Using the Pubmed and Clinicaltrials.gov databases, articles describing the spectrum and biological activity of FGFR3 genomic alterations and trials of FGFR3 inhibitors in UC were identified. Search terms included 'FGFR3 genomic alterations' and 'urothelial cancer' or 'bladder cancer'. Genomic alterations, including translocations and activating mutations, are increasingly described in advanced and metastatic UC. The majority of clinical trials have been performed in unselected populations; however, recent studies have reported encouraging preliminary data. We argue that routine use of molecular genomic tumour analysis in UC may inform selection of patients for appropriate trials and we further investigate the potential of FGFR3 as a meaningful clinical target for this difficult disease.

T-oligo as an anticancer agent in colorectal cancer.

In the United States, there will be an estimated 96,830 new cases of colorectal cancer (CRC) and 50,310 deaths in 2014. CRC is often detected at late stages of the disease, at which point there is no effective chemotherapy. Thus, there is an urgent need for effective novel therapies that have minimal effects on normal cells. T-oligo, an oligonucleotide homologous to the 3'-telomere overhang, induces potent DNA damage responses in multiple malignant cell types, however, its efficacy in CRC has not been studied. This is the first investigation demonstrating T-oligo-induced anticancer effects in two CRC cell lines, HT-29 and LoVo, which are highly resistant to conventional chemotherapies. In this investigation, we show that T-oligo may mediate its DNA damage responses through the p53/p73 pathway, thereby inhibiting cellular proliferation and inducing apoptosis or senescence. Additionally, upregulation of downstream DNA damage response proteins, including E2F1, p53 or p73, was observed. In LoVo cells, T-oligo induced senescence, decreased clonogenicity, and increased expression of senescence associated proteins p21, p27, and p53. In addition, downregulation of POT1 and TRF2, two components of the shelterin protein complex which protects telomeric ends, was observed. Moreover, we studied the antiproliferative effects of T-oligo in combination with an EGFR tyrosine kinase inhibitor, Gefitinib, which resulted in an additive inhibitory effect on cellular proliferation. Collectively, these data provide evidence that T-oligo alone, or in combination with other molecularly targeted therapies, has potential as an anti-cancer agent in CRC.

RGS3 controls T lymphocyte migration in a model of Th2-mediated airway inflammation.

T cell migration toward sites of antigen exposure is mediated by G protein signaling and is a key function in the development of immune responses. Regulators of G protein signaling (RGS) proteins modulate G protein signaling; however, their role in the regulation of adaptive immune responses has not been thoroughly explored. Herein we demonstrated abundant expression of the Gi/Gq-specific RGS3 in activated T cells, and that diminished RGS3 expression in a T cell thymoma increased cytokine-induced migration. To examine the role of endogenous RGS3 in vivo, mice deficient in the RGS domain (RGS3(ΔRGS)) were generated and tested in an experimental model of asthma. Compared with littermate controls, the inflammation in the RGS3(ΔRGS) mice was characterized by increased T cell numbers and the striking development of perivascular lymphoid structures. Surprisingly, while innate inflammatory cells were also increased in the lungs of RGS3(ΔRGS) mice, eosinophil numbers and Th2 cytokine production were equivalent to control mice. In contrast, T cell numbers in the draining lymph nodes (dLN) were reduced in the RGS3(ΔRGS), demonstrating a redistribution of T cells from the dLN to the lungs via increased RGS3(ΔRGS) T cell migration. Together these novel findings show a nonredundant role for endogenous RGS3 in controlling T cell migration in vitro and in an in vivo model of inflammation.

Regulation of myofibroblast differentiation and bleomycin-induced pulmonary fibrosis by adrenomedullin.

Myofibroblast differentiation induced by transforming growth factor-ß (TGF-ß) is characterized by the expression of smooth muscle α-actin (SMA) and extracellular matrix proteins. We and others have previously shown that these changes are regulated by protein kinase A (PKA). Adrenomedullin (ADM) is a vasodilator peptide that activates cAMP/PKA signaling through the calcitonin-receptor-like receptor (CRLR) and receptor-activity-modifying proteins (RAMP). In this study, we found that recombinant ADM had little effect on cAMP/PKA in quiescent human pulmonary fibroblasts, whereas it induced a profound activation of cAMP/PKA signaling in differentiated (by TGF-ß) myofibroblasts. In contrast, the prostacyclin agonist iloprost was equally effective at activating PKA in both quiescent fibroblasts and differentiated myofibroblasts. TGF-ß stimulated a profound expression of CRLR with a time course that mirrored the increased PKA responses to ADM. The TGF-ß receptor kinase inhibitor SB431542 abolished expression of CRLR and attenuated the PKA responses of cells to ADM but not to iloprost. CRLR expression was also dramatically increased in lungs from bleomycin-treated mice. Functionally, ADM did not affect initial differentiation of quiescent fibroblasts in response to TGF-ß but significantly attenuated the expression of SMA, collagen-1, and fibronectin in pre-differentiated myofibroblasts, which was accompanied by decreased contractility of myofibroblasts. Finally, sensitization of ADM signaling by transgenic overexpression of RAMP2 in myofibroblasts resulted in enhanced survival and reduced pulmonary fibrosis in the bleomycin model of the disease. In conclusion, differentiated pulmonary myofibroblasts gain responsiveness to ADM via increased CRLR expression, suggesting the possibility of using ADM for targeting pathological myofibroblasts without affecting normal fibroblasts.

RGS expression in cancer: oncomining the cancer microarray data.

Heterotrimeric G proteins mediate myriads of cell functions including control of cancer cell proliferation and migration. The family of the Regulators of G protein Signaling (RGS) proteins, in turn, controls the activity of G proteins through the acceleration of GTPase activity of the alpha subunits of G proteins. Increasing evidence suggest that the expression of certain RGS proteins is changed dramatically in various cancers, and in some instances, the control of cancer cell proliferation or migration by RGS proteins has been demonstrated. We assessed if common trends might exist in the expression of various RGS proteins in several types of cancer by examining microarray data using the Oncomine database. We focused on the largest R4 sub-family of RGS proteins, containing RGS1, RGS2, RGS3, RGS4, RGS5, RGS8, RGS13, RGS16 and RGS18. This analysis suggests that a number (up to 6) of RGS transcripts are exclusively downregulated in certain cancers, while being exclusively upregulated in other cancer types. Furthermore, significant changes in the expression of certain RGS proteins trended toward the same direction across various cancers. To illustrate, RGS1 is largely upregulated, whereas RGS2 is downregulated in the majority of solid tumors, whereas RGS5 transcripts are greatly increased in eight subtypes of lymphoma with no reports of downregulation in hematological malignancies. Together, these data suggest that (i) RGS proteins may have a combined and cell-specific role in a control of cancer cell function, and (ii) a given RGS protein may regulate the progression of various cancers through a common mechanism.

A finer tuning of G-protein signaling through regulated control of RGS proteins.

Regulators of G-protein signaling (RGS) proteins are GTPase-activating proteins (GAP) for various Gα subunits of heterotrimeric G proteins. Through this mechanism, RGS proteins regulate the magnitude and duration of G-protein-coupled receptor signaling and are often referred to as fine tuners of G-protein signaling. Increasing evidence suggests that RGS proteins themselves are regulated through multiple mechanisms, which may provide an even finer tuning of G-protein signaling and crosstalk between G-protein-coupled receptors and other signaling pathways. This review summarizes the current data on the control of RGS function through regulated expression, intracellular localization, and covalent modification of RGS proteins, as related to cell function and the pathogenesis of diseases.

Non-canonical functions of RGS proteins.

Regulators of G protein signalling (RGS) proteins are united into a family by the presence of the RGS domain which serves as a GTPase-activating protein (GAP) for various Galpha subunits of heterotrimeric G proteins. Through this mechanism, RGS proteins regulate signalling of numerous G protein-coupled receptors. In addition to the RGS domains, RGS proteins contain diverse regions of various lengths that regulate intracellular localization, GAP activity or receptor selectivity of RGS proteins, often through interaction with other partners. However, it is becoming increasingly appreciated that through these non-RGS regions, RGS proteins can serve non-canonical functions distinct from inactivation of Galpha subunits. This review summarizes the data implicating RGS proteins in the (i) regulation of G protein signalling by non-canonical mechanisms, (ii) regulation of non-G protein signalling, (iii) signal transduction from receptors not coupled to G proteins, (iv) activation of mitogen-activated protein kinases, and (v) non-canonical functions in the nucleus.

Myocardin-dependent activation of the CArG box-rich smooth muscle gamma-actin gene: preferential utilization of a single CArG element through functional association with the NKX3.1 homeodomain protein.

Serum response factor (SRF) is a ubiquitously expressed transcription factor that binds a 10-bp element known as the CArG box, located in the proximal regulatory region of hundreds of target genes. SRF activates target genes in a cell- and context-dependent manner by assembling unique combinations of cofactors over CArG elements. One particularly strong SRF cofactor, myocardin (MYOCD), acts as a component of a molecular switch for smooth muscle cell (SMC) differentiation by activating cytoskeletal and contractile genes harboring SRF-binding CArG elements. Here we report that the human ACTG2 promoter, containing four conserved CArG elements, displays SMC-specific basal activity and is highly induced in the presence of MYOCD. Stable transfection of a non-SMC cell type with Myocd elicits elevations in endogenous Actg2 mRNA. Gel shift and luciferase assays reveal a strong bias for MYOCD-dependent transactivation through CArG2 of the human ACTG2 promoter. Substitution of CArG2 with other CArGs, including a consensus CArG element, fails to reconstitute full MYOCD-dependent ACTG2 promoter stimulation. Mutation of an adjacent binding site for NKX3.1 reduces MYOCD-dependent transactivation of the ACTG2 promoter. Co-immunoprecipitation, glutathione S-transferase pulldown, and luciferase assays show a physical and functional association between MYOCD and NKX3.1; no such functional relationship is evident with the related NKX2.5 transcription factor despite its interaction with MYOCD. These results demonstrate the ability of MYOCD to discriminate among several juxtaposed CArG elements, presumably through its novel partnership with NKX3.1, to optimally transactivate the human ACTG2 promoter.

Phosphorylation of myocardin by extracellular signal-regulated kinase.

The contractile phenotype of smooth muscle (SM) cells is controlled by serum response factor (SRF), which drives the expression of SM-specific genes including SM alpha-actin, SM22, and others. Myocardin is a cardiac and SM-restricted coactivator of SRF that is necessary for SM gene transcription. Growth factors inducing proliferation of SM cells inhibit SM gene transcription, in a manner dependent on the activation of extracellular signal-regulated kinases ERK1/2. In this study, we found that ERK1/2 phosphorylates mouse myocardin (isoform B) at four sites (Ser(812), Ser(859), Ser(866), and Thr(893)), all of which are located within the transactivation domain of myocardin. The single mutation of each site either to alanine or to aspartate has no effect on the ability of myocardin to activate SRF. However, the phosphomimetic mutation of all four sites to aspartate (4xD) significantly impairs activation of SRF by myocardin, whereas the phosphodeficient mutation of all four sites to alanine (4xA) has no effect. This translates to a reduced ability of the 4xD (but not of 4xA) mutant of myocardin to stimulate expression of SM alpha-actin and SM22, as assessed by corresponding promoter, mRNA, or protein assays. Furthermore, we found that phosphorylation of myocardin at these sites impairs its interaction with acetyltransferase, cAMP response element-binding protein-binding protein, which is known to promote the transcriptional activity of myocardin. In conclusion, we describe a novel mode of modulation of SM gene transcription by ERK1/2 through a direct phosphorylation of myocardin.

Phosphorylation of beta-catenin by PKA promotes ATP-induced proliferation of vascular smooth muscle cells.

Extracellular ATP stimulates proliferation of vascular smooth muscle cells (VSMC) through activation of G protein-coupled P2Y purinergic receptors. We have previously shown that ATP stimulates a transient activation of protein kinase A (PKA), which, together with the established mitogenic signaling of purinergic receptors, promotes proliferation of VSMC (Hogarth DK, Sandbo N, Taurin S, Kolenko V, Miano JM, Dulin NO. Am J Physiol Cell Physiol 287: C449-C456, 2004). We also have shown that PKA can phosphorylate beta-catenin at two novel sites (Ser552 and Ser675) in vitro and in overexpression cell models (Taurin S, Sandbo N, Qin Y, Browning D, Dulin NO. J Biol Chem 281: 9971-9976, 2006). beta-Catenin promotes cell proliferation by activation of a family of T-cell factor (TCF) transcription factors, which drive the transcription of genes implicated in cell cycle progression including cyclin D1. In the present study, using the phosphospecific antibodies against phospho-Ser552 or phospho-Ser675 sites of beta-catenin, we show that ATP can stimulate PKA-dependent phosphorylation of endogenous beta-catenin at both of these sites without affecting its expression levels in VSMC. This translates to a PKA-dependent stimulation of TCF transcriptional activity through an increased association of phosphorylated (by PKA) beta-catenin with TCF-4. Using the PKA inhibitor PKI or dominant negative TCF-4 mutant, we show that ATP-induced cyclin D1 promoter activation, cyclin D1 protein expression, and proliferation of VSMC are all dependent on PKA and TCF activities. In conclusion, we show a novel mode of regulation of endogenous beta-catenin through its phosphorylation by PKA, and we demonstrate the importance of this mechanism for ATP-induced proliferation of VSMC.