Anti-tumor effects of RTX-240: an engineered red blood cell expressing 4-1BB ligand and interleukin-15.

Recombinant agonists that activate co-stimulatory and cytokine receptors have shown limited clinical anticancer utility, potentially due to narrow therapeutic windows, the need for coordinated activation of co-stimulatory and cytokine pathways and the failure of agonistic antibodies to recapitulate signaling by endogenous ligands. RTX-240 is a genetically engineered red blood cell expressing 4-1BBL and IL-15/IL-15Rα fusion (IL-15TP). RTX-240 is designed to potently and simultaneously stimulate the 4-1BB and IL-15 pathways, thereby activating and expanding T cells and NK cells, while potentially offering an improved safety profile through restricted biodistribution. We assessed the ability of RTX-240 to expand and activate T cells and NK cells and evaluated the in vivo efficacy, pharmacodynamics and tolerability using murine models. Treatment of PBMCs with RTX-240 induced T cell and NK cell activation and proliferation. In vivo studies using mRBC-240, a mouse surrogate for RTX-240, revealed biodistribution predominantly to the red pulp of the spleen, leading to CD8 + T cell and NK cell expansion. mRBC-240 was efficacious in a B16-F10 melanoma model and led to increased NK cell infiltration into the lungs. mRBC-240 significantly inhibited CT26 tumor growth, in association with an increase in tumor-infiltrating proliferating and cytotoxic CD8 + T cells. mRBC-240 was tolerated and showed no evidence of hepatic injury at the highest feasible dose, compared with a 4-1BB agonistic antibody. RTX-240 promotes T cell and NK cell activity in preclinical models and shows efficacy and an improved safety profile. Based on these data, RTX-240 is now being evaluated in a clinical trial.

Subclonal cooperation drives metastasis by modulating local and systemic immune microenvironments.

Most human tumours are heterogeneous, composed of cellular clones with different properties present at variable frequencies. Highly heterogeneous tumours have poor clinical outcomes, yet the underlying mechanism remains poorly understood. Here, we show that minor subclones of breast cancer cells expressing IL11 and FIGF (VEGFD) cooperate to promote metastatic progression and generate polyclonal metastases composed of driver and neutral subclones. Expression profiling of the epithelial and stromal compartments of monoclonal and polyclonal primary and metastatic lesions revealed that this cooperation is indirect, mediated through the local and systemic microenvironments. We identified neutrophils as a leukocyte population stimulated by the IL11-expressing minor subclone and showed that the depletion of neutrophils prevents metastatic outgrowth. Single-cell RNA-seq of CD45+ cell populations from primary tumours, blood and lungs demonstrated that IL11 acts on bone-marrow-derived mesenchymal stromal cells, which induce pro-tumorigenic and pro-metastatic neutrophils. Our results indicate key roles for non-cell-autonomous drivers and minor subclones in metastasis.

IL-1ß inflammatory response driven by primary breast cancer prevents metastasis-initiating cell colonization.

Lack of insight into mechanisms governing breast cancer metastasis has precluded the development of curative therapies. Metastasis-initiating cancer cells (MICs) are uniquely equipped to establish metastases, causing recurrence and therapeutic resistance. Using various metastasis models, we discovered that certain primary tumours elicit a systemic inflammatory response involving interleukin-1ß (IL-1ß)-expressing innate immune cells that infiltrate distant MIC microenvironments. At the metastatic site, IL-1ß maintains MICs in a ZEB1-positive differentiation state, preventing MICs from generating highly proliferative E-cadherin-positive progeny. Thus, when the inherent plasticity of MICs is impeded, overt metastases cannot be established. Ablation of the pro-inflammatory response or inhibition of the IL-1 receptor relieves the differentiation block and results in metastatic colonization. Among patients with lymph node-positive breast cancer, high primary tumour IL-1ß expression is associated with better overall survival and distant metastasis-free survival. Our data reveal complex interactions that occur between primary tumours and disseminated MICs that could be exploited to improve patient survival.

Loss of RasGAP Tumor Suppressors Underlies the Aggressive Nature of Luminal B Breast Cancers.

Luminal breast cancers are typically estrogen receptor-positive and generally have the best prognosis. However, a subset of luminal tumors, namely luminal B cancers, frequently metastasize and recur. Unfortunately, the causal events that drive their progression are unknown, and therefore it is difficult to identify individuals who are likely to relapse and should receive escalated treatment. Here, we identify a bifunctional RasGAP tumor suppressor whose expression is lost in almost 50% of luminal B tumors. Moreover, we show that two RasGAP genes are concomitantly suppressed in the most aggressive luminal malignancies. Importantly, these genes cooperatively regulate two major oncogenic pathways, RAS and NF-κB, through distinct domains, and when inactivated drive the metastasis of luminal tumors in vivo Finally, although the cooperative effects on RAS drive invasion, NF-κB activation triggers epithelial-to-mesenchymal transition and is required for metastasis. Collectively, these studies reveal important mechanistic insight into the pathogenesis of luminal B tumors and provide functionally relevant prognostic biomarkers that may guide treatment decisions. SIGNIFICANCE: The lack of insight into mechanisms that underlie the aggressive behavior of luminal B breast cancers impairs treatment decisions and therapeutic advances. Here, we show that two RasGAP tumor suppressors are concomitantly suppressed in aggressive luminal B tumors and demonstrate that they drive metastasis by activating RAS and NF-κB. Cancer Discov; 7(2); 202-17. ©2016 AACR.See related commentary by Sears and Gray, p. 131This article is highlighted in the In This Issue feature, p. 115.

Stromal EGF and igf-I together modulate plasticity of disseminated triple-negative breast tumors.

The causes for malignant progression of disseminated tumors and the reasons recurrence rates differ in women with different breast cancer subtypes are unknown. Here, we report novel mechanisms of tumor plasticity that are mandated by microenvironmental factors and show that recurrence rates are not strictly due to cell-intrinsic properties. Specifically, outgrowth of the same population of incipient tumors is accelerated in mice with triple-negative breast cancer (TNBC) relative to those with luminal breast cancer. Systemic signals provided by overt TNBCs cause the formation of a tumor-supportive microenvironment enriched for EGF and insulin-like growth factor-I (IGF-I) at distant indolent tumor sites. Bioavailability of EGF and IGF-I enhances the expression of transcription factors associated with pluripotency, proliferation, and epithelial-mesenchymal transition. Combinatorial therapy with EGF receptor and IGF-I receptor inhibitors prevents malignant progression. These results suggest that plasticity and recurrence rates can be dictated by host systemic factors and offer novel therapeutic potential for patients with TNBC.

Identification of luminal breast cancers that establish a tumor-supportive macroenvironment defined by proangiogenic platelets and bone marrow-derived cells.

UNLABELLED: Breast cancer recurrence rates vary following treatment, suggesting that tumor cells disseminate early from primary sites but remain indolent indefinitely before progressing to symptomatic disease. The reasons why some indolent disseminated tumors erupt into overt disease are unknown. We discovered a novel process by which certain luminal breast cancer (LBC) cells and patient tumor specimens (LBC "instigators") establish a systemic macroenvironment that supports outgrowth of otherwise-indolent disseminated tumors ("responders"). Instigating LBCs secrete cytokines that are absorbed by platelets, which are recruited to responding tumor sites where they aid vessel formation. Instigator-activated bone marrow cells enrich responding tumor cell expression of CD24, an adhesion molecule for platelets, and provide a source of VEGF receptor 2(+) tumor vessel cells. This cascade results in growth of responder adenocarcinomas and is abolished when platelet activation is inhibited by aspirin. These findings highlight the macroenvironment as an important component of disease progression that can be exploited therapeutically. SIGNIFICANCE: Currently, processes that mediate progression of otherwise indolent tumors are not well understood, making it difficult to accurately predict which cancer patients are likely to relapse. Our findings highlight the macroenvironment as an important component of disease progression that can be exploited to more accurately identify patients who would benefit from adjuvant therapy.

The bed and the bugs: interactions between the tumor microenvironment and cancer stem cells.

Tumors have been increasingly recognized as organs with a complexity that approaches, and may even exceed, that of healthy tissues. When viewed from this perspective, the biology of a tumor can be understood only by studying tumor cell heterogeneity and the microenvironment that is constructed during the course of tumorigenesis and malignant progression. Recent work has revealed the existence of cancer stem cells, the "bugs", with the capacity for self-renewal and tumor propagation. In addition, it is now recognized that the tumor microenvironment, the "bed", plays a critical role in supporting cancer stem cells and also may promote neoplasia and malignant progression. The interdependence of the cell-intrinsic features of cancer, including the cancer stem cell "bugs" and the tumor microenvironment "bed", is only beginning to be understood. In this review, we highlight the rapidly evolving concepts about the interactions between tumor stem cells and their microenvironment, the insights gained from studying their normal tissue counterparts, and the questions and controversies surrounding this area of research, with an emphasis on breast and lung cancer. Finally, we address evidence supporting the notion that eliminating the bed as well as the bugs should lead to more effective and personalized cancer treatments that improve patient outcome.

The tumor macroenvironment and systemic regulation of breast cancer progression.

Breast cancer is the most common malignancy among women worldwide and is the most common cause of death for women between 35 and 50 years of age. Women with breast cancer are at risk of developing metastases for their entire lifetime and, despite local and systemic therapies, approximately 30% of breast cancer patients will relapse (Jemal et al., 2010). Nearly all breast cancer related deaths are due to metastatic disease, even though metastasis is considered to be an inefficient process. In some cases, tumor cells disseminate from primary sites at an early stage, but remain indolent for protracted periods of time before becoming overt, life-threatening tumors. Little is known about the mechanisms that cause these indolent tumors to grow into malignant disease. Because of this gap in our understanding, we are unable to predict which breast cancer patients are likely to experience disease relapse or develop metastases years after treatment of their primary tumor. A better understanding of the mechanisms and signals involved in the exit of tumor cells from dormancy would not only allow for more accurate selection of patients that would benefit from systemic therapy, but could also lead to the development of more targeted therapies to inhibit the signals that promote disease progression. In this review, we address the systemic, or "macroenvironmental", contribution to tumor initiation and progression and what is known about how a pro-tumorigenic systemic environment is established.

The neuron-specific isoform of glycogen synthase kinase-3beta is required for axon growth.

Glycogen synthase kinase-3 (GSK-3) has become an important target for the treatment of mood disorders and neurodegenerative disease. It comprises three enzymes, GSK-3alpha, beta and the neuron-specific isoform, beta2. GSK-3 regulates axon growth by phosphorylating microtubule-associated proteins including Tau. A genetic polymorphism that leads to an increase in the ratio of GSK-3beta1 to GSK-3beta2 interacts with Tau haplotypes to modify disease risk in Parkinson's and Alzheimer's disease. We have examined the roles of each isoform of GSK-3 in neurons. Silencing of GSK-3beta2 inhibited retinoic acid-induced neurite outgrowth in SH-SY5Y neuroblastoma cells and axon growth in rat cortical neurons. Inhibition of neurite outgrowth was prevented by co-expression of GSK-3beta2 but not by co-expression of GSK-3alpha or GSK-3beta1. Ectopic expression GSK-3beta2 enhanced the effects of retinoic acid on neurite length and induced neurite formation in the absence of retinoic acid. GSK-3beta2 phosphorylated Tau at a subset of those sites phosphorylated by GSK-3beta1. In addition, Axin, which regulates responses to Wnt signals, associated more readily with GSK-3beta1 than with GSK-3beta2. Our results suggest that GSK-3 inhibitors that target the Axin-binding site in GSK-3 will preserve the beneficial effects of GSK-3beta2 on axon growth.

Expression of alpha CP-4 inhibits cell cycle progression and suppresses tumorigenicity of lung cancer cells.

The protein alpha CP-4 (also known as hnRNP E4) is an RNA binding protein encoded by a gene at 3p21, one of the most common altered regions in lung cancer. It has been proposed that alpha CP-4 may function as a lung tumor suppressor. Lack of alpha CP-4 expression is frequent in highly proliferative lung tumors and correlates with alpha CP-4 allele losses. The aim of this study was to evaluate the effect of alpha CP-4 on the tumorigenic capacity of lung cancer cells. alpha CP-4 expression was induced by transient transfection or stable infection with recombinant retroviruses. Induction of alpha CP-4 expression caused cell cycle arrest in G(2)/M in 3 out of the 7 lung cancer cell lines studied, while no effect on apoptosis was observed. Anchorage-independent growth and invasion capacity of H1299 cells were significantly reduced by alpha CP-4 induction. Tumorigenicity of H1299 cells in nude mice was greatly inhibited by the expression of alpha CP-4. Moreover, induction of alpha CP-4 expression in already established tumors resulted in a sudden growth arrest. Immunocytochemistry analysis of the xenograft tumors revealed an in vivo effect of alpha CP-4 on cell proliferation and no effect on apoptosis. Finally, alpha CP-4 showed a subcellular localization different from alpha CP-4a, a splice variant that does not affect cell proliferation. In conclusion, expression of alpha CP-4 can inhibit proliferation and tumorigenesis of lung cancer cells, both in vivo and in vitro, by delaying the progression of the cell cycle.

Expression of complement factor H by lung cancer cells: effects on the activation of the alternative pathway of complement.

The complement system is important in immunosurveillance against tumors. However, malignant cells are usually resistant to complement-mediated lysis. In this study, we examine the expression of factor H, an inhibitor of complement activation, and factor H-like protein 1 (FHL-1), its alternatively spliced form, in lung cancer. We also evaluate the potential effect of factor H/FHL-1 in the protection of lung cancer cells against the activation of the complement cascade. By Northern blot analysis we demonstrate a high expression of factor H and FHL-1 in most non-small cell lung cancer cell lines, although neuroendocrine pulmonary tumors (small cell lung carcinoma and carcinoid cell lines) had undetectable levels. Western blot analysis of conditioned medium showed the active secretion of factor H and FHL-1 by cells that were positive by Northern blot. Expression of factor H/FHL-1 mRNA was also shown in a series of non-small cell lung cancer biopsies by in situ hybridization. Interestingly, many cultured lung cancer cells were able to bind fluorescence-labeled factor H to their surfaces. Deposition of C3 fragments from normal human serum on H1264, a lung adenocarcinoma cell line, was more efficient when factor H/FHL-1 activity was blocked by specific antibodies. Blocking factor H/FHL-1 activity also enhanced the release of anaphylatoxin C5a and moderately increased the susceptibility of these cells to complement-mediated cytotoxicity. In summary, we demonstrate the expression of factor H and FHL-1 by some lung cancer cells and analyze the contribution of these proteins to the protection against complement activation.

Alpha CP-4, encoded by a putative tumor suppressor gene at 3p21, but not its alternative splice variant alpha CP-4a, is underexpressed in lung cancer.

alpha CP-4 is an RNA-binding protein coded by PCBP4, a gene mapped to 3p21, a common deleted region in lung cancer. In this study we characterized the expression of alpha CP-4 and alpha CP-4a, an alternatively spliced variant of alpha CP-4, in lung cancer cell lines and non-small cell lung cancer (NSCLC) samples from early stage lung cancer patients. In NSCLC biopsies, an immunocytochemical analysis showed cytoplasmic expression of alpha CP-4 and alpha CP-4a in normal lung bronchiolar epithelium. In contrast, alpha CP-4 immunoreactivity was not found in 47% adenocarcinomas and 83% squamous cell carcinomas, whereas all of the tumors expressed alpha CP-4a. Besides, lack of alpha CP-4 expression was associated with high proliferation of the tumor (determined by Ki67 expression). By fluorescence in situ hybridization, >30% of NSCLC cell lines and tumors showed allelic losses at PCBP4, correlating with the absence of the protein. On the other hand, no mutations in the coding region of the gene were found in any of the 24 cell lines analyzed. By Northern blotting and real-time reverse transcription-PCR, we detected the expression of alpha CP-4 and alpha CP-4a messages in NSCLC and small cell lung cancer cell lines. Our data demonstrate an abnormal expression of alpha CP-4 in lung cancer, possibly associated with an altered processing of the alpha CP-4 mRNA leading to a predominant expression of alpha CP-4a. This may be considered as an example of alternative splicing involved in tumor suppressor gene inactivation. Finally, induction of alpha CP-4 expression reduced cell growth, in agreement with its proposed role as a tumor suppressor, and suggesting an association of this RNA-binding protein with lung carcinogenesis.