Zafira Castaño

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.

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.

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

αCP-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 αCP-4 and αCP-4a, an alternatively spliced variant of αCP-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 αCP-4 and αCP-4a in normal lung bronchiolar epithelium. In contrast, αCP-4 immunoreactivity was not found in 47% adenocarcinomas and 83% squamous cell carcinomas, whereas all of the tumors expressed αCP-4a. Besides, lack of αCP-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 αCP-4 and αCP-4a messages in NSCLC and small cell lung cancer cell lines. Our data demonstrate an abnormal expression of αCP-4 in lung cancer, possibly associated with an altered processing of the αCP-4 mRNA leading to a predominant expression of αCP-4a. This may be considered as an example of alternative splicing involved in tumor suppressor gene inactivation. Finally, induction of αCP-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.

Loss of RasGAP Tumor Suppressors Underlie 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.

IL-1β 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-1β (IL-1β)-expressing innate immune cells that infiltrate distant MIC microenvironments. At the metastatic site, IL-1β 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-1β 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.Castaño et al. show that primary breast tumours drive an IL-1β -mediated inflammatory response that inhibits cellular plasticity and metastatic colonization of metastasis-initiating cells.

Abstract PR13: Understanding the systemic interactions between primary tumors and disseminated tumor initiating cells

The causes for breast cancer recurrence in the form of metastatic disease and the reasons why less than 1% of disseminated tumor cells form metastases are unknown. A number of studies have demonstrated that the aggressive cancer cell population capable of driving metastasis feature properties of the epithelial-mesenchymal transition (EMT) and tumor initiation (TI). We previously reported novel mechanisms by which systemic and microenvironmental factors enrich tumors for EMT and TI genes and demonstrated that recurrence rates are not strictly due to tumor cell intrinsic properties (Castano et al., Cancer Discovery 2013). Using a preclinical model of breast cancer during the early phases of metastatic disease, when patients harbor disseminated tumor initiating cells (TICs) in the periphery at the time of their primary diagnosis (McAllister SS et al., Cell 2008), we recently made a surprising, and seemingly contrasting discovery. Specifically, we found that certain primary tumors can inhibit progression of disseminated TICs into overt tumors, maintaining them in the TI state and slowing their proliferation and differentiation to form tumor tissue mass. We established that the primary tumor induce mobilization of IL1beta-expressing monocytes that are recruited to sites where TICs reside. At those sites, signaling via the IL1 receptor maintains the EMT/TI state of the disseminated cells, thus promoting disease indolence. Importantly, surgical removal of the primary tumor enables TICs at the secondary sites to exit the EMT/TI state and to produce robustly growing tumors. Confirming our xenograft results, we established that stromal expression of IL1β in patient tumors is associated with poor outcome. Collectively, these data highlight the profound impact a primary tumor can exert on metastasizing cells - in this case, by altering the systemic environment to the detriment of secondary tumor growth. Moreover, our data highlight a central role for IL1β in modulating tumor cell plasticity and suggest it may provide a novel avenue for targeting recurrent disease. Citation Format: Zafira Castano, Christine L. Chaffer, Asaf Spiegel, Ayush Pant, Andrea L. Richardson, Ferenc Reinhardt, Timothy Marsh, Susanne Janssen, Ann M. Gifford, Robert A. Weinberg, Sandra S. McAllister. Understanding the systemic interactions between primary tumors and disseminated tumor initiating cells. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr PR13.

Abstract C94: Systemic and stromal support of triple-negative breast cancer progression is prevented by EGF and IGF-1 inhibitors

By the time metastatic breast cancer is detected, patients are no longer treated with curative intent. Women with triple-negative breast cancer (TNBC) are particularly at risk for early recurrence. Clinical evidence indicates that in women with metastatic TNBC, the primary tumor and disseminated cells co-existed for an indefinite period of time. We demonstrate that outgrowth of distant tumors that would otherwise remain indolent in cancer-free mice is accelerated in mice with TNBC. Systemic signals provided by TNBCs cause formation of a tumor-supportive microenvironment at distant tumor sites. This microenvironment is enriched for EGF and IGF-1, which convert indolent tumors to a malignant state, defined by expression of factors associated with pluripotency, proliferation, and epithelial-mesenchymal transition. Inhibition of both EGFR and IGF1R maintains these tumors in the indolent state. These results suggest that recurrence rates can be dictated by host systemic factors and offer novel therapeutic potential for TNBC patients. Citation Format: Zafira Castano, Tim Marsh, Ramya Tadipatri, Hanna S. Kuznetsov, Fatima Al-Shahrour, Mahanaz Paktinat, April Greene-Colozzi, Bjorn Nilsson, Andrea L. Richardson, Sandra S. McAllister. Systemic and stromal support of triple-negative breast cancer progression is prevented by EGF and IGF-1 inhibitors. [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 C94.

Abstract PR2: Identification of luminal breast cancers that establish a tumor supportive macroenvironment defined by proangiogenic platelets and bone marrow derived cells

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 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 (BMCs) enrich responding tumor cell expression of CD24, an adhesion molecule for platelets, and provide a source of VEGFR2+ 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. This abstract is also presented as Poster B6. Citation Format: Timothy Marsh, Hanna Kuznetsov, Beth Markens, Zafira Castano, April Greene-Colozzi, Samantha Hay, Victoria Brown, Andrea Richardson, Sabina Signoretti, Elisabeth Battinelli, Sandra McAllister. Identification of luminal breast cancers that establish a tumor supportive macroenvironment defined by proangiogenic platelets and bone marrow derived cells. [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 PR2.

Abstract 4861: Triple-negative breast cancers establish a systemic environment that drives malignant progression of otherwise indolent disseminated tumors via EGF and IGF

Breast cancer recurrence rates are variable, suggesting that tumor cells disseminate from primary sites at an early stage but remain indolent for extended periods of time before progressing to symptomatic disease. Little is known about mechanisms that cause otherwise dormant tumors to become overt cancers, making it difficult to predict which breast cancer patients are likely to relapse and therefore likely to benefit from preemptive therapy. To address these challenges, we use a human breast tumor xenograft model in which aggressively growing breast cancers (“instigators”) promote the growth of otherwise indolent breast cancers (“responders”) that have disseminated to distant sites. These pro-tumorigenic systemic effects are established by instigating tumors, in large part, through their mobilization of bone marrow cells (BMCs) that are subsequently recruited to the responding breast tumors, where they create a tumor-supportive microenvironment. We report here that the mechanisms of establishing tumor-supportive systemic environments depend upon the molecular-histopathological subtype of the instigating breast cancer. Instigating cell lines and primary tumor surgical specimens, derived from patients with either triple-negative breast cancer (TNBC) or luminal breast cancer (LBC), established different subtype-specific systemic environments that impinged in different ways upon the malignant features of responding tumor cells and their microenvironment. In particular, the TNBC-induced responding tumor microenvironment was characterized by elevated levels of epidermal growth factor (EGF) and insulin-like growth factor 1 (IGF1). EGF and IGF acted on responding tumor cells in a paracrine manner to induce expression of factors that play a role in tumor-initiation, embryonic stem cell maintenance, and epithelial-mesenchymal transition. Pharmacological administration of EGFR and IGF1R inhibitors prevented responding tumors from acquiring the TNBC-induced malignant features, thus maintaining them in an indolent state. Our results demonstrate that microenvironmental composition, as well as tumor cell phenotypic plasticity and molecular properties, are strongly influenced by the host systemic macroenvironment. Our work suggests that the crosstalk between disseminated tumor cells and their microenvironment can be exploited therapeutically to improve outcomes for patients with TNBC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4861. doi:1538-7445.AM2012-4861