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Immune checkpoint inhibitors (ICIs) use antibodies that block cell surface immune checkpoint proteins with great efficacy in treating immunogenic or “immune hot” tumors such as melanoma, kidney, and lung adenocarcinoma. ICIs have limited response rates to other non-immunogenic cancers. The tumor microenvironment (TME) consists of many cell types that collectively promote tumor progression. Cancer therapeutics are commonly designed to target one molecule in one defined cell type. There is growing evidence that long-term therapeutic responses require the targeting of cancer cells and tumor-promoting populations within the TME. The question remains whether we can identify targetable molecules/pathways that are critical for multiple cell types. Here, we will discuss several molecular targets that may fit a “two or multiple birds, one stone” model, including the B-cell lymphoma-2 (BCL-2) family pro-survival factors, transcriptional factors including signal transducer and activator of transcription 3 (STAT3), the nuclear receptor 4A family (NR4A1, NR4A2, NR4A3), as well as epigenetic regulators such as bromodomain and extra-terminal (BET) family proteins, histone deacetylase (HDAC) family, SET domain bifurcated histone lysine methyltransferase 1 (SETDB1) and lysine-specific demethylase 1 (LSD1/KDM1A). We will focus on the rationale of these targets in immune modulation, as well as the strategies for targeting these important proteins for cancer therapy.

Supplementary Table S1. Index of genes upregulated or downregulated in basal-TIC tumors relative to luminal-TIC tumors. Supplementary Table S2. WNT5A CNV by PAM50 molecular subtype from samples with both RNAseq expression data and CNV (n=807). Figure S1: Verification of Illumina Array by reverse transcription real-time PCR analysis (Supplementary for Figure 1). Figure S2: Allelic loss of WNT5A led to decreased WNT5A expression in human breast-cancer specimens and mouse luminal epithelial cells. Figure S3: Schematic depiction of the purification procedure for different populations of mammary epithelial cells. Figure S4: Supplementary information of Figure 4. Figure S5: Heterozygous deletion of Wnt5a has no impact on normal mammary gland development. Figure S6: WNT5A induces the phosphorylation of SMAD2. Figure S7: Differential expression of canonical and noncanonical Wnt receptors in basal and luminal epithelial cells. Figure S8: RYK or SMAD2 expression does not correlate with metastasis-free survival. Figure S9: Schematic depiction of the major conclusions and explained in detail in the Discussion section.

It is not well understood how paracrine communication between basal and luminal cell populations in the mammary gland affects tumorigenesis. During ErbB2-induced mammary tumorigenesis, enriched mammary stem cells that represent a subpopulation of basal cells exhibit enhanced tumorigenic capacity compared with the corresponding luminal progenitors. Transcript profiling of tumors derived from basal and luminal tumor-initiating cells (TIC) revealed preferential loss of the noncanonical Wnt ligand WNT5A in basal TIC-derived tumors. Heterozygous loss of WNT5A was correlated with shorter survival of breast cancer patients. In a mouse model of ErbB2-induced breast cancer, Wnt5a heterozygosity promoted tumor multiplicity and pulmonary metastasis. As a TGFβ substrate, luminal cell-produced WNT5A induced a feed-forward loop to activate SMAD2 in a RYK and TGFβR1-dependent manner to limit the expansion of basal TIC in a paracrine fashion, a potential explanation for the suppressive effect of WNT5A in mammary tumorigenesis. Our results identify the WNT5A/RYK module as a spatial regulator of the TGFβ–SMAD signaling pathway in the context of mammary gland development and carcinogenesis, offering a new perspective on tumor suppression provided by basal–luminal cross-talk in normal mammary tissue. Cancer Res; 75(10); 1972–82. ©2015 AACR.

Regulatory T (Treg) cells are one of the major immunosuppressive cell types in cancer and a potential target for immunotherapy, but targeting tumor-infiltrating (TI) Treg cells has been challenging. Here, using single-cell RNA sequencing of immune cells from renal clear cell carcinoma (ccRCC) patients, we identify two distinct transcriptional fates for TI Treg cells, Fate-1 and Fate-2. The Fate-1 signature is associated with a poorer prognosis in ccRCC and several other solid cancers. CD177, a cell surface protein normally expressed on neutrophil, is specifically expressed on Fate-1 TI Treg cells in several solid cancer types, but not on other TI or peripheral Treg cells. Mechanistically, blocking CD177 reduces the suppressive activity of Treg cells in vitro, while Treg-specific deletion of Cd177 leads to decreased tumor growth and reduced TI Treg frequency in mice. Our results thus uncover a functional CD177+ TI Treg population that may serve as a target for TI Treg-specific immunotherapy., Regulatory T (Treg) cells are important modulators of the tumor microenvironment. Here the authors perform transcriptome profiling of immune cells from patients with renal clear cell carcinoma to find a Treg signature that correlates with poorer prognosis, with CD177 being implicated as the main mediator for related alterations in Treg activity and tumor outcome.

Renal cell carcinoma (RCC) is the 7th most common type of cancer, with over 400,000 new cases each year and 170,000 deaths worldwide. RCCs are grouped into several histological subtypes, with clear cell renal cell carcinoma (ccRCC) being the most common and most aggressive type. The first line of therapy for ccRCC is tyrosine kinase inhibitors (TKIs) which target multiple kinases including the VEGF receptor. The treatment can be used alone or in combination with immune checkpoint inhibitors. However, most patients develop resistance to the TKIs within six months of treatment. Therefore, our aim is to develop novel single or combinatorial therapies and discover ways to overcome the resistance to TKIs. By analyzing TCGA and published datasets from GEO, we found that angiopoietin-like 4 (ANGPTL4) is significantly elevated in both the WT and TKI-resistant ccRCC tumors. ANGPTL4 has been shown to participate in angiogenesis in several other tumor models, and blocking ANGPTL4 with an antibody in vitro, reduces the angiogenic potential of 786-O and RCC4 human ccRCC cell lines, suggesting a pro-angiogenic effect of ANGPTL4 in ccRCC. Knockout of ANGPTL4 in Caki-1 xenograft tumor model significantly reduced tumor angiogenesis and increased vessel normalization, as evidenced by increased expression of VEGFR2 and adhesion molecules on endothelial cells. The increased normalization of blood vessels is an indication of the therapeutic potential of combing anti-ANGPTL4 therapy with immune checkpoint inhibitors. Furthermore, both knockout of ANGPTL4 in 786-O cells and treatment of 786-O tumor-bearing mice with an ANGPTL4 blocking antibody significantly reduced tumor growth. To illustrate the mechanism of ANGPTL4-induced angiogenesis, we performed live cell receptor capturing to find cANGPTL4 binding partners on the cell membrane of human endothelial cells. After the mass spectrometry analysis, several novel receptors were found, and the top candidate is endothelial cell surface chemotaxis receptor (ECSCR), which is highly expressed on endothelial and adipocytes. Previous studies have shown that ECSCR can potentiate angiogenic signaling, which is similar to our data showing that ANGPTL4 can synergistically increase FGF2-induced angiogenesis. Thus, ANGPTL4 may promote angiogenesis in ccRCC by potentiating other angiogenic signaling via interaction with ECSCR. To summarize, we demonstrated the pro-angiogenic potential of ANGPTL4 in ccRCC and its critical role in tumor development. Knockout or antibody-mediated inhibition of ANGPTL4 reduced tumor angiogenesis and growth and increased vessel normalization. This data supports the therapeutic potential of ANGPTL4 targeted treatment in ccRCC. Citation Format: Zeng Jin, Umasankar De, Lei Wang, Jeremy Kleberg, Weizhou Zhang, Ryan Kolb. Angiopoietin-like 4 as a potential therapeutic target for clear cell renal carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 571.

Aiming to identify immune molecules with a novel function in cancer pathogenesis, we found the cluster of differentiation 177 (CD177), a known neutrophil antigen, to be positively correlated with relapse-free (RFS), metastasis-free (MFS) or overall survival (OS) in breast cancer. Additionally, CD177 expression is correlated with good prognosis in several other solid cancers including prostate, cervical, and lung. Focusing on breast cancer, we found that CD177 is expressed in normal breast epithelial cells and is significantly reduced in invasive cancers. Loss of CD177 leads to hyperproliferative mammary epithelium and contributes to breast cancer pathogenesis. Mechanistically, we found that CD177-deficiency is associated with an increase in β-Catenin signaling. Here we identified CD177 as a novel regulator of mammary epithelial proliferation and breast cancer pathogenesis likely via the modulation of Wnt/β-Catenin signaling pathway, a key signaling pathway involved in multiple cancer types.

Obesity is a risk factor for breast cancer and also predicts poor clinical outcomes regardless of menopausal status. Contributing to the poor clinical outcomes is the suboptimal efficacy of standard therapies due to dose limiting toxicities and obesity-related complications, highlighting the need to develop novel therapeutic approaches for treating obese patients. We recently found that obesity leads to an increase in tumor-infiltrating macrophages with activated NLRC4 inflammasome and increased interleukin (IL)−1β production. IL-1β, in turn, leads to increased angiogenesis and cancer progression. Using Next Generation RNA sequencing, we identified an NLRC4/IL-1β-dependent upregulation of angiopoietin-like 4 (ANGPTL4), a known angiogenic factor in cancer, in tumors from obese mice. ANGPTL4-deficiency by genetic knockout or treatment with a neutralizing antibody led to a significant reduction in obesity-induced angiogenesis and tumor growth. At a mechanistic level, ANGPTL4 expression is induced by IL-1β from primary adipocytes in a manner dependent on NF-κB- and MAP kinase-activation, which is further enhanced by hypoxia. This report shows that adipocyte-derived ANGPTL4 drives disease progression under obese conditions and is a potential therapeutic target for treating obese breast cancer patients. Ministry of Education (MOE) Accepted version RK: NIH T32 AI007260; WZ: NIH R01 grants CA200673 and CA203834, Oberley Award (National Cancer Institute Award P30CA086862) from Holden Comprehensive Cancer Center at the University of Iowa; NB: NIH F30 CA206255; AK: Mark Stinski Developmental Grant from the Department of Microbiology, University of Iowa; BD: NIH R01HL130146; NST: grant from Ministry of Education, Singapore (MOE2014-T2-1-012); FSS: NIH R01AI118719.

Regulatory T cells (Tregs) play an important role in maintaining immune homeostasis and, within tumors, their upregulation is common and promotes an immunosuppressive microenvironment. Therapeutic strategies that can eliminate Tregs in the tumor (i.e., therapies that do not run the risk of affecting normal tissues), are urgently needed for the development of cancer immunotherapies. Here we report our discovery of B-cell lymphoma extra-large (BCL-XL) as a potential molecular target of tumor-infiltrating (TI) Tregs. We show that pharmacological degradation of BCL-XL using a newly developed platelet-sparing BCL-XL Proteolysis-targeting chimera (PROTAC) induces the apoptosis of TI-Tregs and the activation of TI-CD8+ T cells. Moreover, these activities result in an effective suppression of syngeneic tumor growth in immunocompetent, but not in immunodeficient or CD8+ T cell-depleted mice. Notably, treatment with BCL-XL PROTAC does not cause detectable damage within several normal tissues or thrombocytopenia. These findings identify BCL-XL as a target in the elimination of TI-Tregs as a component of cancer immunotherapies, and that the BCL-XL-specific PROTAC has the potential to be developed as a therapeutic for cancer immunotherapy., Targeting regulatory T cells (Treg) represents a therapeutic option to abrogate tumor-associated immune suppression. Here the authors show that pharmacological degradation of BCL-XL preferentially induces apoptosis of tumor-infiltrating Treg, promoting CD8 T cell activation and anti-tumor immune responses in preclinical cancer models.

Regulatory T cells (Tregs) are critical in maintaining immune homeostasis under various pathophysiological conditions. A growing body of evidence demonstrates that Tregs play an important role in cancer progression and that they do so by suppressing cancer-directed immune responses. Tregs have been targeted for destruction by exploiting antibodies against and small-molecule inhibitors of several molecules that are highly expressed in Tregs-including immune checkpoint molecules, chemokine receptors, and metabolites. To date, these strategies have had only limited antitumor efficacy, yet they have also created significant risk of autoimmunity because most of them do not differentiate Tregs in tumors from those in normal tissues. Currently, immune checkpoint inhibitor (ICI)-based cancer immunotherapies have revolutionized cancer treatment, but the resistance to ICI is common and the elevation of Tregs is one of the most important mechanisms. Therapeutic strategies that can selectively eliminate Tregs in the tumor (i.e. therapies that do not run the risk of causing autoimmunity by affecting normal tissue), are urgently needed for the development of cancer immunotherapies. This chapter discusses specific properties of human Tregs under the context of cancer and the various ways to target Treg for cancer immunotherapy.

Interleukin 1 (IL-1) has long been known for its pleiotropic effects on inflammation that plays a complex, and sometimes contrasting, role in different stages of cancer development. As a major proinflammatory cytokine, IL-1β is mainly expressed by innate immune cells. IL-1α, however, is expressed by various cell types under physiological and pathological conditions. IL-1R1 is the main receptor for both ligands and is expressed by various cell types, including innate and adaptive immune cell types, epithelial cells, endothelial cells, adipocytes, chondrocytes, fibroblasts, etc. IL-1 and IL-1R1 receptor interaction leads to a set of common signaling pathways, mainly the NF-kB and MAP kinase pathways, as a result of complex positive and negative regulations. The variety of cell types with IL-1R1 expression dictates the role of IL-1 signaling at different stages of cancer, which under certain circumstances leads to contrasting roles in tumor development. Recent availability of IL-1R1 conditional knockout mouse model has made it possible to dissect the role of IL-1/IL-1R1 signaling transduction in different cell types within the tumor microenvironment. This chapter will focus on the role of IL-1/IL-1R1 in different cell types within the tumor microenvironment and discuss the potential of targeting this pathway in cancer therapy.

Interleukin 1 (IL-1) has long been known for its pleiotropic effects on inflammation that plays a complex, and sometimes contrasting, role in different stages of cancer development. As a major proinflammatory cytokine, IL-1β is mainly expressed by innate immune cells. IL-1α, however, is expressed by various cell types under physiological and pathological conditions. IL-1R1 is the main receptor for both ligands and is expressed by various cell types, including innate and adaptive immune cell types, epithelial cells, endothelial cells, adipocytes, chondrocytes, fibroblasts, etc. IL-1 and IL-1R1 receptor interaction leads to a set of common signaling pathways, mainly the NF-kB and MAP kinase pathways, as a result of complex positive and negative regulations. The variety of cell types with IL-1R1 expression dictates the role of IL-1 signaling at different stages of cancer, which under certain circumstances leads to contrasting roles in tumor development. Recent availability of IL-1R1 conditional knockout mouse model has made it possible to dissect the role of IL-1/IL-1R1 signaling transduction in different cell types within the tumor microenvironment. This chapter will focus on the role of IL-1/IL-1R1 in different cell types within the tumor microenvironment and discuss the potential of targeting this pathway in cancer therapy.

E-cadherin is conventionally considered to be a good prognostic marker in cancer. The loss of E-cadherin is one of the key hallmarks of epithelial-to-mesenchymal transition, a biological process that promotes cancer cell invasiveness and metastasis. Recent evidence has cast doubt on the importance of epithelial-to-mesenchymal transition in metastasis. The availability of protein-level data in the Cancer Genome Atlas allows for the quantitative analysis of protein and prognosis. The prognostic values of E-cadherin and β-catenin were revisited across 19 cancer types, and high E-cadherin was found to correlate with good prognosis in most cancers. Conversely, higher E-cadherin and β-catenin correlated with shorter survival in invasive breast carcinoma. Stratifying breast cancers by histologic subtype revealed that the poor prognosis of E-cadherin and β-catenin proteins was characteristic of infiltrating ductal, but not lobular, carcinomas. To further corroborate the protein findings and examine cellular localization, immunohistochemistry was used for E-cadherin and β-catenin in 163 breast patient samples from the Iowa cohort. Most previous studies showing that reduced or absent E-cadherin and β-catenin was inversely associated with tumor stages in ductal carcinomas were confirmed. Taken together, these results lead us to question the prognostic values of E-cadherin and β-catenin in ductal carcinomas and indicate a complicated role of E-cadherin and β-catenin in breast cancer progression.

Among all breast cancer types, basal-like breast cancer (BLBC) represents an aggressive subtype that lacks targeted therapy. We and others have found that receptor tyrosine kinase-like orphan receptor 1 (ROR1) is overexpressed in BLBC and other types of cancer and that ROR1 is significantly correlated with patient prognosis. In addition, using primary patient-derived xenografts (PDXs) and ROR1-knockout BLBC cells, we found that ROR1+ cells form tumors in immunodeficient mice. We developed an anti-ROR1 immunotoxin and found that targeting ROR1 significantly kills ROR1+ cancer cells and slows down tumor growth in ROR1+ xenografts. Our bioinformatics analysis revealed that ROR1 expression is commonly associated with the activation of FGFR-mediated signaling pathway. Further biochemical analysis confirmed that ROR1 stabilized FGFR expression at the posttranslational level by preventing its degradation. CRISPR/Cas9-mediated ROR1 knockout significantly reduced cancer cell invasion at cellular levels by lowering FGFR protein and consequent inactivation of AKT. Our results identified a novel signaling regulation from ROR1 to FGFR and further confirm that ROR1 is a potential therapeutic target for ROR1+ BLBC cells.

Regulatory T cells (Tregs) are a population of T cells that exert a suppressive effect on a variety of immune cells and non-immune cells. The suppressive effects of Tregs are detrimental to anti-tumor immunity. Recent investigations into cancer-associated Tregs have identified common expression patterns for tumor-infiltration, however the functional heterogeneity in tumor-infiltrating (TI) Treg is largely unknown. We performed single-cell sequencing on immune cells derived from renal clear cell carcinoma (ccRCC) patients, isolating 160 peripheral-blood (PB) Tregs and 574 TI Tregs. We identified distinct transcriptional TI Treg cell fates, with a suppressive subset expressing CD177. We demonstrate CD177+ TI-Tregs have preferential suppressive effects in vivo and ex vivo. Gene signatures derived the CD177+ Treg subset had superior ability to predict survival in ccRCC and seven other cancer types. Further investigation into the development and regulation of TI-Treg heterogeneity will be vital to the application of tumor immunotherapies that possess minimal side effects.

Obesity is a growing public health problem and affects 35% US adults. Obesity increases the risk of many cancer types and is associated with poor outcomes. Clinical management of cancer patients has been essentially the same between normal weight and obese individuals. Understanding causal mechanisms by which obesity drives cancer initiation and progression is essential for the development of novel precision therapy for obese cancer patients. One caveat is that various mechanisms have been proposed for different cancer types for their progression under obesity. Since obesity is known to have global impact on inflammation, here we will summarize recent literature and discuss the potential of inflammation being the common causal mechanism to promote cancer promotion across cancer types.

Immune checkpoints are a diverse set of inhibitory signals to the immune system that play a functional role in adaptive immune response and self-tolerance. Dysregulation of these pathways is a vital mechanism in the avoidance of immune destruction by tumor cells. Immune checkpoint blockade (ICB) refers to targeted strategies to disrupt the tumor co-opted immune suppression to enhance anti-tumor immunity. Cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and programmed cell death 1 (PD-1) are two immune checkpoints that have the widest range of antibody-based therapies. These therapies have gone from promising approaches to Food and Drug Administration-approved first- and second-line agents for a number of immunogenic cancers. The burgeoning investigations of ICB efficacy in blood and solid cancers have underscored the importance of identifying the predictors of response and resistance to ICB. Identification of response correlates is made complicated by the observations of mixed reactions, or different responses in multiple lesions from the same patient, and delayed responses that can occur over a year after the induction therapy. Factors that can influence response and resistance in ICB can illuminate underlying molecular mechanisms of immune activation and suppression. These same response predictors can guide the identification of patients who would benefit from ICB, reduce off-target immune-relate adverse events, and facilitate the use of combinatorial therapies to increase efficacy. Here we review the underlying principles of immune checkpoint therapy and results of single-agent ICB clinical trials, and summarize the predictors of response and resistance.

E-cadherin is conventionally considered to be a good prognostic marker in cancer. The loss of E-cadherin is one of the key hallmarks of epithelial-to-mesenchymal transition, a biological process that promotes cancer cell invasiveness and metastasis. Recent evidence has cast doubt on the importance of epithelial-to-mesenchymal transition in metastasis. The availability of protein-level data in the Cancer Genome Atlas allows for the quantitative analysis of protein and prognosis. The prognostic values of E-cadherin and β-catenin were revisited across 19 cancer types, and high E-cadherin was found to correlate with good prognosis in most cancers. Conversely, higher E-cadherin and β-catenin correlated with shorter survival in invasive breast carcinoma. Stratifying breast cancers by histologic subtype revealed that the poor prognosis of E-cadherin and β-catenin proteins was characteristic of infiltrating ductal, but not lobular, carcinomas. To further corroborate the protein findings and examine cellular localization, immunohistochemistry was used for E-cadherin and β-catenin in 163 breast patient samples from the Iowa cohort. Most previous studies showing that reduced or absent E-cadherin and β-catenin was inversely associated with tumor stages in ductal carcinomas were confirmed. Taken together, these results lead us to question the prognostic values of E-cadherin and β-catenin in ductal carcinomas and indicate a complicated role of E-cadherin and β-catenin in breast cancer progression.

Breast cancer is the leading cause of cancer-related mortality for females worldwide [1]. Improving early screening strategies and understanding the events that lead to tumor initiation have led to demonstrable improvements in clinical outcome. Our previous work revealed a variance in the tumorigenic capacity between different mammary epithelial cell populations in an MMTV-ErbB2 mouse model. In order to greater understand how different mammary epithelial cells influence the tumorigenic capacity in ErbB2-induced breast cancer, we transplanted different cell populations from pre-neoplastic MMTV-ErbB2 female mice into recipient mice for tumorigenic study. We found that different mammary epithelial cells bear different tumorigenic potentials even when induced by the same ErbB2 proto-oncogene. To understand the difference in tumors formed from different epithelial cells, we performed gene expression profiling using these tumors (GSE64487). Several genes were further validated using real-time reverse transcription polymerase chain reaction (RT-PCR). Here we provide further details on the experimental methods and microarray analysis. This data provides a resource to further understanding how different mammary cell populations can initiate ErbB2-driven tumors and the role of these cell populations as putative tumor-initiating cells (TICs).

Chronic inflammatory responses have long been observed to be associated with various types of cancer and play decisive roles at different stages of cancer development. Inflammasomes, which are potent inducers of interleukin (IL)-1β and IL-18 during inflammation, are large protein complexes typically consisting of a Nod-like receptor (NLR), the adapter protein ASC, and Caspase-1. During malignant transformation or cancer therapy, the inflammasomes are postulated to become activated in response to danger signals arising from the tumors or from therapy-induced damage to the tumor or healthy tissue. The activation of inflammasomes plays diverse and sometimes contrasting roles in cancer promotion and therapy depending on the specific context. Here we summarize the role of different inflammasome complexes in cancer progression and therapy. Inflammasome components and pathways may provide novel targets to treat certain types of cancer; however, using such agents should be cautiously evaluated due to the complex roles that inflammasomes and pro-inflammatory cytokines play in immunity.

Obesity is associated with an increased risk of developing breast cancer and is also associated with worse clinical prognosis. The mechanistic link between obesity and breast cancer progression remains unclear, and there has been no development of specific treatments to improve the outcome of obese cancer patients. Here we show that obesity-associated NLRC4 inflammasome activation/ interleukin (IL)-1 signalling promotes breast cancer progression. The tumour microenvironment in the context of obesity induces an increase in tumour-infiltrating myeloid cells with an activated NLRC4 inflammasome that in turn activates IL-1β, which drives disease progression through adipocyte-mediated vascular endothelial growth factor A (VEGFA) expression and angiogenesis. Further studies show that treatment of mice with metformin inhibits obesity-associated tumour progression associated with a marked decrease in angiogenesis. This report provides a causal mechanism by which obesity promotes breast cancer progression and lays out a foundation to block NLRC4 inflammasome activation or IL-1β signalling transduction that may be useful for the treatment of obese cancer patients., Obesity is associated with higher breast cancer risk and poor prognosis. Here, the authors show that obesity promotes breast cancer through the recruitment of macrophages with activated NLRC4 inflammasome, which activate IL-1β production, resulting in VEGFA expression in adipocytes and angiogenesis.

Receptor Tyrosine Kinase-Like Orphan Receptor 1 (ROR1) is an oncofetal protein and has gained attention in cancer therapy since its initial discovery as a relatively specific surface antigen on B cell chronic lymphocytic leukemia (CLL) in 2008. The list of cancer types with ROR1 expression keeps growing, comprising, among others, malignant melanoma, breast cancer, and prostate cancer. It has been shown that ROR1 mediates several oncogenic pathways in a cancer type- and context-dependent manner. There are several ways to target ROR1 molecule, some of which have been in preclinical and clinical trials. We briefly summarize the oncogenic signaling pathways related to ROR1, as well as the update on ROR1-targeted therapies.

Diabetic nephropathy (DN) is the leading cause of end-stage kidney disease worldwide but current treatments remain suboptimal. The role of inflammation in DN has only recently been recognized. It has been shown that the NLRP3-inflammasome contributes to DN development by inducing interleukin (IL)-1β processing and secretion. In an effort to understand other IL-1β activating mechanism during DN development, we examined the role of the NLRC4-inflammasome in DN and found that NLRC4 is a parallel mechanism, in addition to the NLRP3-inflammasome, to induce pro-IL-1β processing and activation. We found that the expression of NLRC4 is elevated in DN kidneys. NLRC4-deficiency results in diminished DN disease progression, as manifested by a decrease in blood glucose and albumin excretion, as well as preserved renal histology. We further found that DN kidneys have increased F4/80+ macrophages, increased IL-1β production, and other signaling pathways related to kidney pathology such as activation of NF-κB and MAP kinase pathways, all of which were rescued by NLRC4-deficiency. This study demonstrates NLRC4-driven IL-1β production as critical for the progression of DN, which underscores the importance to target this pathway to alleviate this devastating disease.

G2/M checkpoint activation after DNA damage results in G2/M cell cycle arrest that allows time for DNA repair before the entry of cells into mitosis. Activation of G2/M checkpoint involves a series of signaling events, which include activation of ataxia telangiectecia-mutated and Rad3-related (ATR) and Chk1 kinases and inhibition of Cdc2/Cyclin B activity. Studies presented in this report show that serine (Ser)/threonine (Thr) protein phosphatase 2A (PP2A) has an important role in G2/M checkpoint activation in response to gamma-irradiation (IR) exposure. Using PP2A inhibitors, as well as siRNA targeting various forms of Ser/Thr protein phosphatases, results presented in this report show that specific PP2A inhibition abrogates IR-induced activation of ATR and Chk1 kinases, as well as phosphorylation of Cdc2-Tyr15, and attenuates IR-induced G2/M arrest. These results suggest an important regulation of PP2A on IR-induced G2/M checkpoint signaling response.

To better treat breast cancer patients, it is imperative to be able to predict breast cancer relapse and metastasis. Here, we identified the cluster of differentiation 177 (CD177) expression is positively correlated with relapse-free and metastasis-free survival of breast cancer patients. CD177 protein is highly expressed in normal breast epithelial cells and is significantly reduced in invasive cancers. Using mouse models, we found that CD177 suppresses breast cancer, primarily by forming a complex with E-Cadherin and b-Catenin at adherens junctions. The physical interaction prevents the activation of b-Catenin-mediated transcription by the canonical WNT signaling as demonstrated by a TOP-flash dual luciferase assay. In conclusion, we have identified CD177 as a novel breast cancer suppressor and uncovered a new regulatory mechanism for the canonical WNT/b-Catenin-mediated oncogenic signaling transduction. Citation Format: Paige N. Kluz, Ryan Kolb, Qing Xie, Nicholas Borcherding, Linna Wang, James P. De Andrade, Phillip M. Spanheimer, Wei Li, Katherine N. Gibson-Corley, Andy W. Tao, Sonia Sugg, Ronald J. Weigel, Weizhou Zhang. CD177 suppresses breast cancer progression by regulating the canonical WNT β-Catenin pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5529. doi:10.1158/1538-7445.AM2017-5529

Neutrophils play an essential role in the innate immune response to infection. Neutrophils migrate from the vasculature into the tissue in response to infection. Recently, a neutrophil cell surface receptor, CD177, was shown to help mediate neutrophil migration across the endothelium through interactions with PECAM1. We examined a publicly available gene array dataset of CD177 expression from human neutrophils following pulmonary endotoxin instillation. Among all 22,214 genes examined, CD177 mRNA was the most upregulated following endotoxin exposure. The high level of CD177 expression is also maintained in airspace neutrophils, suggesting a potential involvement of CD177 in neutrophil infiltration under infectious diseases. To determine the role of CD177 in neutrophils in vivo, we constructed a CD177-genetic knockout mouse model. The mice with homozygous deletion of CD177 have no discernible phenotype and no significant change in immune cells, other than decreased neutrophil counts in peripheral blood. We examined the role of CD177 in neutrophil accumulation using a skin infection model with Staphylococcus aureus. CD177 deletion reduced neutrophil counts in inflammatory skin caused by S. aureus. Mechanistically we found that CD177 deletion in mouse neutrophils has no significant impact in CXCL1/KC- or fMLP-induced migration, but led to significant cell death. Herein we established a novel genetic mouse model to study the role of CD177 and found that CD177 plays an important role in neutrophils. Electronic supplementary material The online version of this article (doi:10.1007/s13238-014-0109-1) contains supplementary material, which is available to authorized users.

Inflammation plays a critical role in tumorigenesis and can contribute to oncogenic mutations, tumor promotion, and angiogenesis. Tumor promoting inflammation is driven by many factors including the presence of the pro-inflammatory cytokines interleukin (IL)-1β and IL-18. One major source of IL-1β and IL-18 secretion is through the activation of inflammasomes. Inflammasomes are multiprotein complexes that upon activation lead to the processing and secretion of IL-1β and IL-18 mediated by the cysteine protease caspase-1. Several inflammasomes, including NLRP3, NLRC4, and NLRP6, have been implicated in tumorigenesis. However, inflammasomes play divergent roles in different types of cancer reflecting the complexity of inflammation during tumorigenesis. Understanding the role of inflammasome activation during specific stages of tumorigenesis and also during cancer immunotherapy will help identify novel therapeutic targets that could improve treatment strategies for cancer patients. Here we will discuss recent advances in understanding the mechanism by which NLRs regulate carcinogenesis.

Previous studies from our laboratory have shown that the activation of G2-M checkpoint after exposure of MCF-7 breast cancer cells to γ-irradiation (IR) is dependent on the activation of extracellular signal-regulated kinase 1/2 (ERK1/2) signaling. Studies presented in this report indicate that IR exposure of MCF-7 cells is associated with a marked increase in expression of breast cancer 1 (BRCA1) tumor suppressor, an effect that requires ERK1/2 activation and involves posttranscriptional control mechanisms. Furthermore, reciprocal coimmunoprecipitation, as well as colocalization studies, indicate an interaction between BRCA1 and ERK1/2 in both nonirradiated and irradiated cells. Studies using short hairpin RNA targeting BRCA1 show that BRCA1 expression is necessary for IR-induced G2-M cell cycle arrest, as well as ERK1/2 activation in MCF-7 cells. Although BRCA1 expression is not required for IR-induced phosphorylation of ataxia telangiectasia mutated (ATM)–Ser1981, it is required for ATM-mediated downstream signaling events, including IR-induced phosphorylation of Chk2-Thr68 and p53-Ser20. Moreover, BRCA1 expression is also required for IR-induced ATM and rad3 related activation and Chk1 phosphorylation in MCF-7 cells. These results implicate an important interaction between BRCA1 and ERK1/2 in the regulation of cellular response after IR-induced DNA damage in MCF-7 cells. [Cancer Res 2008;68(13):5113–21]

Stromal cell-derived factor 1 alpha (SDF-1alpha, CXCL12) and its receptor CXCR4 play an important role in the central nervous system (CNS) development and adulthood by mediating cell migration, enhancing precursor cell proliferation, assisting in neuronal circuit formation, and possibly regulating migration during repair. The expression pattern of CXCR4 and CXCL12 during neurogenesis has not been thoroughly elucidated. In this study, we investigated the expression of CXCL12 and CXCR4 during neural progenitor cells (NPC) differentiation by microarray analysis and reverse transcriptase-polymerase chain reaction (RT-PCR) using human fetal NPC as a model system. The production of CXCL12 was measured by enzyme-linked immunosorbent assay (ELISA). CXCR4 expression was determined by florescence-activated cell sorting (FACS) analysis, immunocytochemical staining, and CXCR4-mediated inhibition of cyclic AMP (cAMP) accumulation. Our data demonstrated that CXCR4 expression is significantly upregulated when NPC are differentiated into neuronal precursors, whereas CXCL12 is upregulated when differentiated into astrocytes. We also provide evidence that CXCR4 localization changes as neurons mature. In neuronal precursors, CXCR4 is localized in both neuronal processes and the cell body, whereas in mature neurons, it is primarily expressed on axons and dendrites. This differential expression of CXCR4 and CXCL12 may be important for the temporal regulation of neuronal migration and circuit formation during development and possibly in adult neurogenesis and repair.

Obesity is not only a known risk factor for the development of ER+ breast cancer in postmenopausal woman, but is also associated with a poor clinical outcome across all subtypes of breast cancer. Obesity is also one of the only known risk factors for the development of invasive ductal carcinoma (IDC) versus ductal carcinoma in-situ (DCIS; non-invasive). A number of hypotheses have been proposed to explain the association between obesity and breast cancer progression. One such hypothesis is that the chronic inflammation in adipose tissue associated with obesity, especially adipose tissue in the mammary glands, creates a microenvironment that promotes breast cancer progression by acting as a reservoir of pro-inflammatory cytokines (including interleukin IL-1β and IL-6). One source of pro-inflammatory cytokines in adipose tissue is activated inflammasomes in infiltrating macrophages. Inflammasomes are multiprotein complexes whose main function is the activation of IL-1β and IL-18 during inflammation via Caspase 1-mediated proteolytic cleavage. Chronic inflammation, NLRP3 inflammasome activation and IL-1β are thought to be a major cause of obesity-associated insulin resistance and are involved in many human diseases including type-2 diabetes and cancer. However, the mechanism linking obesity and breast cancer progression remains unknown. We hypothesize that obesity-induced inflammasome activation in mammary adipose tissue results in active IL-1β which in turn promotes breast cancer progression. Using a syngeneic orthotopic transplant model of breast cancer in C57Bl/6 mice (Py8119 cells and E0771 cells) we have shown that diet-induced obese (DIO) mice (mice fed a high fat diet) have increased tumor growth and metastasis compared to control mice (mice fed normal chow). Tumors from DIO mice have increased macrophage infiltration and caspase-1 activation compared to tumors from control mice. Additional studies have shown that treating DIO mice with an IL1R1 (the receptor for IL-1) blocking antibody reduces tumor growth to that observed in control mice. Furthermore, NLRC4 deficient DIO mice had reduced tumor growth (compared to wild-type DIO) mice, while there was no decrease in tumor growth in NLRP3 deficient DIO mice, indicating that NLRC4 inflammasome activation promotes breast cancer growth in obese mice. Finally, tumors from DIO mice had increased vascularization (as evidenced by an increase in CD31+ cells) compared to control mice, which was decreased in NLRC4 deficient mice or mice treated with the anti-IL1R1 antibody. This data indicates that obesity induced NLRC4 inflammasome activation in infiltrating macrophages in the mammary gland promotes breast cancer progression possibly through increased angiogenesis in the tumor microenvironment. Thus, targeting inflammasome activation or the IL-1/IL1R1 axis represents a rational target for the treatment of breast cancer in obese patients. Citation Format: Ryan Kolb, Nicholas Borcherding, Yinghong Liu, Fang Yuan, Qing Xie, Fayyaz Sutterwala, Weizhou Zhang. The NLRC4 inflammasome promotes breast cancer progression in diet-induced obese mice. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3173. doi:10.1158/1538-7445.AM2015-3173

Mammary glands contain luminal and basal epithelial cells. We recently found that both luminal and basal epithelial cells can initiate malignant mammary tumors with forced ErbB2/Her2/c-Neu expression (referred to as luminal and basal tumor-initiating cells, respectively). Basal tumor-initiating cell (TIC)-derived tumors had a greater incidence and larger volume than luminal TIC-derived tumors. Transcriptomic analysis was performed to evaluate the underlying transcriptional difference between ErbB2-driven luminal and basal TIC-derived tumors. CD177 was found to be downregulated within basal TIC-derived tumors. CD177 is an important membrane glycoprotein on neutrophils and serves as a marker for myeloproliferative diseases. The role of CD177 in solid tumors is unknown. Our initial analysis revealed that loss of CD177 expression is linked to higher metastasis incidence and shorter survival in breast cancer patients, but not other diagnostic markers like estrogen-, progesterone-, or HER2-receptors. Silencing CD177 in 4T1 cells, a mouse mammary tumor cell line, led to a significant increase for colonic growth in soft agar and pulmonary metastasis in mice. Overexpressing CD177 in MCF7 cells, a human luminal mammary tumor cell line, resulted in a significant decrease for colonic growth in soft agar. We also found that genetic deletion of CD177 in female mice results in significantly increased Lineage-CD24+CD49fhi basal epithelial cells, the major tumor-initiating cells for ErbB2-induced breast cancer. Collectively, we identified a novel potential metastasis-suppressor for human breast cancer with important prognostic value. Future studies will involve in the mechanistic understanding of how CD177 regulates metastasis of breast cancer. Citation Format: Qing Xie, Nicholas Borcherding, Ryan Kolb, Weizhou Zhang. Cd177, a novel metastasis suppressor of breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2470. doi:10.1158/1538-7445.AM2014-2470

Triple negative breast cancer (TNBC) accounts for 15-20% of total invasive breast cancer. These tumors are more aggressive and have a worse prognosis relative to other types of breast cancer, partially due to the lack of FDA-approved targeted therapy. Utilizing the RNA-seq data for invasive breast cancer specimens deposited in the Cancer Genome Atlas (TCGA), we found that the mRNA expression of receptor tyrosine kinase-like orphan receptor 1 (ROR1) decreased in the luminal A (n=231), luminal B (n=128) and Her2+ (n=58) subtypes relative to their corresponding normal controls. In contrast, ROR1 expression increased in all the pathologic stages of TNBC samples relative to normal specimens and ROR1 expression correlates positively with progression of TNBC (n=78). ROR1 is a receptor tyrosine kinase that is involved in embryonic patterning, but lacks expression in adult tissues. We undertook a series of mechanistic studies to understand the role of ROR1 in TNBC and evaluate ROR1 as a potential target for TNBC therapy. Using complex bioinformatics analysis, we found that ROR1 expression correlates with signature genes of TNBC, cancer stem cells, and TGFβ/SMAD pathways, which, in addition to recent observation of potential crosstalk between WNT5A/ROR1 signaling with TGFβ/SMAD, prompted us to examine if this crosstalk contributes to TNBC. Indeed, we found that WNT5a promotes SMAD2/3 activation in a ROR1- and TGFβR1-dependent manner. Inhibiting ROR1 by neutralizing antibody shifted cancer stem cells of TNBC to a more luminal-type, indicating a critical of ROR1 in self-renewal of cancer stem cells. SNAI1/2, master regulators of epithelial to mesenchymal transition (EMT), are potentially involved in the WNT5a/ROR1-mediated self-renewal of cancer stem cells. In agreement with these findings, TNBC cell lines with the highest surface expression of ROR1 had a greater cancer stem cell (CSC) population (CD24-CD44high). In collaboration with Speed Biosystems LLC, we developed an anti-ROR1 immunotoxin that specifically kills ROR1-high TNBC cells but not ROR1-low breast cancer cells or normal epithelial cells. Our findings suggest a role of WNT5a/ROR1 signaling in the progression of TNBC, through its role in potentiating TGFβ/SMAD/SNAI1/2 pathway thus promoting EMT and stem-like phenotype. ROR1 represents a novel therapeutic target for TNBC, especially for late-stage diseases. Citation Format: Nicholas Borcherding, Magdalene Ameka, Ryan Kolb, Qing Xie, Weizhou Zhang. WNT5a/ROR1 axis in triple-negative breast cancer progression and potential therapy. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2083. doi:10.1158/1538-7445.AM2014-2083

The purpose of this research is to determine the role of inflammasome-mediated production of interleukin(IL)-1β in obesity-associated breast cancer progression. Previous studies have shown that obesity is a risk factor for both developing breast cancer and breast cancer progression. Obesity is also associated with the development of invasive ductal carcinoma (IDC) versus ductal carcinoma in-situ (DCIS; non-invasive). A number of hypotheses have been proposed to explain the association between obesity and breast cancer progression. It has been hypothesized that the chronic inflammation in adipose tissue associated with obesity, especially adipose tissue in the mammary glands, creates a microenvironment that promotes breast cancer progression by acting as a reservoir of pro-inflammatory cytokines (including IL-1β and IL-6). One source of pro-inflammatory cytokines in adipose tissue is activated inflammasomes in infiltrating macrophages. Inflammasomes are multiprotein complexes whose main function is the activation of IL-1β and IL-18 during inflammation via Caspase 1 (Casp1)-mediated proteolytic cleavage. Chronic inflammation, Nlrp3 inflammasome activation and IL-1β are thought to be a major cause of obesity-associated insulin resistance and are involved in many human diseases including type-2 diabetes and cancer. However, the mechanism linking obesity and breast cancer progression remains unknown. We hypothesize that obesity-induced inflammasome activation in mammary adipose tissue results in active IL-1β which in turn promotes breast cancer progression. To determine the role of inflammasome activation in obesity-associated breast cancer progression, we will use inflammasome-deficient mice or neutralizing antibodies to inhibit IL-1 signaling in a syngeneic orthotopic transplant model of Py8119 cells in obese mice (mice fed a high fat diet). Our results show that Py8119 tumors have increased tumor growth and metastasis in obese mice compared to lean mice (mice fed normal chow). Furthermore, tumors from obese mice had increased expression of IL-1β and its receptor IL1 receptor 1 (IL1R1) as well as increased Casp1. Tumors from obese mice also had increase in M1 (which can secrete IL-1β) and M2 (which can be recruited in response to IL-1β signaling) tumor-infiltrating macrophages. Finally, inhibition of IL-1β signaling using an IL1R1 neutralizing antibody or Casp1 deficiency results in reduced tumor growth in obese mice. In conclusion, our results suggest that obesity-induced inflammasome activation promotes breast cancer progression in obese mice. Targeting inflammasome activation or neutralizing the IL-1/IL1R axis represents a rational therapy to treat obese patients with invasive breast cancer. Citation Format: Ryan Kolb, Yinghong Liu, Qing Xie, Nicholas Borcherding, Wei Li, Weizhou Zhang. Inflammasome activation in obesity-associated breast cancer progression. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4098. doi:10.1158/1538-7445.AM2014-4098

Gamma-irradiation (IR)-induced double strand DNA breaks frequently results in G2/M cell cycle arrest, allowing time for DNA repair prior to re-entering cell cycle. Previous studies from our laboratory have shown that the G2/M checkpoint activation following exposure of MCF-7 breast cancer cells to IR is dependent on the activation of extracellular signal-regulated protein kinase 1 and 2 (ERK1/2) signaling. Studies presented in this report indicate that IR exposure of several breast cancer cell lines (MCF-7, MDA-MB-231, SKBR3 and T47D) is associated with a marked increase in HER2/neu phosphorylation and that the inhibition of HER2/neu using specific inhibitors abrogates the IR-induced activation of both MEK1/2 and ERK1/2 in these cells. Furthermore, the HER2/neu inhibition also results in attenuation of IR-induced G2/M arrest and concomitant diminution of IR-induced activation of ATM, ATR, Chk1 and Chk2 kinases. In contrast, inhibition of HER1/EGFR using specific inhibitors has no detectable effect on IR-induced increase of ERK1/2 phosphorylation as well as the induction of G2/M arrest following IR exposure. Collectively, results in this report suggest that HER2/neu plays an essential role in the activation of IR-induced of MEK/ERK signaling and subsequent G2/M checkpoint response. 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 2041. doi:1538-7445.AM2012-2041