Leah M. Cook

Primary Cilia Are Decreased in Breast Cancer: Analysis of a Collection of Human Breast Cancer Cell Lines and Tissues

Primary cilia (PC) are solitary, sensory organelles that are critical for several signaling pathways. PC were detected by immunofluorescence of cultured cells and breast tissues. After growth for 7 days in vitro, PC were detected in ∼70% of breast fibroblasts and in 7–19% of epithelial cells derived from benign breast (184A1 and MCF10A). In 11 breast cancer cell lines, PC were present at a low frequency in four (from 0.3% to 4% of cells), but were absent in the remainder. The cancer cell lines with PC were all of the basal B subtype, which is analogous to the clinical triple-negative breast cancer subtype. Furthermore, the frequency of PC decreased with increasing degree of transformation/progression in the MCF10 and MDA-MB-435/LCC6 isogenic models of cancer progression. In histologically normal breast tissues, PC were frequent in fibroblasts and myoepithelial cells and less common in luminal epithelial cells. Of 26 breast cancers examined, rare PC were identified in cancer epithelial cells of only one cancer, which was of the triple-negative subtype. These data indicate a decrease or loss of PC in breast cancer and an association of PC with the basal B subtype. This manuscript contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials.

Genistein and resveratrol, alone and in combination, suppress prostate cancer in SV-40 tag rats†

Chemoprevention utilizing dietary agents is an effective means to slow the development of prostate cancer. We evaluated the potential additive and synergistic effects of genistein and resveratrol for suppressing prostate cancer in the Simian Virus‐40 T‐antigen (SV‐40 Tag) targeted probasin promoter rat model, a transgenic model of spontaneously developing prostate cancer.

Metastasis suppressors and the tumor microenvironment.

The most lethal and debilitating attribute of cancer cells is their ability to metastasize. Throughout the process of metastasis, tumor cells interact with other tumor cells, host cells and a variety of molecules. Tumor cells are also faced with a number of insults, such as hemodynamic sheer pressure and immune selection. This brief review explores how metastasis suppressor proteins regulate interactions between tumor cells and the microenvironments in which tumor cells find themselves.

Integrating new discoveries into the "vicious cycle" paradigm of prostate to bone metastases

In prostate to bone metastases, the “vicious cycle” paradigm has been traditionally used to illustrate how metastases manipulate the bone forming osteoblasts and resorbing osteoclasts in order to yield factors that facilitate growth and establishment. However, recent advances have illustrated that the cycle is far more complex than this simple interpretation. In this review, we will discuss the role of exosomes and hematopoietic/mesenchymal stem/stromal cells (MSC) that facilitate the establishment and activation of prostate metastases and how cells including myeloid-derived suppressor cells, macrophages, T cells, and nerve cells contribute to the momentum of the vicious cycle. The increased complexity of the tumor–bone microenvironment requires a system level approach. The evolution of computational models to interrogate the tumor–bone microenvironment is also discussed, and the application of this integrated approach should allow for the development of effective therapies to treat and cure prostate to bone metastases.

Homotypic Gap Junctional Communication Associated with Metastasis Suppression Increases with PKA Activity and Is Unaffected by PI3K Inhibition

Loss of gap junctional intercellular communication (GJIC) between cancer cells is a common characteristic of malignant transformation. This communication is mediated by connexin proteins that make up the functional units of gap junctions. Connexins are highly regulated at the protein level and phosphorylation events play a key role in their trafficking and degradation. The metastasis suppressor breast cancer metastasis suppressor 1 (BRMS1) upregulates GJIC and decreases phosphoinositide-3-kinase (PI3K) signaling. On the basis of these observations, we set out to determine whether there was a link between PI3K and GJIC in tumorigenic and metastatic cell lines. Treatment of cells with the well-known PI3K inhibitor LY294002, and its structural analogue LY303511, which does not inhibit PI3K, increased homotypic GJIC; however, we found the effect to be independent of PI3K/AKT inhibition. We show in multiple cancer cell lines of varying metastatic capability that GJIC can be restored without enforced expression of a connexin gene. In addition, while levels of connexin 43 remained unchanged, its relocalization from the cytosol to the plasma membrane was observed. Both LY294002 and LY303511 increased the activity of protein kinase A (PKA). Moreover, PKA blockade by the small molecule inhibitor H89 decreased the LY294002/LY303511-mediated increase in GJIC. Collectively, our findings show a connection between PKA activity and GJIC mediated by PI3K-independent mechanisms of LY294002 and LY303511. Manipulation of these signaling pathways could prove useful for antimetastatic therapy.

An integrated computational model of the bone microenvironment in bone-metastatic prostate cancer.

Bone metastasis will impact most men with advanced prostate cancer. The vicious cycle of bone degradation and formation driven by metastatic prostate cells in bone yields factors that drive cancer growth. Mechanistic insights into this vicious cycle have suggested new therapeutic opportunities, but complex temporal and cellular interactions in the bone microenvironment make drug development challenging. We have integrated biologic and computational approaches to generate a hybrid cellular automata model of normal bone matrix homeostasis and the prostate cancer-bone microenvironment. The model accurately reproduces the basic multicellular unit bone coupling process, such that introduction of a single prostate cancer cell yields a vicious cycle similar in cellular composition and pathophysiology to models of prostate-to-bone metastasis. Notably, the model revealed distinct phases of osteolytic and osteogenic activity, a critical role for mesenchymal stromal cells in osteogenesis, and temporal changes in cellular composition. To evaluate the robustness of the model, we assessed the effect of established bisphosphonate and anti-RANKL therapies on bone metastases. At approximately 100% efficacy, bisphosphonates inhibited cancer progression while, in contrast with clinical observations in humans, anti-RANKL therapy fully eradicated metastases. Reducing anti-RANKL yielded clinically similar results, suggesting that better targeting or dosing could improve patient survival. Our work establishes a computational model that can be tailored for rapid assessment of experimental therapies and delivery of precision medicine to patients with prostate cancer with bone metastases.

Ubiquitous Brms1 expression is critical for mammary carcinoma metastasis suppression via promotion of apoptosis

Morbidity and mortality of breast cancer patients are drastically increased when primary tumor cells are able to spread to distant sites and proliferate to become secondary lesions. Effective treatment of metastatic disease has been limited; therefore, an increased molecular understanding to identify biomarkers and therapeutic targets is needed. Breast cancer metastasis suppressor 1 (BRMS1) suppresses development of pulmonary metastases when expressed in a variety of cancer types, including metastatic mammary carcinoma. Little is known of Brms1 function throughout the initiation and progression of mammary carcinoma. The goal of this study was to investigate mechanisms of Brms1-mediated metastasis suppression in transgenic mice that express Brms1 using polyoma middle T oncogene-induced models. Brms1 expression did not significantly alter growth of the primary tumors. When expressed ubiquitously using a β-actin promoter, Brms1 suppressed pulmonary metastasis and promoted apoptosis of tumor cells located in the lungs but not in the mammary glands. Surprisingly, selective expression of Brms1 in the mammary gland using the MMTV promoter did not significantly block metastasis nor did it promote apoptosis in the mammary glands or lung, despite MMTV-induced expression within the lungs. These results strongly suggest that cell type-specific over-expression of Brms1 is important for Brms1-mediated metastasis suppression.

Predictive computational modeling to define effective treatment strategies for bone metastatic prostate cancer

The ability to rapidly assess the efficacy of therapeutic strategies for incurable bone metastatic prostate cancer is an urgent need. Pre-clinical in vivo models are limited in their ability to define the temporal effects of therapies on simultaneous multicellular interactions in the cancer-bone microenvironment. Integrating biological and computational modeling approaches can overcome this limitation. Here, we generated a biologically driven discrete hybrid cellular automaton (HCA) model of bone metastatic prostate cancer to identify the optimal therapeutic window for putative targeted therapies. As proof of principle, we focused on TGFβ because of its known pleiotropic cellular effects. HCA simulations predict an optimal effect for TGFβ inhibition in a pre-metastatic setting with quantitative outputs indicating a significant impact on prostate cancer cell viability, osteoclast formation and osteoblast differentiation. In silico predictions were validated in vivo with models of bone metastatic prostate cancer (PAIII and C4-2B). Analysis of human bone metastatic prostate cancer specimens reveals heterogeneous cancer cell use of TGFβ. Patient specific information was seeded into the HCA model to predict the effect of TGFβ inhibitor treatment on disease evolution. Collectively, we demonstrate how an integrated computational/biological approach can rapidly optimize the efficacy of potential targeted therapies on bone metastatic prostate cancer.

Characterization of SV-40 Tag rats as a model to study prostate cancer

BackgroundProstate cancer is the second most frequently diagnosed cancer in men. Animal models that closely mimic clinical disease in humans are invaluable tools in the fight against prostate cancer. Recently, a Simian Virus-40 T-antigen (SV-40 Tag) targeted probasin promoter rat model was developed. This model, however, has not been extensively characterized; hence we have investigated the ontogeny of prostate cancer and determined the role of sex steroid receptor and insulin-like growth factor-1 (IGF-1) signaling proteins in the novel SV-40 Tag rat.MethodsThe SV-40 Tag rat was histopathologically characterized for time to tumor development, incidence and multiplicity and in the ventral, dorsal, lateral and anterior lobes of the prostate. Immunoassay techniques were employed to measure cell proliferation, apoptosis, and sex steroid receptor and growth factor signaling-related proteins. Steroid hormone concentrations were measured via coated well enzyme linked immunosorbent assay (ELISA) kits.ResultsProstatic intraepithelial neoplasia (PIN) and well-differentiated prostate cancer developed as early as 2 and 10 weeks of age, respectively in the ventral prostate (VP) followed by in the dorsolateral (DLP). At 8 weeks of age, testosterone and dihydrotestosterone (DHT) concentrations in SV-40 Tag rats were increased when compared to non-transgenic rats. High cell proliferation and apoptotic indices were found in VP and DLP of transgenic rats. Furthermore, we observed increased protein expression of androgen receptor, IGF-1, IGF-1 receptor, and extracellular signal-regulated kinases in the prostates of SV-40 Tag rats.ConclusionThe rapid development of PIN and prostate cancer in conjunction with the large prostate size makes the SV-40 Tag rat a useful model for studying prostate cancer. This study provides evidence of the role of sex steroid and growth factor proteins in prostate cancer development and defines appropriate windows of opportunity for preclinical trials and aids in the rational design of chemoprevention, intervention, regression, and therapeutic studies using prostate cancer rodent models.

Pre-osteoblastic MC3T3-E1 cells promote breast cancer growth in bone in a murine xenograft model

The bones are the most common sites of breast cancer metastasis. Upon arrival within the bone microenvironment, breast cancer cells coordinate the activities of stromal cells, resulting in an increase in osteoclast activity and bone matrix degradation. In late stages of bone metastasis, breast cancer cells induce apoptosis in osteoblasts, which further exacerbates bone loss. However, in early stages, breast cancer cells induce osteoblasts to secrete inflammatory cytokines purported to drive tumor progression. To more thoroughly evaluate the role of osteoblasts in early stages of breast cancer metastasis to the bones, we used green fluorescent protein-labeled human breast cancer cell lines MDA-MB-231 and MDA-MB-435, which both induce osteolysis after intra-femoral injection in athymic mice, and the murine pre-osteoblastic cell line MC3T3-E1 to modulate osteoblast populations at the sites of breast cancer metastasis. Breast cancer cells were injected directly into the femur with or without equal numbers of MC3T3-E1 cells. Tumors grew significantly larger when co-injected with breast cancer cells and MC3T3-E1 cells than injected with breast cancer cells alone. Osteolysis was induced in both groups, indicating that MC3T3-E1 cells did not block the ability of breast cancer cells to cause bone destruction. MC3T3-E1 cells promoted tumor growth out of the bone into the extraosseous stroma. These data suggest that breast cancer cells and osteoblasts communicate during early stages of bone metastasis and promote tumor growth.