Inflammatory Breast Cancer: The Immune Perspective

Inflammatory breast cancer (IBC) is the most insidious form of locally advanced disease. Although rare and less than 2% of all breast cancer, IBC is responsible for up to 10% of all breast cancer deaths. Despite the name, very little is known about the role of inflammation or immune mediators in IBC. Therefore, we analyzed blood samples from IBC patients and non-IBC patients, as well as healthy donor controls to establish an IBC-specific profile of peripheral blood leukocyte phenotype and function of T cells and dendritic cells and serum inflammatory cytokines. Emerging evidence suggests that host factors in the microenviromement may interact with underlying IBC genetics to promote the aggressive nature of the tumor. An integral part of the metastatic process involves epithelial to mesenchymal transition (EMT) where primary breast cancer cells gain motility and stem cell-like features that allow distant seeding. Interestingly, the IBC consortium microarray data found no clear evidence for EMT in IBC tumor tissues. It is becoming increasingly evident that inflammatory factors can induce EMT. However, it is unknown if EMT-inducing soluble factors secreted by activated immune cells in the IBC microenvironment canπ account for the absence of EMT in studies of the tumor cells themselves. We hypothesized that soluble factors from immune cells are capable of inducing EMT in IBC. We tested the ability of immune conditioned media to induce EMT in IBC cells. We found that soluble factors from activated immune cells are able to induce the expression of EMT-related factors in IBC cells along with increased migration and invasion. Specifically, the pro-inflammatory cytokines TNF-α, IL-6 and TGF-β were able to induce EMT and blocking these factors in conditioned media abated the induction of EMT. Surprisingly, unique to IBC cells, this process was related to increased levels of E-cadherin expression and adhesion, reminiscent of the characteristic tightly packed tumor emboli seen in IBC samples. This data offers insight into the unique pathology of IBC by suggesting that tumor immune interactions in the tumor microenvironment contribute to the aggressive nature of IBC implying that immune induced inflammation can be a novel therapeutic target. Specifically, we showed that soluble factors secreted by activated immune cells are capable of inducing EMT in IBC cells and may mediate the persistent E-cadherin expression observed in IBC. This data suggests that immune mediated inflammation may contribute to the highly aggressive nature of IBC and represents a potential therapeutic target that warrants further investigation.

INVESTIGATING THE ROLES OF THE p63 ISOFORMS IN THE MICRORNA BIOGENESIS PATHWAY

MicroRNAs play roles in various biological processes like development, tumorigenesis, metastasis and pluripotency. My thesis work has demonstrated roles for p63, a p53 family member, in the upstream regulation of microRNA biogenesis. The p63 gene has a complex gene structure and has multiple isoforms. The TAp63 isoforms contain an acidic transcription activation domain. The ΔNp63 isoforms, lack the TA domain, but have a proline rich region critical for gene transactivation. To understand the functions of these isoforms, the Flores lab generated TAp63 and ΔNp63 conditional knock out mice. Using these mice and tissues and cells from these mice we have found that TAp63 transcriptionally regulates Dicer while ΔNp63 transcriptionally regulates DGCR8. TAp63 -/- mice are highly tumor prone. These mice develop metastatic mammary adenocarcinomas, squamous cell carcinomas, and lung adenocarcinomas to distant sites including the liver, lungs, and brain. I found that TAp63 suppresses metastasis by transcriptionally activating Dicer. TAp63 and Dicer levels were very low or lost in high grade human tumors like mammary adenocarcinomas, squamous cell carcinomas, and lung adenocarcinomas. Expression of Dicer in these tumor cell lines reduced their invasiveness. Using ΔNp63 -/- mice, I found that ΔNp63 transcriptionally activates DGCR8, resulting in a miRNA profile that is critical to reprogram cells to pluripotency. Analysis of epidermal cells derived from ΔNp63 -/- mice revealed that these cells expressed markers of pluripotency, including Sox2, Oct 4 and Nanog; however, genome-wide analysis revealed a novel profile of genes that are common between ΔNp63 -/- epidermal cells and embryonic stem cells. I also found that mouse cells depleted of ΔNp63 form chimeric mice and teratomas in SCID mice, demonstrating that ΔNp63 deficient cells are pluripotent. Further, I found that restoration of DGCR8 in ΔNp63 -/- epidermal cells reduces their pluripotency and induces terminal differentiation. I also demonstrated that iMS (induced multipotent stem) cells could be generated using human keratinocytes by knockdown of ∆Np63 or DGCR8. Taken together, my work has placed p63 and its isoforms at a critical node in controlling miRNA biogenesis.

Hemato-lymphoid in vivo reconstitution potential of subpopulations derived from in vitro differentiated embryonic stem cells

During differentiation in vitro, embryonic stem (ES) cells generate progenitors for most hemato-lymphoid lineages. We studied the developmental potential of two ES cell subpopulations that share the fetal stem cell antigen AA4.1 but differ in expression of the lymphoid marker B220 (CD45R). Upon transfer into lymphoid deficient mice, the B220+ population generated a single transient wave of IgM+ IgD+ B cells but failed to generate T cells. In contrast, transfer of the B220− fraction achieved long-term repopulation of both T and B lymphoid compartments and restored humoral and cell-mediated immune reactions in the recipients. To assess the hemato-lymphopoietic potential of ES cell subsets in comparison to their physiological counterparts, cotransplantation experiments with phenotypically homologous subsets of fetal liver cells were performed, revealing a more potent developmental capacity of the latter. The results suggest that multipotential and lineage-committed lymphoid precursors are generated during in vitro differentiation of ES cells and that both subsets can undergo complete final maturation in vivo.

Murine hematopoietic stem cells committed to macrophage/dendritic cell formation: Stimulation by Flk2-ligand with enhancement by regulators using the gp130 receptor chain

The stimulation by Flk2-ligand (FL) of blast colony formation by murine bone marrow cells was selectively potentiated by the addition of regulators sharing in common the gp130 signaling receptor–leukemia inhibitory factor (LIF), oncostatin M, interleukin 11, or interleukin 6. Recloning of blast colony cells indicated that the majority were progenitor cells committed exclusively to macrophage formation and responding selectively to proliferative stimulation by macrophage colony-stimulating factor. Reculture of blast colony cells initiated by FL plus LIF in cultures containing granulocyte/macrophage colony-stimulating factor plus tumor necrosis factor α indicated that at least some of the cells were capable of maturation to dendritic cells. The cells forming blast colonies in response to FL plus LIF were unrelated to those forming blast colonies in response to stimulation by stem cell factor and appear to be a distinct subset of mature hematopoietic stem cells.

Increased expression of preprotachykinin-I and neurokinin receptors in human breast cancer cells: Implications for bone marrow metastasis

Neuropeptides are implicated in many tumors, breast cancer (BC) included. Preprotachykinin-I (PPT-I) encodes multiple neuropeptides with pleiotropic functions such as neurotransmission, immune/hematopoietic modulation, angiogenesis, and mitogenesis. PPT-I is constitutively expressed in some tumors. In this study, we investigated a role for PPT-I and its receptors, neurokinin-1 (NK-1) and NK-2, in BC by using quantitative reverse transcription–PCR, ELISA, and in situ hybridization. Compared with normal mammary epithelial cells (n = 2) and benign breast biopsies (n = 21), BC cell lines (n = 7) and malignant breast biopsies (n = 25) showed increased expression of PPT-I and NK-1. NK-2 levels were high in normal and malignant cells. Specific NK-1 and NK-2 antagonists inhibited BC cell proliferation, suggesting autocrine and/or intercrine stimulation of BC cells by PPT-I peptides. NK-2 showed no effect on the proliferation of normal cells but mediated the proliferation of BC cells. Cytosolic extracts from malignant BC cells enhanced PPT-I translation whereas extracts from normal mammary epithelial cells caused no change. These enhancing effects may be protein-specific because a similar increase was observed for IL-6 translation and no effect was observed for IL-1α and stem cell factor. The data suggest that PPT-I peptides and their receptors may be important in BC development. Considering that PPT-I peptides are hematopoietic modulators, these results could be extended to understand early integration of BC cells in the bone marrow, a preferred site of metastasis. Molecular signaling transduced by PPT-I peptides and the mechanism that enhances translation of PPT-I mRNA could lead to innovative strategies for BC treatments and metastasis.

Expansion in vitro of adult murine hematopoietic stem cells with transplantable lympho-myeloid reconstituting ability

Elucidation of mechanisms that regulate hematopoietic stem cell self-renewal and differentiation would be facilitated by the identification of defined culture conditions that allow these cells to be amplified. We now demonstrate a significant net increase (3-fold, P < 0.001) in vitro of cells that are individually able to permanently and competitively reconstitute the lymphoid and myeloid systems of syngeneic recipient mice when Sca-1+lin− adult marrow cells are incubated for 10 days in serum-free medium with interleukin 11, flt3-ligand, and Steel factor. Moreover, the culture-derived repopulating cells continued to expand their numbers in the primary hosts at the same rate seen in recipients of noncultured stem cells. In the expansion cultures, long-term culture-initiating cells increased 7- ± 2-fold, myeloid colony-forming cells increased 140- ± 36-fold, and total nucleated cells increased 230- ± 62-fold. Twenty-seven of 100 cultures initiated with 15 Sca-1+lin− marrow cells were found to contain transplantable stem cells 10 days later. This frequency of positive cultures is the same as the frequency of transplantable stem cells in the original input suspension, suggesting that most had undergone at least one self-renewal division in vitro. No expansion of stem cells was seen when Sca-1+TER119− CD34+ day 14.5 fetal liver cells were cultured under the same conditions. These findings set the stage for further investigations of the mechanisms by which cytokine stimulation may elicit different outcomes in mitotically activated hematopoietic stem cells during ontogeny and in the adult.

Anti-PSCA mAbs inhibit tumor growth and metastasis formation and prolong the survival of mice bearing human prostate cancer xenografts

Prostate stem-cell antigen (PSCA) is a cell-surface antigen expressed in normal prostate and overexpressed in prostate cancer tissues. PSCA expression is detected in over 80% of patients with local disease, and elevated levels of PSCA are correlated with increased tumor stage, grade, and androgen independence, including high expression in bone metastases. We evaluated the therapeutic efficacy of anti-PSCA mAbs in human prostate cancer xenograft mouse models by using the androgen-dependent LAPC-9 xenograft and the androgen-independent recombinant cell line PC3-PSCA. Two different anti-PSCA mAbs, 1G8 (IgG1κ) and 3C5 (IgG2aκ), inhibited formation of s.c. and orthotopic xenograft tumors in a dose-dependent manner. Furthermore, administration of anti-PSCA mAbs led to retardation of established orthotopic tumor growth and inhibition of metastasis to distant sites, resulting in a significant prolongation in the survival of tumor-bearing mice. These studies suggest PSCA as an attractive target for immunotherapy and demonstrate the therapeutic potential of anti-PSCA mAbs for the treatment of local and metastatic prostate cancer.

Long-term culture of lymphohematopoietic stem cells.

Pluripotent hematopoietic stem cells (PHSCs) show self-renewal and give rise to all blood cell types. The extremely low number of these cells in primary hematopoietic organs and the lack of culture systems that support proliferation of undifferentiated PHSCs have precluded the study of both the biology of these cells and their clinical application. We describe here cell lines and clones derived from PHSCs that were established from hematopoietic cells from the fetal liver or bone marrow of normal and p53-deficient mice with a combination of four growth factors. Most cell lines were Sca-1+, c-Kit+, PgP-1+, HSA+, and Lin- (B-220-, Joro 75-, 8C5-, F4/80-, CD4-, CD8-, CD3-, IgM-, and TER 119-negative) and expressed three new surface markers: Joro 177, Joro 184, and Joro 96. They did not synthesize RNA transcripts for several genes expressed at early stages of lymphocyte and myeloid/erythroid cell development. The clones were able to generate lymphoid, myeloid, and erythroid hematopoietic cells and to reconstitute the hematopoietic system of irradiated mice for a long time. The availability of lymphohematopoietic stem cell lines should facilitate the analysis of the molecular mechanisms that control self-renewal and differentiation and the development of efficient protocols for somatic gene therapy.

Human semaphorins A(V) and IV reside in the 3p21.3 small cell lung cancer deletion region and demonstrate distinct expression patterns.

Semaphorins and collapsins make up a family of conserved genes that encode nerve growth cone guidance signals. We have identified two additional members of the human semaphorin family [human semaphorin A(V) and human semaphorin IV] in chromosome region 3p21.3, where several small cell lung cancer (SCLC) cell lines exhibit homozygous deletions indicative of a tumor suppressor gene. Human semaphorin A(V) has 86% amino acid homology with murine semaphorin A, whereas semaphorin IV is most closely related to murine semaphorin E, with 50% homology. These semaphorin genes are approximately 70 kb apart flanking two GTP-binding protein genes, GNAI-2 and GNAT-1. In contrast, other human semaphorin gene sequences (human semaphorin III and homologues of murine semaphorins B and C) are not located on chromosome 3. Human semaphorin A(V) is translated in vitro into a 90-kDa protein, which accumulates at the endoplasmic reticulum. The human semaphorin A(V) (3.4-kb mRNA) and IV (3.9- and 2.9-kb mRNAs) genes are expressed abundantly but differentially in a variety of human neural and nonneural tissues. Human semaphorin A(V) was expressed in only 1 out of 23 SCLCs and 7 out of 16 non-SCLCs, whereas semaphorin IV was expressed in 19 out of 23 SCLCs and 13 out of 16 non-SCLCs. Mutational analysis in semaphorin A(V) revealed mutations (germ line in one case) in 3 of 40 lung cancers. Our data suggest the need to determine the function of human semaphorins A(V) and IV in nonneural tissues and their role in the pathogenesis of lung cancer.

Targeted disruption of vinculin genes in F9 and embryonic stem cells changes cell morphology, adhesion, and locomotion.

Vinculin, a major constituent of focal adhesions and zonula adherens junctions, is thought to be involved in linking the microfilaments to areas of cell-substrate and cell-cell contacts. To test the role of vinculin in cell adhesion and motility, we used homologous recombination to generate F9 embryonal carcinoma and embryonic stem cell clones homozygous for a disrupted vinculin gene. When compared to wild-type cells, vinculin-mutant cells displayed a rounder morphology and a reduced ability to adhere and spread on plastic or fibronectin. Decreased adhesion of the mutant cells was associated with a reduction in lamellipodial extensions, as observed by time-lapse video microscopy. The locomotive activities of control F9 and the vinculin-null cells were compared in two assays. Loss of vinculin resulted in a 2.4-fold increase in cell motility. These results demonstrate an important role for vinculin in determining cell shape, adhesion, surface protrusive activity, and cell locomotion.

Single-cell derived clones from human adipose stem cells present different immunomodulatory properties

Human adipose mesenchymal stem cells are a heterogeneous population, where cell cultures derived from single cell-expanded clones present varying degrees of differential plasticity. This work focuses on the immunomodulatory/anti-inflammatory properties of these cells. To this end, 5 single cell clones were isolated (generally called 1.X and 3.X) from 2 volunteers. Regarding the expression level of the lineage-characteristic surface antigens, clones 1.10 and 1.22 expressed the lowest amounts, while clones 3.10 and 3.5 expressed more CD105 than the rest and clone 1.7 expressed higher amounts of CD73 and CD44. Regarding cytokine secretion, all clones were capable of spontaneously releasing high levels of IL-6 and low to moderate levels of IL-8. These differences can be explained in part by the distinct methylation profile exhibited by the clones. Furthermore and after lipopolysaccharide stimulation, clone 3.X produced the highest amounts of pro-inflammatory cytokines such as IL-1β, while clones 1.10 and 1.22 highly expressed IL-4 and IL-5. In co-culture experiments, clones 1.X are altogether more potent inhibitors than clones 3.X for proliferation of total, CD3+T, CD4+T and CD8+T lymphocytes and NK cells. The results of this work indicates that adipose stem cell population is heterogeneous in cytokine production profile, and that isolation, characterization and selection of the appropriate cell clone is a more exact method for the possible treatment of different patients or pathologies.

Angiogenesis in cancer - general pathways and their therapeutic implications

A vast amount of data shows that angiogenesis has a pivotal role in tumor growth, progression, invasiveness and metastasis. This is a complex process involving essential signaling pathways such as vascular endothelial growth factor (VEGF) and Notch in vasculature, as well as additional players such as bone marrow-derived endothelial progenitor cells. Primary tumor cells, stromal cells and cancer stem cells strongly influence vessel growth in tumors. Better understanding of the role of the different pathways and the crosstalk between different cells during tumor angiogenesis are crucial factors for developing more effective anticancer therapies. Targeting angiogenic factors from the VEGF family has become an effective strategy to inhibit tumor growth and so far the most successful results are seen in metastatic colorectal cancer (CRC), renal cell carcinoma (RCC) and non-small cell lung cancer (NSCLL). Despite the initial enthusiasm, the angiogenesis inhibitors showed only moderate survival benefit as monotherapy, along with a high cost and many side effects. Obviously, other important pathways may affect the angiogenic switch, among them Notch signaling pathway attracted a large interest because its ubiquitous role in carcinogenesis and angiogenesis. Herein we present the basics for VEGF and Notch signaling pathways and current advances of targeting them in antiangiogenic, antitumor therapy.

Columnar cell lesions of the breast: The missing link in breast cancer progression? A morphological and molecular analysis

Columnar cell lesions (CCLs) of the breast are a spectrum of lesions that have posed difficulties to pathologists for many years, prompting discussion concerning their biologic and clinical significance. We present a study of CCL in context with hyperplasia of usual type (HUT) and the more advanced lesions ductal carcinoma in situ (DCIS) and invasive ductal carcinoma. A total of 81 lesions from 18 patients were subjected to a comprehensive morphologic review based upon a modified version of Schnitt's classification system for CCL, immunophenotypic analysis (estrogen receptor [ER], progesterone receptor [PgR], Her2/neu, cytokeratin 5/6 [CK5/6], cytokeratin 14 [CK14], E-cadherin, p53) and for the first time, a whole genome molecular analysis by comparative genomic hybridization. Multiple CCLs from 3 patients were studied in particular detail, with topographic information and/or showing a morphologic spectrum of CCL within individual terminal duct lobular units. CCLs were ER an PgR positive, CK5/6 and CK14 negative, exhibit low numbers of genetic alterations and recurrent 16q loss, features that are similar to those of low grade in situ and invasive carcinoma. The molecular genetic profiles closely reflect the degree of proliferation and atypia in CCL, indicating some of these lesions represent both a morphologic and molecular continuum. In addition, overlapping chromosomal alterations between CCL and more advanced lesions within individual terminal duct lobular units suggest a commonality in molecular evolution. These data further support the hypothesis that CCLs are a nonobligate, intermediary step in the development of some forms of low grade in situ and invasive carcinoma. Copyright: © 2005 Lippincott Williams & Wilkins, Inc.

Selection of conserved epitopes from hepatitis C virus for pan-populational stimulation of T-cell responses

The hepatitis C virus (HCV) is able to persist as a chronic infection, which can lead to cirrhosis and liver cancer. There is evidence that clearance of HCV is linked to strong responses by CD8 cytotoxic T lymphocytes (CTLs), suggesting that eliciting CTL responses against HCV through an epitope-based vaccine could prove an effective means of immunization. However, HCV genomic plasticity as well as the polymorphisms of HLA I molecules restricting CD8 T-cell responses challenges the selection of epitopes for a widely protective vaccine. Here, we devised an approach to overcome these limitations. From available databases, we first collected a set of 245 HCV-specific CD8 T-cell epitopes, all known to be targeted in the course of a natural infection in humans. After a sequence variability analysis, we next identified 17 highly invariant epitopes. Subsequently, we predicted the epitope HLA I binding profiles that determine their potential presentation and recognition. Finally, using the relevant HLA I-genetic frequencies, we identified various epitope subsets encompassing 6 conserved HCV-specific CTL epitopes each predicted to elicit an effective T-cell response in any individual regardless of their HLA I background. We implemented this epitope selection approach for free public use at the EPISOPT web server. © 2013 Magdalena Molero-Abraham et al.

A Randomized Phase II Crossover Study of Imatinib or Rituximab for Cutaneous Sclerosis after Hematopoietic Cell Transplantation.

PURPOSE: Cutaneous sclerosis occurs in 20% of patients with chronic graft-versus-host disease (GVHD) and can compromise mobility and quality of life. EXPERIMENTAL DESIGN: We conducted a prospective, multicenter, randomized, two-arm phase II crossover trial of imatinib (200 mg daily) or rituximab (375 mg/m(2) i.v. weekly × 4 doses, repeatable after 3 months) for treatment of cutaneous sclerosis diagnosed within 18 months (NCT01309997). The primary endpoint was significant clinical response (SCR) at 6 months, defined as quantitative improvement in skin sclerosis or joint range of motion. Treatment success was defined as SCR at 6 months without crossover, recurrent malignancy or death. Secondary endpoints included changes of B-cell profiles in blood (BAFF levels and cellular subsets), patient-reported outcomes, and histopathology between responders and nonresponders with each therapy. RESULTS: SCR was observed in 9 of 35 [26%; 95% confidence interval (CI); 13%-43%] participants randomized to imatinib and 10 of 37 (27%; 95% CI, 14%-44%) randomized to rituximab. Six (17%; 95% CI, 7%-34%) patients in the imatinib arm and 5 (14%; 95% CI, 5%-29%) in the rituximab arm had treatment success. Higher percentages of activated B cells (CD27(+)) were seen at enrollment in rituximab-treated patients who had treatment success (P = 0.01), but not in imatinib-treated patients. CONCLUSIONS: These results support the need for more effective therapies for cutaneous sclerosis and suggest that activated B cells define a subgroup of patients with cutaneous sclerosis who are more likely to respond to rituximab.