Cancer and Pregnancy: Parallels in Growth, Invasion, and Immune Modulation and Implications for Cancer Therapeutic Agents

Abstract

Many proliferative, invasive, and immune tolerance mechanisms that support normal human pregnancy are also exploited by malignancies to establish a nutrient supply and evade or edit the host immune response. In addition to the shared capacity for invading through normal tissues, both cancer cells and cells of the developing placenta create a microenvironment supportive of both immunologic privilege and angiogenesis. Systemic alterations in immunity are also detectable, particularly with respect to a helper T cell type 2 polarization evident in advanced cancers and midtrimester pregnancy. This review summarizes the similarities between growth and immune privilege in cancer and pregnancy and identifies areas for further investigation. Our PubMed search strategy included combinations of terms such as immune tolerance, pregnancy, cancer, cytokines, angiogenesis, and invasion. We did not place any restrictions on publication dates. The knowledge gained from analyzing similarities and differences between the physiologic state of pregnancy and the pathologic state of cancer could lead to identification of new potential targets for cancer therapeutic agents.

CTL = CD8⁺ T cytotoxic lymphocyte; DC = dendritic cell; EVT = extravillous trophoblast; HLA = human leukocyte antigen; IL = interleukin; NK = natural killer; TH1 = helper T cell type 1; TH2 = helper T cell type 2; Treg = regulatory T cell; uNK = uterine NK

A substantial body of literature exists describing the mechanisms cancer cells use to escape apoptosis and migrate through normal structures while evading a host immune response. What is not well known, however, is how these complex and interrelated mechanisms are orchestrated, starting with modulation of the immune response within the tumor microenvironment and ending with migration and proliferation of cancer cells at distant sites. One potential model to further study how a single malignant cell could proliferate and then metastasize undetected within a host is that of normal human pregnancy, in which the developing placenta invades the uterus and a semiallogeneic fetus escapes rejection from the maternal immune system.¹ A multitude of immunomodulatory properties of the fetomaternal interface (placenta) have evolved to allow the survival of the immunologically distinct fetus to parturition without an attack from the maternal immune system. The similarities between the mechanisms involved in fetomaternal and tumor-associated immunologic tolerance are intriguing and suggest a common pattern; however, neither system of immune evasion is perfect. A clear example of placental failure to protect the fetus against maternal immunity is that of Rh incompatibility. In multiparous women sensitized against fetal Rh antigens, re-exposure to fetal Rh antigens with subsequent pregnancy may lead to hemolytic disease of the newborn and fetal death.² Such imperfections of shared mechanisms of immune tolerance between pregnancy and cancer suggest that cancer rejection via immunologic means may be possible, even considering the myriad mechanisms extending immunologic privilege to the fetus as well as cancer cells.

This review summarizes the parallels in proliferation, invasion, and immune privilege between cancer and pregnancy by first detailing shared characteristics of fetal-derived trophoblast cells of the placenta and tumor cells. It then describes the similarities between tolerogenic systems within the tumor microenvironment and the fetomaternal interface. Finally, it provides an overview of the evidence for systemic immune modulation in cancer and pregnancy and suggests the implications of these similarities in designing an integrated approach to cancer therapy. Our PubMed search strategy included combinations of terms such as immune tolerance, pregnancy, cancer, cytokines, angiogenesis, and invasion. We also searched for articles on cellular subsets, including natural killer (NK) cells, dendritic cells (DCs), regulatory T cells (T_reg), and other lymphocyte populations with respect to their presence and function in pregnancy and cancer. We did not place any restrictions on publication dates. A better understanding of how the maternal immune system is altered during the normal processes of implantation, gestation, and labor may translate into individualized, novel therapies aimed at restoring immune competency in patients with advanced malignancies.

SHARED CHARACTERISTICS OF TROPHOBLAST CELLS AND TUMOR CELLS

Five days after fertilization, the human zygote forms into a structure consisting of 2 primary cell lines: the inner cell mass (or embryoblast) and the trophoblast.³ Trophoblast cells constitute the outer layer of the blastocyst, rapidly proliferating and invading the maternal endometrial decidua around day 7. A monolayer of cytotrophoblast cells surrounds the embryonic disc as the embryo completely embeds beneath the uterine decidua. By day 9, cytotrophoblast cells have differentiated into 2 distinct cell types: the syncytiotrophoblast and the extravillous trophoblast (EVT). The multinucleated syncytiotrophoblast cells form the external layer and are terminally differentiated. These cells are involved in fetomaternal nutrient exchanges and endocrine functions (such as β-human chorionic gonadotropic production). In contrast, EVT cells have a proliferative and invasive phenotype, migrating through the syncytiotrophoblast into the uterine wall to anchor the placenta beginning around day 14 after implantation.⁴ These EVT cells display a phenotype strikingly similar to cancer cells with their capacity for proliferation, migration, and establishment of a blood supply, making them a compelling model for oncologic comparison (Figure). This review highlights several shared characteristics of trophoblast and tumor cells and discusses them in the context of existing or developmental targeted cancer therapeutics (Table 1).

FIGURE.

Similarities between the fetomaternal interface and tumor microenvironment. For expansion of all gene symbols, see Glossary of Genetics Terminology at the end of the article. HLA = human leukocyte antigen; IL = interleukin; VM = vasculogenic mimicry.

TABLE 1.

Tumorlike Attributes of the Human Trophoblast Cells and a Selection of Representative Targeted Cancer Therapeutic Strategies in Use or Development^a

PROLIFERATION

Like tumor cells, trophoblast cells have a very high proliferative capacity and exhibit molecular characteristics found in rapidly dividing cancer cells.⁵⁴ For example, increased telomerase activity, typically not observed to a substantial degree in normal somatic cells, is detectable in 85% of human cancers.⁵⁵ In fact, the intracellular concentration of telomerase is exponentially related to the proliferative capacity of a cell.⁵⁶ In human pregnancy, telomerase activity is highest during the first trimester and decreases with maturation of the placenta.⁵⁷ Survivin, a protein that promotes proliferation and inhibits apoptosis, is overexpressed in many cancers⁵⁸ and is also up-regulated by trophoblast cells.⁵⁹ Inhibition of survivin by knockdown with small interfering RNA leads to a marked decrease in proliferation in trophoblast cell lines.⁶⁰ A similar decrease in proliferation is seen with survivin in small interfering RNA treatment of prostate,⁶¹ glioma,⁶² non-Hodgkin lymphoma,⁶³ cervical cancer cells, and breast cancer cells.⁶⁴ Both survivin and telomerase levels are dramatically higher in hydatidiform moles than in normal placentas, providing insight into the potential involvement of these 2 different mechanisms in neoplastic transformation.⁵⁹

Another pathway supportive of both trophoblast and cancer cell proliferation is the IGF pathway (for expansion of all gene symbols, see Glossary of Genetics Terminology at the end of the article). By binding to the IGF1R on cytotrophoblast cells, IGF stimulates proliferation through the MAPK pathway and survival via activation of the PI3K pathway.⁶⁵ Normally, levels of IGF are tightly regulated by IGF-binding proteins and protease pregnancy-associated plasma protein A, a binding protein.⁶⁶ Loss of binding protein regulation may contribute to the malignant phenotype.⁶⁷ In cancer cells, the IGF1R pathway is not only mitogenic and antiapoptotic but is involved in protecting cancer cells from damaging effects of chemotherapy and radiation, potentially as a result of its effects on downstream signaling pathways.⁶⁸ Additionally, the fetal form of the insulin receptor IR-A, which is highly expressed in fetal tissues and responsive to IGF2, is also a member of the IGF-signaling system.⁶⁹ In many cancers, including those of the breast and ovary, dysregulation of this fetal form of the insulin receptor becomes the predominant isoform leading to IGF2-stimulated proliferation and survival.^70,71

INVASION

The sine qua non of both a successful pregnancy and the growth of cancer is the establishment of a blood and nutrient supply, and invasion through normal tissues is required for this process. However, whereas cancer cells spread throughout the host and then engage in local proliferation, trophoblasts follow an organized pattern of differentiation from proliferation to invasion without distant metastasis.⁷² Some of the molecular switches involved in this differentiation pattern and their relevance for cancer therapeutic agents are discussed in the sections that follow.

As EVT cells migrate down the cytotrophoblast cell columns into the maternal decidua (Figure), they encircle and erode into the maternal spiral arteries and differentiate from a proliferative phenotype into an invasive phenotype.⁷³ This differentiation occurs at about 10 to 12 weeks of gestation and is associated with opening of the intervillous space and exposure to maternal blood. Many parallels can be observed between invasive EVT cells and cancer cells. Some of these similarities are highlighted in the sections to follow; for a more in-depth discussion, readers should refer to excellent reviews by Soundararajan and Rao⁷⁴ and Ferretti et al.⁷⁵

Requirements for cellular invasion include changes in cell adhesion molecules, secretion of proteases, and availability of growth factors. An example of a cellular program used by both cancer cells⁷⁶ and trophoblast cells⁷⁷ to promote invasion is epithelial-mesenchymal transition, which results in loss of cell-to-cell contact inhibition. Associated with this program are changes in integrin expression and loss of E cadherin, allowing loss of polarity and enhanced motility.^78,79 Both trophoblast and cancer cells secrete proteases to degrade extracellular matrix proteins required for dispersal through tissues. The cytoplasm of migratory EVT cells express HSP27, which is correlated with MMP2 activity.⁸⁰ Basal HSP27 levels are unusually high in cancer cells, protecting them from apoptotic stimuli,⁸¹ and are associated with metastatic potential.⁸² Finally, growth factors such as epidermal growth factor stimulate motility of EVT cells through phosphorylation of p42 and p44 MAPKs and the PI3K-dependent proteins, AKT and p38.⁸³ Epidermal growth factor is associated with tumor cell invasiveness through expression of MMPs.⁸⁴

Switches involved in triggering trophoblast and cancer cell molecular programs for invasion are not completely understood. The Wnt pathway, a system highly conserved across species involved in cellular proliferation and motility, has recently been implicated in switching trophoblast cells from a proliferative to an invasive phenotype.⁸⁵ Activation of the Wnt pathway is aberrant in many cancers, resulting in escape of β-catenin from proteosomal degradation, with subsequent β-catenin translocation into the cell nucleus and activation of multiple target genes.⁸⁶ Although direct activation of β-catenin alone has shown no effect on motility of EVT cells, inhibition of the Wnt—β-catenin pathway can block blastocyst implantation.⁸⁷ In EVT cells, activation of PAR1 (also known as the thrombin receptor) also stabilizes β-catenin and is associated with a proliferative and invasive capacity, whereas application of PAR1-silencing RNA inhibits EVT invasion.⁸⁸ Consistent with the need for tight regulation of invasive trophoblast cells, PAR1 is expressed in EVT cells between the 7th and 10th gestational week but is abruptly shut off by the 12th week.⁸⁹ Constitutive increased expression of PAR1 can be seen in cancer cells, especially in cells lacking normal p53 activity.⁹⁰ In vitro assays have shown PAR1 antagonism to inhibit MMP1-induced endothelial cell activation in tumor—endothelial cell communication.⁹¹ Whether this system could successfully be targeted for cancer therapy is under investigation. Other signal transduction pathways common in both trophoblast and cancer cell invasion include the JAK-STAT pathway,⁹² FAKs, G proteins, Rho-associated kinase, MAPKs, PI3K, and SMAD family proteins.⁷³ All of these pathways represent areas of current anticancer therapeutic development.

As EVTs acquire an invasive phenotype during placental development, they become polyploid (4N-8N) by switching from mitotic division to endoreduplication,⁹³ a process in which G2 or M phase (4N) cells replicate DNA without undergoing mitosis. In trophoblast cell lines, polyploid trophoblast giant cells are relatively resistant to the DNA-damaging effects of radiation,⁹⁴ illustrating a mechanism by which survival is promoted in invasive trophoblast cells. This process can also be observed in cancer cells treated with DNA-damaging agents. Endoreduplication can be induced in tumor cells on exposure to genotoxic agents such as paclitaxel⁹⁵ and cisplatin; a nonproliferative, senescent state in a small population of cells is induced in the latter case. The polyploid tumor cells can undergo depolyploidization to form diploid, cisplatin-resistant escape cells.⁹⁶ In cells with an impaired p53 system, treatment with the Aurora kinase inhibitor VX-680 leads to endoreduplication followed by apoptosis.²² However, in 2 wild-type p53 cancer cell lines, stabilization of p53 by Nutlin-3a, an inhibitor of the p53-binding protein MDM2, leads to initial endoreduplication followed by the emergence of stable radiation- and cisplatin-resistant tetraploid clones.²¹ A better understanding of the EVT endoreduplication process may lead to the development of targeted drugs to maintain tumor cell chemotherapeutic sensitivity.

VASCULOGENIC MIMICRY

As trophoblasts invade maternal spiral arteries, they further differentiate to display a vascular phenotype in a process termed vasculogenic mimicry, in which cells other than endothelial cells form vascular structures.⁹⁷ Vasculogenic mimicry can also be observed in aggressive cancers, and the genes and signaling pathways involved with the process of vasculogenic mimicry may be shared between EVT and cancer cells.⁹⁸ For example, the matrix glycoprotein—binding galectin 3 is highly expressed in EVT cells.⁹⁹ Galectin-3 also appears to be a key factor in the development of an endothelial phenotype and the tube formation well described in aggressive melanomas.¹⁰⁰ Galectin inhibitors are in preclinical testing as cancer therapeutic agents.¹⁰¹ Mig-7 was found in circulating tumor cells and tumor tissue (regardless of tissue of origin) from more than 200 patients with cancer; notably, it was absent from healthy controls.¹⁰² Mig-7 expression is associated with invasion and vasculogenic mimicry in cancer cells and also has recently been demonstrated in invasive embryonic cytotrophoblasts, peaking when EVT cells invade maternal decidua and remodel the vasculature during early placental development.¹⁰³ This finding represents the only known expression of Mig-7 in noncancerous cells. Cancers with an endothelial phenotype have not been shown to be responsive to antiangiogenic therapies.¹⁰⁴ Because cancer therapy aimed at proliferating cells is less likely to be effective in invading cells,¹⁰⁵ galactin-3, Mig-7, and other pathways involved in vasculogenic mimicry may also be important targets for cancer therapy.

ANGIOGENESIS

Molecular circuits involved in neoangiogenesis separate from vasculogenic mimicry are also likely shared between EVT and tumor cells. Angiopoietins and VEGF family members are extremely important in both spiral artery remodeling in placentation¹⁰⁶ and the growth of many tumor types.¹⁰⁷ Inhibition of VEGF has become an important therapeutic strategy in many cancers, although resistance can develop,¹⁰⁸ resulting from the induction of an angiogenic rescue program characterized by the up-regulation of multiple angiogenic genes in hypoxic tumor cells and supporting stroma.^109,110 Another member of the VEGF family, PGF, is a part of the VEGF blockade—associated rescue program that is involved in the response to pathologic conditions, such as wounds, ischemia, inflammation, or cancer.¹¹¹ Both VEGF and PGF are highly expressed in trophoblast cells.¹¹² It is interesting that serum levels of PGF increase after treatment of patients with cancer with the anti-VEGF monoclonal antibody bevacizumab.¹¹³ Preclinical studies indicate that PGF blockade reduces neoangiogenesis and lymphangiogenesis, hampers recruitment of intratumoral macrophages, and is not associated with the typical anti-VEGF adverse effects (thrombosis, hypertension, proteinuria, and microvascular pruning) in healthy mice.¹¹⁰

Also important for angiogenesis is the oxygen-sensitive MTOR pathway.¹¹⁴ Central to controlling trophoblast cell proliferation in response to nutrients and growth factors,¹¹⁵ MTOR is expressed on the transporting epithelium of intact human placenta.¹¹⁶ It is downstream of the PI3K/AKT pathway; controls cell cycle progression and cell size and mass; is involved in angiogenesis via the VEGF, IGF, and HIF-1α—signaling pathways; and is constitutively activated in many malignancies.^114,117 The MTOR inhibitor everolimus has antiangiogenic properties.¹¹⁸ A better understanding of the PI3K/AKT/MTOR pathway and other molecular circuits used by trophoblast cells in proliferation, invasion, and endothelial interactions may lead to the development of targeted therapies for cancer.⁷⁵ Overall, we are in our infancy of understanding the complexity, redundancy, and interrelatedness of these molecular pathways in both placentation and neoplasia.

IMMUNOLOGIC PROPERTIES OF THE FETOMATERNAL INTERFACE AND TUMOR MICROENVIRONMENT

In addition to sharing many proliferative and invasive features, the cells of the trophoblast, like cancer cells, actively modulate the host immune response to develop and sustain a nutrient supply. Historically, the placenta was considered an inert, mechanical barrier protecting the semiallogeneic fetus from maternal immunologic attack.¹¹⁹ Current evidence, however, supports just the opposite—many maternal and placental immunomodulatory factors are required for adequate placental invasion. Around 40% of decidual cells are cells of the innate immune system (eg, NK cells, macrophages, and DCs), a substantial proportion considering that the uterus is a nonlymphoid organ.¹²⁰ Likewise, although cancer previously has been considered immunologically invisible to the host, many recent studies support the notion that cancer cells actively engage immune cells; for example, the presence of tumor-infiltrating lymphocytes has been well described in the literature.¹²¹ The main components of the maternal immune response at the fetomaternal interface and the similarities to the tumor microenvironment are discussed in the sections that follow.

The most abundant immune cell present at the fetomaternal interface is the uterine NK (uNK) cell, which constitutes approximately 70% of all immune cells found in this tissue.¹²² Uterine NK cells are thought to be recruited from peripheral blood when interleukin (IL)-15 is secreted by endometrial stromal cells.¹²³ They are distinct from peripheral blood NK cells in that they do not express CD16, the FcRγIIIA receptor required for antibody-dependent cell-mediated cytotoxicity.¹²⁰ The mechanisms associated with this loss of CD16 are unclear but may be related to high levels of TGF-β within the microenvironment.¹²⁴ Also, in contrast to peripheral blood NK cells, uNK cells are more immunomodulatory than cytotoxic, secreting galectin 1 to induce tolerogenic DCs¹²⁵ as well as angiogenic factors VEGF and PGF that are important for decidual remodeling.¹²⁶ An improper balance of cytotoxic to regulatory NK cells could contribute to recurrent miscarriage and pre-eclampsia.¹²⁷ Expression of IL-15 and NK cell infiltration have been reported in many different malignancies,¹²⁸ including renal cell carcinoma,¹²⁹ with variable prognostic implications. Recently, tumor-infiltrating CD16-NK cells have also been characterized and appear to behave similarly to uNK cells with respect to cytokine production and reduced cytotoxic activity.¹³⁰ A closer look at factors that determine the balance of killer and regulatory NK cells during pregnancy may help identify mechanisms that shift immunity toward NK cytotoxic activity in patients with cancer.

Also infiltrating the decidua, albeit in smaller numbers than uNK cells, are macrophages, T_reg, and DCs. Macrophages phagocytose apoptotic EVT cells and secrete IL-10 and IDO, contributing to the tolerogenic T_H2 milieu.¹³¹ Gene expression profiling of decidual macrophages supports an immunosuppressive/anti-inflammatory phenotype with higher expression of CCL18, IGF1, IDO, neuropilin 1, and other genes associated with M2-polarized macrophages.¹³² Tumor-associated macrophages can be both inflammatory and immunosuppressive, and T_H1/T_H2 polarization is effected through the activation of NF-κB (also known as NFKB1).¹³³ In fact, in vitro studies suggest that tumor-associated macrophages may be re-educated to display a classically activated rather than an M2 phenotype by inhibition of inhibitory kappa B kinase β, the major activator of NF-κB.¹³⁴

Regulatory T cells are additional important mediators of tolerance in both pregnancy and cancer. Immunophenotypically, these cells express surface CD4, CD25, and FOXP3, and they expand in both decidua¹³⁵ and peripheral blood¹³⁶ during normal pregnancies. This expansion is antigen-specific and is induced by paternal/fetal alloantigens¹³⁷ and not simply by hormonal changes in pregnancy.¹³⁸ A decrease in this lymphocyte subset is associated with spontaneous abortion¹³⁹ and pre-eclampsia.¹⁴⁰ Regulatory T cells are also expanded in cancer and are implicated in impaired antitumor immunity,¹⁴¹ suppression of effector T lymphocyte proliferation,¹⁴² and increased tumor blood vessel density,¹⁴³ suggesting an important link between immunity and angiogenesis. Regulatory T cells in patients with cancer also recognize tumor-specific antigens and proliferate in response to antigenic stimulation.¹⁴⁴ Targeting the T_reg population to boost antitumor immunity is under investigation with agents such as denileukin diftitox (IL2/diphtheria fusion protein) or LMB-2 (Fv fragment of CD25 antibody/Pseudomonas endotoxin A fusion protein) and CTLA-4 inhibitors.^145,146 Some of the benefit of cytotoxic chemotherapy may be derived from concomitant impairment of the immunosuppressive T_reg proliferation driven by the cancer.¹⁴⁷

Antigen-presenting CD83⁺ DCs are involved in the maintenance of the T_H2-predominant state in decidual tissues,¹⁴⁸ as well as at other mucosal surfaces.¹⁴⁹ However, the role of the DC is likely more complex than antigen presentation and secretion of immunosuppressive cytokines. Ablation of uterine DCs leads to decidualization failure and embryo resorption in mice; this occurs even with syngeneic pregnancy in mice in which alloantigens are absent.¹⁵⁰ Dendritic cells also represent another link between immunity and angiogenesis, secreting soluble FLT1 (also known as VEGFR1) and TGF-β1 required for endothelial cell survival and vascular maturation. In the absence of DCs, angiogenesis is severely impaired. In cancer, DCs also play a role that is more than immunoregulatory through their production of potent angiogenic growth factors. Moreover, cancer cells can secrete substances that suppress maturation of DCs, including VEGF, TGF-β, hepatocyte growth factor, and osteopontin, thereby maintaining a proangiogenic, immature DC phenotype.¹⁵¹

Expression of certain cell surface molecules on both trophoblast and cancer cells can also confer immunologic protection. Among the most important of these molecules is the nonpolymorphic, highly conserved class I human leukocyte antigen (HLA) molecules such as HLAG¹⁵²; in contrast, the highly diverse classical HLA class I proteins A, B, and C are essential in cell-mediated immune responses. In fact, in trophoblast cells, interferon-γ fails to stimulate classical HLA class I expression.¹⁵³ A similar property of down-regulated or absent classical HLA class I expression can cloak cancer cells from the host's immune system.¹⁵⁴ Cancer treatment modalities including gamma irradiation,²⁹ radiopharmaceutical samarium-153-ethylenediaminetetramethylenephosphonate,³⁰ and chemotherapeutic agents such as 5-fluorouracil¹⁵⁵ and hypomethylating agents¹⁵⁶ increase HLA class I expression.

Expression of HLAG on trophoblast cells and cancer cells has important immunomodulatory effects. In the placenta, HLAG expression is most evident on EVTs at the fetomaternal interface, with lower expression at the proliferative area of the villous column and increased expression with invasive, interstitial, and endovascular EVT cells.¹⁵⁷ On the basis of sequence homologies, HLAG has been proposed as the ancestral MHC class I gene and has only a few known sequence variations in humans, in sharp contrast to the profound allelic diversity (measured in the hundreds of allelic variants) of classical MHC class I genes.¹⁵⁸ Human leukocyte antigen-G interacts with NK cells via inhibitory receptors, such as CD94/NKG2A, ILT2, and killer cell immunoglobulin-like receptor KIR2DL4.¹²⁰ The role of HLAG is to suppress cytolytic killing by both NK and cytotoxic T cells, induce apoptosis of immune cells, regulate cytokine production in blood mononuclear cells, and reduce stimulatory capacity and impair maturation of DCs (reviewed in Hunt et al¹⁵⁹). Within the tumor microenvironment, the generation of HLAG⁺—suppressive NK cells occurs by trogocytosis (ie, the rapid cell-to-cell contact-dependent transfer of membranes and associated molecules from one cell to another), leading to the inhibition of other HLAG⁺ (cross-inhibition) or HLAG^- NK cells through HLAG and ILT2 cross-linking.¹⁶⁰ Expression of HLAG is associated with a poor prognosis in patients with lymphoproliferative disorders,¹⁶¹ melanoma,¹⁶² mesothelioma,¹⁶³ breast carcinoma,¹⁶³ ovarian carcinoma,¹⁶⁴ renal cell carcinoma,¹⁶⁵ squamous esophageal cancer,¹⁶⁶ gastric carcinoma,¹⁶⁷ cervical cancer,¹⁶⁸ non—small cell lung cancer,¹⁶⁹ bladder cancer,¹⁷⁰ prostate cancer,¹⁷¹ endometrial cancer,¹⁷² colorectal cancer,¹⁷³ and myeloid malignancies, including acute myeloid leukemia.^174,175 However, relatively little is known about the regulation of the expression of this important immunomodulatory molecule.¹⁷⁴ Regulation of HLAG expression may be at the epigenetic level, with transcription of HLAG being detectable in acute myeloid leukemia cell lines after treatment with 5-aza-2′-deoxycytidine.³² Some preliminary evidence also supports a micro-RNA regulatory mechanism.¹⁷⁶ Clearly, HLAG represents an attractive target for immune-based cancer therapies given its preferential expression in many malignancies as well as limited expression in normal tissues.¹⁷⁷ Targeting HLAG with a peptide-based vaccine strategy to develop a cytotoxic T-cell response against tumor cells bearing the molecule has proved feasible,¹⁷⁸ although much work remains before other methods of HLAG inhibition could lead to restoration of antitumor immunity.

Other cell surface tolerance signals common between trophoblasts and cancer cells include CD200 (OX-2) and CEACAM-1. Trophoblast cells expressing CD200 can inhibit CD8⁺ T cytotoxic lymphocyte (CTL) generation and shift the cytokine balance toward T_H2 in vitro.¹⁷⁹ Expression of CD200 is a negative prognostic factor in patients with multiple myeloma¹⁸⁰ and acute myeloid leukemia,¹⁸¹ and it has been shown to down-regulate T_H1 cytokines in vitro in solid tumors, including melanomas, ovarian carcinomas, and renal cell carcinomas.¹⁸² As a potential cancer stem cell marker, CD200 may be a promising target for these cells that survive conventional chemotherapy.¹⁸³ CEACAM-1 (CD66a), expressed on both trophoblasts and IL-2-activated decidual leukocytes, plays a role in inhibiting NK-mediated cytolysis.¹⁸⁴ Colocalization of osteopontin on EVT cells is associated with an invasive phenotype important for successful placentation.¹⁸⁵ CEACAM-1 expression in cancer is associated with increased angiogenesis in non-small cell lung cancer¹⁸⁶; in melanoma, it has been shown to be predictive of the development of metastatic disease.¹⁸⁷ Expression of other immunomodulatory molecules, including components of the extrinsic apoptotic pathway such as FAS, TNF superfamily receptors,¹⁸⁸ TRAIL,¹⁸⁹ and B7 family members such as B7H1 (or programmed death ligand 1, PDL-1),¹⁹⁰ is also common between trophoblast and cancer cells (Table 1).

Chemokines and cytokines also play a role in promoting a tolerogenic environment in placentation and the tumor microenvironment. Implantation of the blastocyst occurs in a T_H1-predominant (inflammatory) milieu, but the fetomaternal interface must transition to a T_H2-polarized (immunologically tolerant) state for pregnancy to continue (for an excellent review, refer to van Mourik et al¹⁹¹). However, before implantation can occur, the endometrial lining must be receptive in the so-called window of implantation, in which many immunomodulatory genes are up-regulated monthly during the midsecretory phase of the menstrual cycle.¹⁹¹ Under the influence of progesterone, the endometrial epithelium up-regulates decay-accelerating factor and osteoponin expression, and the endometrial stroma increases IL-15 expression.^192,193 Expression of complement regulatory proteins (eg, decay-accelerating factor) is a well-established immunomodulatory mechanism used by many cancers to escape complement-mediated cell death and evade an immune response by inhibiting T-cell proliferation.¹⁹⁴ Osteopontin has T_H1 cytokine functions and is chemotactic for macrophages, T cells, and DCs, the last of which it induces to secrete IL-12 and tumor necrosis factor α (TNF-α).^195,196 Osteopontin is overexpressed in many cancers and is associated with metastatic potential.¹⁹⁷ Additionally, tissues that physiologically express high levels of osteopontin, such as bone, lung, and liver, may create a receptive microenvironment for metastasis via interaction with osteopontin receptor CD44 on the surface of cancer cells.¹⁹⁸

RANTES (CCL5) is a chemokine produced by trophoblasts that may play a role in apoptosis of potentially harmful maternal CD3⁺ cells.¹⁹⁹ Melanoma cells can induce tumor-infiltrating lymphocytes to secrete RANTES and subsequently undergo apoptosis as another mechanism to evade an immune response.²⁰⁰ Trophoblast cells also secrete chemoattractant cytokines, such as GRO-α, MCP1, and IL-8, to actively recruit the CD14⁺ monocytes to the fetomaternal interface.²⁰¹ GRO-α is an oncogenic and angiogenic cytokine driven by RAS, which is inappropriately activated in most cancers.²⁰² Capable of inducing vascular permeability along with mononuclear cell recruitment, MCP1 is associated with angiogenesis and malignant pleural effusions.²⁰³ Inhibition of MCP1 can lead to reduced malignant angiogenesis and recruitment of tumor-associated macrophages in a mouse model of melanoma.²⁰⁴ Finally, the IL-8 pathway is well known to be a central immune and angiogenic factor within the tumor microenvironment and is important in stress-induced chemotherapeutic resistance.²⁰⁵

A tryptophan-catabolizing enzyme, IDO is important in promoting tolerance by inhibiting proliferation of lymphocytes both at the fetomaternal interface²⁰⁶ and tumor microenvironment.²⁰⁷ Tryptophan levels have been observed to decrease in pregnancy with a return to normal, nonpregnant levels in the puerperium,²⁰⁸ possibly a result of tryptophan degradation by IDO-expressing trophoblast cells. Expression of HLAG on DCs can be induced by IDO, indicating potential cooperation in immune suppression between these 2 molecules.²⁰⁹ Tumor-derived PGE2 secretion can increase IDO expression in local DCs.²¹⁰ Antigen-expressing cells and IDO-expressing tumor cells might also contribute to local immunosuppression in tumor-draining lymph nodes.²¹¹ Pharmacologic inhibitors of IDO are under development and in early-stage clinical trials as anticancer agents.²⁰⁷ Induction of IDO can also be blocked in vitro by cyclooxygenase 2 inhibitors.²¹² When murine breast cancer vaccine recipients received the oral cyclooxygenase 2 inhibitor celecoxib, an increase in tumor-specific CTLs was observed.³⁵

Trophoblast invasion and spiral artery remodeling are tightly controlled processes, likely kept in check both by molecular programming of trophoblast cells and by paracrine immune factors.²¹³ We have much to gain in terms of developing novel immunologic interventions for our patients with cancer by closely examining both the similarities and differences of the intimate cross-talk that occurs within the tumor and placental microenvironments.

EVIDENCE FOR SYSTEMIC IMMUNE MODULATION

Similar to the increasing antigenic burden of progressive cancer,²¹⁴ fetal DNA can be found circulating in maternal blood by the second trimester in the height of the tolerogenic cytokine milieu.²¹⁵ Although its immunologic consequences have not been fully elucidated, this circulating DNA likely contributes to tolerance and eventual exhaustion of antigen-specific CTLs. This phenomenon is well described for the human immunodeficiency virus, chronic infection with which leads to progressive HIV-specific T cell dysfunction.²¹⁶ In addition to circulating nucleic acids, cellular fragments, known as microparticles or exosomes, can be detected in the peripheral blood of pregnant women in the third trimester.²¹⁷ Trophoblast-derived microparticles are proinflammatory, activate the coagulation system, can cause endothelial dysfunction, and are circulating at higher levels in pre-eclamptic vs normal pregnancies.²¹⁸ These microparticles are also involved in down-regulation of T-cell activity and deletion of activated T cells through interactions with FAS or TRAIL on the microparticle surface.²¹⁹ A similar phenomenon of cancer cell-derived microparticles contributing to the hypercoagulable state and impaired antitumor immunity of patients with cancer has been described (reviewed in Amin et al²²⁰). Microparticles derived from melanoma cells have been shown to express HLAG, likely contributing to their immunomodulatory properties.²²¹

Just as circulating tumor cells have been identified in patients with early-stage malignancies,²²² intact trophoblast cells are also known to circulate in the maternal peripheral blood as early as the ninth week of pregnancy.²²³ These fetally derived cells can engraft in the mother irrespective of HLA disparity and establish a long-term microchimerism that persists for decades after parturition.²²⁴ Rates of fetal microchimerism are decreased in female patients with cancer (34%) compared with healthy controls (57%), and the immunomodulatory implications of this decrease are unclear.²²⁵ An increased number of fetal microchimeric cells in aggressive breast carcinoma²²⁶ and melanoma²²⁷ during pregnancy have been observed. Whether these cells were recruited to the tumor microenvironment by inflammation and behave as innocent bystanders or whether they participate in tumor progression by providing angiogenic or tolerogenic signals is unclear at this time.

Many additional immunomodulatory proteins are secreted by trophoblast cells and can be found circulating in maternal peripheral blood. Among these molecules, soluble HLAG may be the most extensively studied.²²⁸ Soluble HLAG impairs NK/DC cross-talk, promotes proinflammatory cytokine secretion from both uterine and peripheral blood mononuclear cells,²²⁹ and induces apoptosis of CD8⁺ cells through CD8 ligation²³⁰ and FAS-FASL interaction.²³¹ Soluble HLAG has been well documented in malignancies,¹⁷⁴ including acute leukemia,²³² multiple myeloma,²³³ lymphoproliferative disorders,²³⁴ breast and ovarian carcinoma,¹⁶³ renal cell carcinoma,^165,235 lung cancer,²³⁶ gliomas,²³⁷ and melanoma.²³⁸ Cancer cells can also trigger monocytes to release HLAG, further down-regulating antitumor immunity.²³⁹ Whether HLAG can be targeted to break cancer-specific tolerance remains to be investigated.

A search for other immunomodulatory molecules from conditioned media of placental tissue has yielded interesting results. Surprisingly, no interleukins were identified by either proteomic analysis or sensitive radioimmunoassays; rather, in addition to pregnancy-associated hormones, substances including PSG1, glycodelin, TGF-β2, thrombospondin-1, PEDF, MIF, and galectin 1 were identified as important immunoregulators in pregnancy.²⁴⁰ Many of these substances have been identified in cancer as well. For example, PSGs may not be pregnancy specific at all. Pregnancy-specific glycoprotein 9 deregulation is an early event in colorectal carcinogenesis.²⁴¹ Expressed frequently in lung carcinomas,¹⁶⁹ PSG1 is associated with estrogen receptor negativity and a higher risk of death in early-stage breast cancer.²⁴² Glycodelin may be involved in tumor angiogenesis by increasing VEGF release in many cell lines.²⁴³ An inhibitor of TGF-β2 (overexpressed in many cancers) is in phase 1/2 cancer clinical trials.¹³ Thrombospondin 1 is an endogenous angiogenesis inhibitor, although its expression in tumor stroma may render tumor cells insensitive to VEGF and help maintain tumor cell dormancy.²⁴⁴ Another endogenous angiogenesis inhibitor, PEDF, may have anti-invasive effects on tumor cells.²⁴⁵ MIF can stabilize HIF-1α, a factor central to cellular response to hypoxia.²⁴⁶ Galectin1 expression within tumors and the stromal tissues is positively correlated with cancer aggressiveness²⁴⁷ and a diminished T-cell response.²⁴⁸

Another soluble immunomodulator, soluble CD30, a member of the tumor necrosis superfamily of receptors and marker of T_H2 polarization, is increased in women with normal pregnancies and reduced in those with preeclampsia and intrauterine growth retardation.²⁴⁹ In addition to being prognostic in patients with CD30⁺ classical Hodgkin lymphoma,²⁵⁰ soluble CD30 is a potential marker of chronic B cell hyperactivation and can predict those at risk of AIDS-associated non-Hodgkin lymphoma.²⁵¹ The identification of common immunomodulators helps expand the concept of tolerance in pregnancy and cancer beyond T_H2 and toward a more complete understanding of chronic inflammation, angiogenesis, and immunologic privilege.

IMPLICATIONS FOR CANCER THERAPEUTICS

As a healthy pregnancy progresses toward parturition, several changes within the mother reflect a restoration of active, T_H1-predominant immunity. Although T_reg levels stay constant until the postpartum period,²⁵² a gradual return of CD16⁺ NK cells is observed in late pregnancy.²⁵³ Suppressed earlier in pregnancy, circulating cytotoxic γδ-T cells increase with the onset of labor.²⁵⁴ Interleukin 2 levels decrease while granulocyte macrophage colony—stimulating factor and interferon-γ increase through the third trimester and even more markedly at the onset of labor.²⁵⁵ Increased expression of genes associated with acute inflammation and neutrophil and monocyte influx has been observed in human fetal membranes at parturition.^256,257 Concomitant with an increase in the potent uterine contractile prostanoid PGF-2α, proinflammatory cytokines and MMPs prepare the uterus for labor.²⁵⁸ Markedly down-regulated at term compared with midgestation are genes involved in angiogenesis, such as angiopoietin 2.²⁵⁹ Taken together, these changes support a transition from a T_H2 to a T_H1 polarity during the third trimester.

In contrast, patients with advanced malignancies continue to experience a progressive failure of antitumor immunity, which has been associated with a T_H2-polarization and VEGF-driven chronic inflammation.²⁶⁰ We have identified the expression of immunomodulatory genes known to be supportive of pregnancy in our own patients with metastatic melanoma via gene-expression profiling (unpublished data). We have also verified that these immunomodulatory genes are differentially expressed in melanoma vs benign melanocytic nevi in 2 independent publically available datasets from the National Center for Biotechnology Information/GenBank GEO database: GSE4587,²⁶¹ which was analyzed on the GeneChip Human Genome U133 Plus 2.0 Array platform (Affymetrix, Santa Clara, CA), and GSE3189,²⁶² which was analyzed on the GeneChip Human Genome U133 Array Set HG-U133A platform (Affymetrix). We selected approximately 70 immunomodulatory genes on the basis of our critical review of the obstetrics literature, log-transformed the raw data, and performed an analysis of variance on this gene set on Partek 6.4 software. A summary of results is listed in Table 2. Osteopontin and other important components of innate immunity such as TLR2 and TLR4 and PTX3 were significantly up-regulated in melanoma compared with benign nevi. Galectins 1 and 9 were also significantly up-regulated compared with nevi. Notably down-regulated in melanoma were genes known to be up-regulated in term placenta,^259,263 including LPL, FABP4, and FZD10 (a Wnt receptor). Overall, this pattern is supportive of our theory that tumor cells use similar mechanisms of immune escape as those cells of the developing placenta, although these similarities have not yet been studied in a systematic fashion. Given what we have learned about the similarities between the placenta and tumor microenvironment, we plan to next comprehensively evaluate changes in systemic immune homeostasis in pregnancy vs cancer in order to prioritize potential therapeutic targets. In particular, identifying immunologic distinctions between pregnancy and cancer will be critical for this process.

TABLE 2.

Immunomodulatory Genes Differentially Expressed in Melanoma vs Benign Melanocytic Lesions^a

CONCLUSION

By comparing immunologic patterns throughout healthy pregnancies, and in particular the return to T_H1-polarized immunity through the third trimester, with those patterns observed in advanced malignancies, we have an opportunity to learn potential mechanisms to overcome the burden of long-term antigenic exposure and immunologic exhaustion in patients with cancer. The challenge for investigators in this field will be to extend our observations beyond the T_H1/T_H2 paradigm in both pregnancy and cancer to a model that can both assess the status and guide treatment of malignancies in an individualized, rational, real-time manner. A critical need exists for the development of treatments aimed at all aspects of cancer: malignant proliferation, invasion, vasculogenic mimicry, angiogenesis, and immune privilege. Studying how all these aspects are orchestrated in the predictable, physiologic process of pregnancy can facilitate the search for novel cancer treatment strategies, from cytotoxic chemotherapy to biologic agents and immunologic adjuncts, in the often unpredictable and arduous fight against the pathologic process of cancer.

Glossary of Genetics Terminology

AKT

v-akt murine thymoma viral oncogene homolog

ALK

anaplastic lymphoma receptor tyrosine kinase

BCL2

B cell chronic lymphocytic leukemia/lymphoma 2

CDK

cyclin dependent kinase

CEACAM1

carcinoembryonic antigen-related cell adhesion molecule 1 (biliary glycoprotein)

CMET (also known as MET)

met proto-oncogene (hepatocyte growth factor) receptor

CSF

colony-stimulating factor

CSF1R

CSF type 1 receptor

CXCR4

chemokine (C-X-C motif) receptor 4

EGF

epidermal growth factor (beta-urogastrone)

ERK

extracellular signal—related kinase

FABP4

fatty acid—binding protein 4

FAK

focal adhesion kinase

FAS

Fas (TNF receptor superfamily, member 6)

FASL

FAS ligand

FCγIIIA

FC gamma receptor III A

FGF

fibroblast growth factor

FLT1

fms-related tyrosine kinase 1 (vascular endothelial growth factor/vascular permeability factor receptor)

FOXP3

forkhead box P3

FZD10

frizzled homolog 10

GRO-α

growth-related oncogene α

HGF

hepatocyte growth factor

HIF-1α

hypoxia inducible factor 1α

HSP27

heat shock protein 27

IDO

indoleamine 2, 3 dioxygenase

IGF

insulinlike growth factor

IGF1R

IGF type 1 receptor

ILT2

Ig-like transcript 2

JAK

janus kinase

LGALS1

galactin 1

LGALS9

galactin 9

LPL

lipoprotein lipase

MAPK

mitogen-activated protein kinase

MCP1

monocyte chemoattractant protein 1

MEK (also known as MAP2K)

MAPK/ERK kinase

MIF

macrophage migration inhibitory factor

Mig-7

migration-induction protein 7

MDM2

mouse double minute 2

MMP

matrix metalloproteinase

MTOR

mammalian target of rapamycin

MUC1

mucin 1

NF-κB

nuclear factor κB

p38

tumor protein 38

PAR1

protease activated receptor 1

PDGF

platelet-derived growth factor

PDGFR

PDGF receptor

PEDF

pigment epithelial—derived factor

PGE2

prostaglandin E2

PGF

placental growth factor

PI3K

phosphoinositide-3 kinase

PSG1

pregnancy-specific glycoprotein 1

PTX3

pentraxin 3

RANTES (also known as CCL5)

regulated on activation, normal T-cell expressed and secreted

RAF

v-raf-1 murine leukemia viral oncogene homolog 1

RAS

rat sarcoma viral oncogene homolog

SDF (also known as CXCL12)

stromal-derived factor 1

SPP1

osteopontin

STAT

signal transducers and activator of transcription

TGF

transforming growth factor

TIM3 (also known as HAVCR2)

T cell immunoglobulin mucin 3

TIMP2

tissue inhibitor of metalloproteinase 2

TLR

toll-like receptor

TNF

tumor necrosis factor

TRAIL

TNF-related apoptosis-inducing ligand

TRAILR

TRAIL receptor

uPA

urokinase plasminogen activator

VEGF

vascular endothelial growth factor

VEGFR

VEGF receptor

waf1/cip1 (also known as CDKN1A)

cyclin-dependent kinase inhibitor 1A

Wnt

wingless/T-cell factor

XIAP

X-link inhibitor of apoptosis protein