4. Role of E-Cadherin: Friend or Foe?

While cEMT is considered essential for successful tumor cell migration and invasion, it is now well accepted that cMErT confers increased survival and aids in successful colonization of metastatic sites by cancer cells [1]. Within this context, E-cadherin, a calcium-dependent cell-cell adhesion protein traditionally viewed as a tumor suppressor, seems to have a powerful role in therapeutic pan-resistance of micrometastases.

Early evidence for the importance of cMErT with regards to metastatic outgrowths came from observations of equal or increased levels of E-cadherin expression in distant metastases compared to their matched primary breast and prostate cancer specimens [39,40,41,42,43]. On top of that, metastatic tumors of invasive ductal adenocarcinoma origin express E-cadherin regardless of the E-cadherin status of the primary breast tumors [42]. Consistently, bone metastases from E-cadherin-negative, poorly differentiated breast carcinomas also exhibit E-cadherin expression [44]. All the above add one more layer of complexity regarding the role of cEMT in the cancer cell adaptation and establishment at the metastatic sites, instead pinpointing to E-cadherin as an essential player for the establishment and protection of metastatic deposits.

An abundance of markers have been associated with cEMP, including mesenchymal markers such as vimentin and N-cadherin, core cEMT-activating transcription factors (SNAIL1/2, ZEB1/2, TWIST1/2), and loss of epithelial protein expression (E-cadherin, EpCAM) (31). Of these, E-cadherin might be the strongest, since its loss is a hallmark of cEMT and strongly correlates with dissemination even in the absence of mesenchymal marker expression [27,45]. This is the basis for the partial epithelial and mesenchymal phenotypes noted in cancer cells.

The role of E-cadherin is extremely important in “shielding” micrometastases from chemotherapy as shown by Ma et al. in two studies in prostate cancer, where it was shown that hepatocytes induce a cMErT of prostate cancer cells via initial suppression of the MAP kinases p38 and ERK, conferring an epithelioid morphology to prostate cancer cells with an increase/re-expression of E-cadherin and other epithelial markers (ZO-1, connexin 43). Upon hepatocyte-dictated E-cadherin upregulation in prostate cancer cells and cell-cell adhesion through homeotypic binding, prostate cancer cells reactivated canonical survival pathways, such as PI3K/Akt, MEK/ERK, and JAK/STAT in response to chemotherapy and/or death signals, gaining resistance to treatment in a proliferation-independent manner [46,47] (Figure 1). The translational impact of these findings is impressive, as these pathways could be targeted in the context of combinatorial therapeutic approaches to synergistically improve the efficacy of traditional chemotherapies by using small doses of MEK inhibitors not for their direct therapeutic effect, but rather for re-sensitizing small micrometastases to existing chemotherapeutic agents.

Figure 1. E-cadherin mediates micrometastatic dormancy and chemoresistance. Hepatocytes induce E-cadherin upregulation in metastatic prostate cancer as well as other epithelial markers, thus promoting a partial cancer-associated mesenchymal-to-epithelial reverting transition (cMErT) by initial suppression of p38 and ERK. Upon cell-cell E-cadherin ligandation, stimulation of metastatic cells with chemotherapy induces activation of canonical pro-survival kinases resulting in increased chemoresistance. This chemoresistance is proliferation-independent and can be targeted with an adjuvant treatment to achieve optimal efficacy with traditional chemotherapies. DTCs: disseminated tumor cells.

 

5. Disseminated Tumor Cell Dormancy

Upon successful extravasation of DTCs into the metastatic site, these either exist as individual cells in a state of quiescent dormancy or form small masses that fail to expand. This cellular dormancy represents one of the most important mechanisms driving the resistance of disseminated tumor cells to chemotherapy and potentially immunotherapy for reasons that will be presented next. As circulating tumor cells arrive at the metastatic site and extravasate, the first thing they encounter is the basement membrane of the endothelium and its supporting stroma, comprising the perivascular niche. Stepping into the perivascular niche drives DTCs to enter into a state of quiescent dormancy and remain dormant for months or years until triggered to start proliferating again (48). With the use of an in vitro engineered microvascular niche model recapitulating key-elements of the pre-metastatic niche, Ghajar et al showed that the proliferative fate of DTCs was determined by their proximity to blood vessels, strongly suggesting that blood vessel-derived cues promoted quiescence of adjacent DTCs (49). Since most chemotherapeutic agents in clinical use target specific components of the proliferative capacity of cancer cells, such as microtubule formation, response to cytotoxic therapies critically depends on cell cycle activity, the cell cycle arrest in dormant cells confers resistance to therapy (50). This logical explanation was first supported by evidence arising from two studies measuring absolute numbers and proliferation indices (Ki-67) of DTCs in bone marrow before and after adjuvant therapy (51-53). DTCs were mostly negative for proliferation markers and moreover, their survival post-chemotherapy was associated with later relapse. Therefore, relapses occurring after months or years are presumed to arise from DTCs lying dormant at least over the period of several months during and after drug administration. Nevertheless, subsequent studies by Carlson et al expanded the concept of the protective role of the perivascular niche even to non-proliferating cells, as endothelial cells protected human basal breast cancer T4-2 cells from apoptosis induced by paclitaxel, doxorubicin, 5-FU or lapatinib, and the chemotherapy-resistant cancer cells comprised of both an mVenus/p27-positive quiescent and an mVenus/p27-negative proliferating population (54). Furthermore, proteomic analysis of the perivascular niche identified integrin-mediated adhesion of cancer cells to the extracellular matrix (ECM) as the main mechanism of perivascular niche-mediated resistance to chemotherapy. Notably, while endothelial cells of mature microvessels deposited thrombospondin-1 (TSP-1) into the basement membrane and promoted dormancy of DTCs, areas of sprouting angiogenesis lacked TSP-1 expression thus exerting an opposing effect to the dormant DTCs (49).It is now widely accepted that the microenvironment influences DTC fate through a variety of different signals. Bragado et al provided the first evidence of the bone marrow being a growth-restrictive ‘’soil’’ where TGFβ2 and p38 signaling induce DTC dormancy (55). Master regulators of hematopoietic stem cell (HSC) dormancy in the bone marrow, including BMP4, BMP7, TGFβ2 and GAS6 could induce residual disease dormancy in many types of cancer, including breast and prostate cancer (56-60). Importantly, many of the aforementioned signals induce ERK inhibition and p38 activation, and thus a low ERK/p38 signaling ratio which drives a G0/G1 arrest and entrance into dormancy (61). From a mechanistic point of view, increased p38 kinase activity induces activation of the unfolded protein response (UPR), which regulates the endoplasmic reticulum stress-related transcription factor ATF6, in turn promoting survival of dormant cells via Rheb upregulation and mTOR signaling (62, 63). Consistently, induction of protein disulphide isomerase and GRP78 in dormant cells secondary to p38 activation results in increased survival of dormant cells when exposed to chemotherapy and nutrient deprivation via BAX inhibition, potentially through BIK upregulation (64).Many studies have shown that systemic cancer spread potentially occurs long before diagnosis, thus acquisition of genetic alterations for DTCs to drive metastasis frequently occurs within the metastatic site (6, 8). Therefore, the presence of a niche promoting acquisition of metastasis-supportive adaptations and providing protection against chemotherapy is beneficial for early DTCs. Direct measurement of local oxygen concentrations in bone marrow revealed hypoxic regions sufficient to trigger an NR2F1-regulated dormancy and chemoresistance program (65). Although identified in primary tumor cells, hypoxic conditions in the bone marrow may induce dormancy in arriving DTCs as well (66). Additionally, this NR2F1-driven program was found to induce expression of pluripotency genes in experimental models of DTCs (67). Given that chemotherapy alters the local oxygen levels in the bone marrow (65), it can potentially enhance stemness traits in dormant DTCs fueling metastatic progression. Overall, adjacent cellular elements combined with non-cellular microenvironmental parameters (oxygenation, ECM) have a dramatic impact on the response to therapy and therapy per se represents a part of the equation between emergence versus dormancy.

 

6. MME and Metastatic Reactivation

The role of MME is fundamental not only for induction of dormancy in DTCs and micrometastases, but also for the successful micrometastatic reactivation and treatment resistance within this context as well. This emergence from dormancy relies not only on the immediately adjacent microenvironment (e.g. endothelial cells, stromal fibroblasts), but also systemic signals and ECM components governing several processes including neoangiogenesis, inflammation and fibrosis (68-71).By co-culturing breast cancer cell lines MCF-7 and MDA-MB-231 with hepatic non-parenchymal cells (NPCs) and endothelial cells, Taylor et al showed that NPCs in the metastatic hepatic niche secrete factors inducing a mesenchymal shift in these epithelial breast cancer cells initiating outgrowth partly via EGFR signaling activation (72). Furthermore, macrophages are also important players regarding metastatic outgrowth as macrophage phenotypic subtypes M1-like and M2-like were shown to diametrically regulate cEMP in metastatic breast cancer cells, with M2-like macrophages conferring a mesenchymal phenotype to breast cancer cells imparting a growth advantage (73). In agreement with the notion that specific signals from the MME promote micrometastatic activation via a bidirectional crosstalk, it has been shown that IL-11 secreted by breast cancer cells induces recruitment and activation of osteoclasts which foster micrometastatic expansion in the bone (74). Moreover, breast cancer cells promote osteolytic bone metastasis by producing Jagged1 and engaging Notch signaling in bone-resident cells (75).The role of a dense, fibrotic MME is also essential for emergence of macrometastases. Fibrosis with enrichment of collagen type I at the metastatic site can act as a critical determinant of cytoskeletal reorganization in dormant breast cancer cells, leading to their transition from dormancy to metastatic growth (76, 77). Additionally, collagen I-mediated signaling through discoidin domain receptor tyrosine kinase 1 (DDR1) is a requirement for the reactivation of dormant breast cancer cells (78). Heavy crosslinking of collagen fibers, including those created by HIF1-induced lysil oxidase, can induce metastatic reactivation by promoting integrin-mediated conversion of mechanical forces to biochemical cues (76, 79-81). In a similar manner, cancer-associated fibroblasts (CAFs) and hepatic stellate cells (Ito cells) have been shown to produce ECM rich in collagen, fibronectin and laminin, thus fostering a dense, fibrotic stroma and subsequent binding of breast cancer cells to these components confers resistance to cytotoxic chemotherapy via apoptosis inhibition (82-84). Finally, a recent study by Albrengues et al showed that stimuli of inflammation, such as bacterial lipopolysaccharide (LPS) or cigarette smoke can activate neutrophils to release their DNA content into the lung parenchyma forming neutrophil extracellular traps (NETs), which are associated with neutrophil degranulation and release of neutrophil elastase and MMP9. While NETs act as scaffolds, these powerful proteases enhance the processing of laminin-111, a major basement membrane component, which subsequently binds α3β1 integrin and activates focal adhesion kinase (FAK) signaling in dormant DTCs. This phenomenon induced proliferation of DTCs in mouse models of cancer, while use of an α3β1 integrin-blocking antibody prevented DTC reactivation in NET-rich lungs (85).Overall, it becomes clear that the induction of a dense ECM rich in collagen, fibronectin and laminin in the MME, which promotes emergence from dormancy and a secondary cMErT, represents one major mechanism of resistance to chemotherapy (Figure 2). Given the association between quiescence and protection from chemotherapy this finding might seem counterintuitive. Nevertheless, it is also true that other mechanisms are critical regulators of resistance, such as the binding of tumor cells to these ECM components activating downstream pathways and triggering inhibition of drug-induced apoptosis and conferring increased resistance even to targeted therapeutics (82-84). On top of these, the matricellular protein tenascin C also provides for survival by tonic signaling via the EGFR (86). Furthermore, the stiffness of the matrix contributes to treatment resistance by hindering the diffusion of chemotherapeutic agents into cancer tissues via increased hydrostatic pressure (87). Based on the observation that tumors originating from ‘’stiff tissues’’ (lung, skin, bone) have significantly higher number of somatic mutations and copy number alterations compared to tumors from ‘’soft tissues’’ (bone marrow, brain), it has been suggested that tumor stiffness could represent even a major driver of genomic instability through increased cell proliferation (increasing the probability of spontaneous mutations), increased frequency of nuclear envelope rupture and higher selective pressure (88). Finally, the role of the immune component of the MME is detrimental but will be discussed in detail below in ‘’immune surveillance’’.

 

Figure 2. Metastatic microenvironment dictates emergence from dormancy and treatment resistance. Reawakening of disseminated tumor cells (DTCs) from dormancy may be triggered by changes in the microenvironment in which these DTCs reside or even changes in immune activity. Key signaling components involved in metastatic reactivation include collagen, laminin and fibronectin interacting with integrins and DDR (Discoidin Domain Receptor). Neutrophils recruited can produce neutrophil extracellular traps (NETs) and the associated proteases permit the integrin-mediated activation of dormant DTCs that proliferate to form macrometastases. Concurrently, binding of tumor cells to these ECM components trigger inhibition of drug-induced apoptosis and resistance to targeted therapies. Tonic activation of EGFR by tenascin C also provides survival signaling. Finally, the density of the matrix impedes the diffusion of chemotherapy agents.

 

7. Immune Surveillance

Over the last years it is becoming increasingly clear how important the role of the immune system is in controlling dormancy, mediating treatment resistance and triggering reactivation of metastatic cancer. This knowledge is driven not only by a multitude of mechanistic studies, but also by the striking success of checkpoint blockade immunotherapy with the use of CTLA-4- and PD-1/PD-L1-blocking antibodies (89). The introduction in the clinic of immune checkpoint blockers has led to sustained long-term control of several types of cancer, including advanced melanoma, renal cell carcinoma, urothelial carcinoma and non-small cell lung cancer, among others, though often only in small subsets of cancers. Nevertheless, outcomes are far from satisfactory with regards to other types of cancer, such as metastatic castration-resistant prostate cancer (mCRPC), and efforts are focused on combinatorial therapeutic interventions to improve efficacy.The effects of the immune system on metastasis is a phenomenon starting early, with the immune cells implicated as critical regulators of all steps of the invasion-metastasis cascade (90, 91). Metastasis-associated macrophages (MAMs), especially those with an M2-like pro-tumorigenic phenotype, together with regulatory T lymphocytes (Tregs) and myeloid derived suppressor cells (MDSCs), aided by a number of cytokines and chemokines, foster a permissible premetastatic niche (92, 93) (Figure 3). All three types of cells contribute to the formation of a protective microenvironment for the incoming DTCs by suppressing the cytotoxic functions of T and NK cells (94-96). These cells are recruited and mobilized to play a pro-metastatic role by soluble factors and extracellular vesicles in the context of primary tumor-driven systemic processes that affect metastatic colonization (97). In models of bladder cancer metastasis to the lung, it has been demonstrated that DTC-derived endothelin-1 (ET-1) and ET_AR activity are necessary conditions for successful lung metastatic colonization, demonstrating not just immune modulation but also secretory support ensures from these cells. This is consistent with findings that macrophage infiltration of the tumor and the pulmonary parenchyma, resulting in secretion of trophic and immunosuppressive factors (MCP-1, IL-6), are also required for this process (98). Similarly, in breast cancer metastasis models, monocytes (recruited to DTCs by CCL2 produced in the lung both by the tumor and the surrounding stroma) differentiate into MAMs that promote DTC proliferation (99-102). Moreover, in a genetic mouse model of pancreatic ductal adenocarcinoma, it has been shown that granulin secretion by MAMs induced transformation of hepatic stellate cells into myofibroblasts, which in turn secreted periostin and induced the formation of a fibrotic microenvironment, favorable for activation of integrin signaling (103).Successful eradication of DTCs is dependent on the effector Ly6C+CD8+ T cell population and the inability of a tumor to induce a suppressive granulocytic MDSC population (104). The presence of MDSCs in metastatic lesions correlate with several negative parameters, including advanced disease stage, poor responses to chemotherapy as well as resistance to immune checkpoint blockade (105-109). The importance of MDSCs is further illustrated by recent work on mCRPC. Signaling mediated by MDSC-secreted IL-23 and its receptor on prostate cancer cells promotes the development of mCRPC, and this dismal phenomenon can be delayed by the use of pharmacologic agents blocking IL-23-mediated signaling (110). Additionally, MDSCs are critical in generating a suppressive tumor microenvironment by induction of PD-L1, iNOS, arginase-1, IDO and osteopontin, thus inhibiting T cell and NK cell tumoricidal activities and stimulating Treg cells (95, 111-114).

 

Figure 3. Roles of immune system in DTC dormancy and reawakening. This simplified schematic diagram shows that dormant DTCs may upregulate UPR activity which results in MHC-I downregulation and thus impaired immunological visibility to NK cells and CD8+ (cytotoxic) T cells. Downregulation of PD-L1 is a potential mechanism through which dormant DTCs may evade immunotherapy based on PD-1/PD-L1-blockade. Emergence of outgrowing metastases is linked to the activities of several immune cells, including regulatory T cells (Treg), myeloid-derived suppressor cells (MDSC) and metastasis-associated macrophages (MAMs), and also upregulation of immune markers such as PD-L1.

 

The immune response is dynamic and tightly regulated, in the sense that signals that enhance anti-tumor immune responses concurrently turn on inhibitory genes that function as ‘’immune breaks’’. For example, initial T cell activation, via T-cell receptor signaling and CD28 co-stimulation, eventually lead to increased expression of the immune checkpoint inhibitor, CTLA-4 (115). Similarly, effector Th1 responses such as increased IFNγ production concurrently lead to increased expression of PD-L1 on multiple cell types, including tumor cells and macrophages, which can engage the PD-1 receptor on T cells to suppress anti-tumor immunity (116, 117). As a consequence, metastases frequently exploit the aforementioned mechanisms to escape immune surveillance, and even the perivascular niche itself can suppress immune responses through expression of checkpoint ligands, such as PD-L1 (118). Recently, combinatorial immunotherapy of immune checkpoint blockade, anti-CTLA-4 or anti-PD-1 antibodies, with multikinase inhibitors targeting MDSCs (cabozatinib and BEZ235) showed robust synergistic response in preclinical models of mCRPC (119).An interesting aspect relevant to DTCs evading immune detection is related to antigen processing and presentation through major histocompatibility complex class I (MHC-I) molecules. Decreased expression of MHC-I in DTCs was initially suggested based on studies of breast, stomach, and colon carcinoma (52, 53). On top of this, breast and lung cancer DTCs were reported to broadly down-regulate ligands for an immune system-activating receptor on NK cells, named ULBP1, thus achieving evasion of NK-cell-mediated clearance. By expressing a Sox-dependent stem-like state and inhibiting WNT signaling in an autocrine manner, DTCs were able to enter quiescence and evade innate immunity to remain latent for extended periods (120). Recently, Pommier et al showed that dormant DTCs activate components of the unfolded protein response (UPR) causing posttranscriptional downregulation MHC-I molecules, thus suppressing antigen presentation to CD8+ T cells (121). This is in line with evidence from quiescent hair follicle stem cells, which although non-neoplastic, reside in specialized niches that control their quiescence, and in this state downregulate MHC-I and evade CD8+ T cell immune recognition (122).The ability of metastatic deposits to evade immune detection is notoriously evident in the case of outgrowing metastases. Immunosuppressive cancer microenvironments are now recognized as a major hurdle for positive clinical outcomes regarding the use of immune checkpoint blockade. Some immunotherapy resistant tumors are characterized by a paucity of neoantigens that are available for presentation (reflecting a low tumor mutational burden) and ultimately this presents an obstacle for antigen recognition (123, 124). This difficulty is particularly evident in mCRPC, a traditionally ‘’immunocold’’ tumor, where multiple clinical trials of immune checkpoint inhibitors failed to produce improvement in progression-free survival or overall survival. In other cases, despite expression of PD-1 and PD-L1, failure of immune checkpoint inhibitors can be explained by somatic JAK1/2 mutations rendering tumor cells insensitive to IFNγ (this can be either a primary or acquired resistance mechanism) or loss of β2-microglobulin impairing tumoral antigen presentation (125, 126). Notably, some innately resistant metastatic tumors have been shown to display a transcriptional signature referred to as IPRES (innate anti-PD-1 resistance), indicating concurrent upregulation of genes involved in mesenchymal transition, cell adhesion, extracellular matrix remodeling, angiogenesis, and wound healing (127).

8. Epigenetic modifications in metastasis

Epigenetic mechanisms of transcriptional regulation include DNA methylation, histone modifications, regulation by non-coding RNAs, and the dynamic binding of various proteins shaping the chromatin compaction status, thus spatially regulating the accessibility of chromatin to transcriptional factors and the ability to form active transcriptional higher order chromatin organization (128, 129). DNA methylation represents the most widely studied epigenetic modification and usually refers to the covalent modification of cytosine at the 5ʹ position at sites preceding guanine (CpG) (130). Epigenetic modifications are increasingly recognized as a major contributor to development of treatment resistance both to targeted agents, but also chemo- and radiotherapy in general and can even predict treatment response (131).Phenotypic differences between tumor cells demonstrating stemness versus those lacking it, can be predominantly attributed to epigenetic changes within the context of cEMT in the former, and this is a critical attribute for establishment of treatment resistance (132). One particularly relevant target of epigenetic regulation is that of E-cadherin downregulation, the sine qua non of cEMT; this target is suppressed at different levels ranging from autocrine signaling-induced internalization and transcriptional repression (such as in prostate cancer) to DNA methylation (as in breast cancer) (27). The value of such epigenetic downregulation rather than genetic mutation or deletion is the reversibility/phenotypic plasticity needed for metastatic seeding (26). The presence of E-cadherin expression provides for a pan-resistance to chemotherapies due to the downstream survival signals as discussed above.It has also been proposed that open chromatin structure incorporates traditional chemotherapeutics such as DNA-damaging agents carboplatin and doxorubicin more easily than compact chromatin, and similar correlations apply to irradiation (133, 134). Within this context, epigenetic targeting drugs have been shown to increase accessibility of DNA-damaging agents via global chromatin decompaction, and consequently trigger DNA damage and apoptosis in breast cancer models (135). With reference to the predictive value of epigenetic changes in treatment response, the most representative example is MGMT (O-6-methylguanine DNA methyltransferase) hypermethylation in gliomas, which represents the best independent predictor of response to carmustine and temozolomide, since MGMT hypermethylation renders tumor cells more sensitive to treatments and is associated with regression of tumor and prolonged overall survival (136, 137).Finally, the role of epigenetics in defining responses to immunotherapy is an area of tremendous interest. Strikingly, melanoma with low PD-L1 expression and global hypermethylation represents a large group of patients exhibiting primary resistance to immune checkpoint blockade therapy and this hypermethylation is associated with overexpression of DNA methyltransferases (DNMTs) and histone methyltransferase EZH2 in the PRC2 repressive complex (138). Consistently, epigenetic therapies (such as DNMT inhibitors) have been increasingly reported to enhance antigen processing and presentation pathways in tumors, especially by upregulating MHC-I, therefore potentiating responses to immunotherapies (139-144). In the years to come, it will be particularly interesting to see the results from clinical trials combining epigenetic therapies with immune checkpoint blockade especially in the case of traditionally immune ‘’cold’’ cancers, such as prostate cancer.

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