• CD95 is a surface receptor that has the capacity to mediate apoptosis induction in cancer cells.

  • To induce apoptosis, CD95 recruits a number of proapoptotic factors including caspase-8 to form the death-inducing signaling complex when stimulated by CD95 ligand (CD95L).

  • Immune cells (i.e., cytotoxic killer and natural killer cells) use CD95L as one mechanism to kill cancer cells or virus-infected cells.

  • Most cancer cells are resistant to CD95-mediated apoptosis.

  • CD95L can not be used systemically for cancer therapy because of the side effects due to apoptosis induction in hepatocytes.

Open Questions

  • Why do most if not all cancer cells express both CD95 and CD95L?

  • Why do cancer cells acquire mutations in CD95 usually only in one allele?

  • Why are the cancer cells that are sensitive to CD95-mediated apoptosis (at least in vitro) among the most sensitive of any cells?

CD95/CD95L in the Immune System

CD95 (Fas/APO-1/TNFRSF6), a cell surface protein that belongs to the tumor necrosis factor receptor family, can mediate apoptosis when bound to its natural ligand, CD95L (CD178/TNFSF6) or stimulated with agonistic antibodies. It is ubiquitously expressed in the body, but is particularly abundant in the thymus, liver, heart, and kidney. CD95L is predominantly expressed in activated T lymphocytes and natural killer cells, and is constitutively expressed in tissues of ‘immune-privilege sites’ such as the testis and eye.1 Experiments with mutant mice have demonstrated the importance of CD95-mediated apoptosis in the maintenance of cell homeostasis and in the deletion of useless or autoreactive T cells.1, 2, 3 A mutation found in the lpr (lymphoproliferation) mouse strain causes defective expression of CD95. Lpr mice develop lymphadenopathy and suffer from a systemic lupus erythematosus-like autoimmune disease.4 A second mouse strain (gld, generalized lymphoproliferative disease) expresses a mutant form of CD95L. Gld mice have an abnormal phenotype similar to lpr mice, which includes lpr and autoimmune disease.5 Complete knockout mice lacking either CD95 or CD95L expression also show an autoimmune phenotype that is more pronounced than the one seen in lpr or gld mice.6, 7, 8 A third mutant mouse strain with an lpr-like phenotype (lprcg) was found to have a point mutation (T to A) in the center of the CD95 cytoplasmic region. This mutation generates a receptor in which the ability to transduce an apoptotic signal is blocked.9 In a related human condition, autoimmune lymphoproliferative syndrome (ALPS),10 ALPS type Ia patients carry dominant-negative mutations in CD95 and type Ib patients have mutations in CD95L, resembling mice with lprcg and gld mutations, respectively.

Canonical Signaling of CD95 in Cancer

CD95 is predominantly located at the cell surface, where it has been shown to pre-associate in homotrimers.11 Similar to all death receptors, CD95 carries a conserved stretch of 80 amino acids in its cytoplasmic tail, the death domain (DD), that is essential for apoptosis initiation.1, 12, 13 Upon binding of CD95L, the CD95 DD assembles the death-inducing signaling complex (DISC) composed of CD95, the adaptor molecule FADD (Fas-associated with a death domain), procaspase-8, procaspase-10, and the caspase-8/10 regulator c-FLIP.13 Activated caspase-8 then initiates the apoptotic program by cleaving various intracellular proteins resulting in the execution of apoptosis.14 Likely, the most established proapoptotic activity of CD95 is to mediate the apoptotic death of either virus-infected or cancer cells when engaged by a CD8+ cytotoxic lymphocyte (CTL; Figure 1). In addition to the perforin/granzyme pathway15 and some indirect mechanisms involving cytokines such as tumor necrosis factor-α (TNFα) and interferon-γ,16, 17, 18, 19, 20, 21, 22, 23 CD95/CD95L is a direct major system that both CTLs as well as CD4+ cytolytic effector T cells use to eliminate neoplastically transformed cells.24, 25, 26, 27, 28, 29 CD95 can also mediate receptor interacting protein (RIP)-1-dependent necroptosis under circumstances of caspase inhibition or knockdown of TRAF2.30, 31 However, the physiological relevance of this activity for cancer has not been established. Expression of CD95 and CD95L by cancer cells implies that they are themselves resistant to CD95-mediated apoptosis. Indeed, most cancer cells are relatively resistant to CD95-induced apoptosis even with high levels of CD95 at the surface of the cells.32 Cancer cells have multiple ways of becoming resistant to a possible apoptotic insult mediated by CD95. A common mechanism used by the cells is to regulate cell surface expression of the receptor.33, 34 The CD95 apoptotic signal can also be inhibited at the level of the DISC via increased expression of cFLIP (cellular FLICE inhibitory protein), which can inhibit the interactions of caspase-8 and -10 with the DISC,35 or via reduced expression of FADD36 or caspase-8.37, 38 Loss of apoptosis signaling through CD95 can also be the consequence of deregulation of the expression of the Bcl-2 family proteins or inhibitor of apoptosis proteins, thereby favoring tumor survival.39

Figure 1
figure 1

The canonical apoptosis-inducing function of the CD95/CD95L system in killing cancer cells. Cancer cells that are recognized by CTLs in an antigen-specific way are being attacked by direct mechanisms: release of perforin/granzyme (shown as GrB) or use of CD95L to engage CD95 on the surface of cancer cells. Alternatively, indirect mechanisms are activated that result in upregulation of cytokines such as TNFα and INFγ, which in turn cause upregulation of CD95 and MHC-I (by IFNγ) or induction of cell death through cancer-expressed TNF receptors (by TNFα). Ag, antigen; CTL, cytotoxic lymphocyte; GrB, granzyme B; IFNγ, interferon γ; MHC-I, major histocompatibility complex I; TCR, T-cell receptor; TNFα, tumor necrosis factor α

Other Activities of the Apoptosis-inducing Receptor CD95

In addition to the activities of CD95 and CD95L in mediating apoptosis induction, mostly in the contest of an immune response,1, 2, 3 it is now established that CD95 has multiple nonapoptotic activities.40, 41, 42, 43 For example, CD95 is required for efficient liver regeneration following partial hepatectomy;44, 45 CD95 activation stimulates renal tubular epithelial cell migration by a β8 integrin-dependent mechanism,46 and CD95 provides a mitogenic signal in quiescent hepatic stellate cells through activating epidermal growth factor receptor (EGFR).47 CD95 is also important for neurite outgrowth.48, 49 CD95 and CD95L have additional, cancer-relevant, activities. We have identified at least five cancer-relevant activities of CD95 that could be targeted for cancer therapy, and one (apoptosis induction through CD95) that should not be (Figure 2).

Figure 2
figure 2

Graphical summary of the role of CD95/CD95L in cancer. Together with the tumor-suppressing ability to trigger apoptosis in (apoptosis sensitive) cancer cells (1), CD95L has a range of tumor-promoting activities, some of which are indirect, such as the suppression of the immune response in the cancer micro-environment by either tumor-generated CD95L (2) or by CD95L expressed by endothelial cells (3), and some of which are direct, such as the promotion of tumor growth and invasiveness (4) or the acquisition of a CSC phenotype (5). Importantly, a low-baseline level of CD95/CD95L signaling is required for survival of cancer cells. Elimination of CD95/CD95L signaling leads to an irreversible and effective type of cell death, DICE, which predominantly affects CSCs (6). CSC, cancer stem cell, CTL; cytotoxic T lymphocyte; IL-10, interleukin 10; EMT, epithelial-to-mesenchymal transition; PGE2, prostaglandin E2; TIL, tumor-infiltrating lymphocyte; VEGF-A, vascular endothelial growth factor A. Stippled arrows indicate hypothetical interactions

Apoptosis induction through CD95

Apoptosis induction is the most well-established activity of CD95, documented by thousands of publications and summarized in numerous review articles (e.g., Nagata,1 Peter and Krammer,13 and Nagata50). In the context of cancer, it is relevant that CD95L is one of only a few molecules that immune cells use to activate apoptosis to kill cancer cells (Figure 1).51 Apoptosis induction as a cancer cell killing strategy is presumed to be accomplished by tumor-infiltrating lymphocytes expressing CD95L (Figure 2-1, apoptosis). Apoptosis induction in cancer cells through CD95 is the only scenario in which recombinant CD95L could be used for cancer therapy. However, given the fact that almost all established cancers express CD95, and the fact that most cancer cells are resistant to apoptosis induction, we would suggest that stimulating CD95 on cancer cells may not be an effective approach to killing cancer cells. In addition, stimulation of CD95 could never be used therapeutically because of major side effects such as massive apoptosis induction in the liver.52 Based on recent data, we propose that inhibiting the activity of CD95L or targeting CD95L mRNA may be more effective for cancer therapy than using CD95L to induce apoptosis in cancer cells:

The tumor strikes back

It has been demonstrated a number of times that expression of CD95L by apoptosis-resistant tumor cells enables a powerful ‘counterattack’ against antitumor immune effector cells, such as cytotoxic killer cells, many of which are themselves sensitive to CD95L-mediated apoptosis53, 54, 55 (Figure 2-2, tumor counterattack). However, while there is some evidence for the occurrence of this counterattack, its existence remains controversial.56 The reported increased concentration of soluble CD95L (sCD95L) in the serum of many cancer patients was often interpreted in the context of the CD95L counterattack theory (Table 1). Upregulation of CD95L in patient sera would suggest a possible immunosuppressive role for this molecule. However, the generalized immune suppression that would be expected from this situation could not be confirmed in cancer patients; thus, it may be that the increase in CD95L expression in tumor tissues has a more direct role in tumor progression.

Table 1 Tumor-promoting activities of CD95 and CD95L in clinical studies

The tumor endothelium expresses CD95L

Recently, the tumor strikes back concept was rediscovered in a different form. CD95L is expressed on the tumor endothelium in mice and humans57, 58 (Figure 2-3, endothelial cell barrier). CD95L was reported to be expressed by tumor epithelium of various human solid cancers but not by normal endothelial cells.59 Tumor cells were found to cause upregulation of membrane-bound (m)CD95L on endothelial cells through the action of interleukin 10, prostaglandin E2, and vascular endothelial growth factor A. Interestingly, mCD95L only induced apoptosis of effector killer T cells but not of regulatory T cells, which were found to be protected by expression of a number of antiapoptotic proteins including cFLIP, Bcl-2, and Bcl-xL. This finding was supported by a syngeneic in vivo mouse model of ovarian cancer, in which it was demonstrated that expression of CD95L on endothelial cells causes reduced CD8 T-cell infiltration into the tumor. Finally, it was shown that mice treated with a neutralizing anti-CD95L antibody show increased infiltration of adoptively transferred tumor vaccine-primed CD8 T cells.59 These data suggest that inhibiting endothelial CD95L expression could be a new therapeutic strategy to enhance the potency of adoptive transfer of antitumor T cells.

The tumor-promoting activities of CD95

Although the concept of inducing apoptosis in cancer cells using death ligands such as CD95L was intriguing, it was unlikely that the only function of CD95 was to induce apoptosis. As early as 1993,60 it was recognized that CD95 also induces proliferation in various cell types such as T cells, liver cells, and neurons.45, 48, 49, 61, 62, 63 In 2004, we reported that stimulation of CD95 on 22 apoptosis-resistant cancer cell lines increases their motility and invasiveness in vitro.64 In a study with cells from ALPS patients, as well as cellular and mouse model systems, we demonstrated that nonapoptotic signaling through CD95 involved activation of NF-κB and the three MAP kinases, Erk1/2, JNK1/2, and p38.64, 65, 66, 67 In addition, we demonstrated in various cancer cell lines that CD95-mediated invasiveness requires activation of NF-κB and ERK, and involves active caspase-8 and urokinase plasminogen activator.64 It is now widely accepted that once cancer cells acquire resistance to CD95-mediated apoptosis, further stimulation of CD95 is tumorigenic (Figure 2-4, invasiveness and growth).64, 68, 69, 70, 71, 72, 73, 74, 75 CD95L is expressed in two flavors, a membrane-bound form and a soluble form that is generated through cleavage of mCD95L by metalloproteinases.76, 77 mCD95L in vivo is essential for apoptosis induction, whereas sCD95L has nonapoptotic activities and may be the predominant tumor-promoting activity in vivo.78 The concept that CD95 can be a tumor promoter has now gained wide acceptance, supported by a number of reports describing marked activities of CD95 in tumor growth and spread (Table 2).

Table 2 Tumor-promoting activities of CD95 and CD95L in vitro and in mouse models

CD95 is coupled to multiple potentially tumorigenic signaling pathways. CD95 was identified in a small hairpin RNA (shRNA) screen as a modifier that renders human lung adenocarcinomas resistant to EGFR tyrosine kinase inhibitors through activation of NF-κB.79 Others have demonstrated that CD95 mediates invasion via the Src/PI3K/GSK3β/MMP (matrix metalloproteinase) pathway;74, 80 however, the transactivation of tyrosine kinases by CD95 is incompletely understood. In colon cancer, it was shown that activated CD95 promotes the formation of cell protrusions through a new signaling pathway involving platelet-derived growth factor receptor-beta mediated phospholipase C-γ activation and phosphatidylinositol (4,5)-bisphosphate hydrolysis.81 The subsequent release of cofilin from the plasma membrane and the continued suppression of LIMK1 by Kras/RAF1 together allow robust activation of the cofilin pathway. Cofilin activation was shown to be required for CD95-stimulated formation of membrane protrusions and increased tumor cell invasion. Recently, metalloproteinase-cleaved CD95L was reported to trigger a motility-inducing signaling complex formation in triple-negative breast cancer cells.82 Most recently, it was shown that CD95-mediated activation of Sck/Shc2 is indispensable for cell cycle progression of metastatic pancreatic ductal adenocarcinoma (PDAC).83 These data suggest that CD95 is connected to a myriad of prosurvival and migratory signaling pathways.

We recently tested the relevance of these nonapoptotic functions of CD95 and CD95L for cancer cells. We knocked down either CD95 or CD95L in numerous cancer cell lines using multiple small interfering RNA (siRNAs) and shRNAs. This resulted in a profound reduction in growth of the cancer cells.44 In addition, we generated tissue-specific knockout mice lacking CD95 expression in the liver or on the surface epithelial cells of the ovaries. Using appropriate tumor mouse models, we found a severe reduction in liver cancer in mice lacking CD95 in hepatocytes (diethylnitrosamine injection model), and mice lacking CD95 in the ovaries barely developed cancer at all (using the KrasD12G/pten−/− endometrioid ovarian cancer model84). Finally, it was shown that mice that only express soluble but not mCD95L suffer from large histiocytic sarcomas in the liver,78 likely owing to a lack of apoptosis induction and a tumorigenic activity of CD95L.

A number of studies reported CD95 as a positive prognostic marker for cancer.85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 This is likely owing to the fact that CD95 is often downregulated during tumor progression because cancer cells need to lower the risk of undergoing apoptosis while benefiting from CD95’s tumorigenic activities. Occasionally, CD95L was also described as a positive prognostic marker for cancer.86, 100, 101 However, the vast majority of reports have shown that disease progression is associated with progressively increased expression of CD95L and sometimes also CD95, and expression of both CD95 and especially of CD95L in most cases act as negative prognostic markers for many cancers (Table 1). In summary, most studies suggest that CD95 and/or CD95L expression promotes tumor growth and favors the establishment of tumor metastases.

Maintenance of CSCs by CD95 and CD95L

The cancer stem cell (CSC) model is an attractive hypothesis that translates properties of normal stem cells into the cancer field, and explains some of the most lethal features of cancers. The CSC model proposes that the cells within a tumor are hierarchically organized, and it predicts the existence of a subpopulation of cells with high tumorigenicity that are able to both self–renew and to generate differentiated cells (non-CSCs).102, 103 One of the most malignant features of cancer is the appearance of relapses, sometimes years after radiotherapy or chemotherapeutical intervention, and this has been related to the occurrence of cells with the CSC phenotype.104 Therefore, elucidating the mechanisms of CSC maintenance is important for understanding tumor cell persistence and relapse, and may enable specific targeting of CSCs, a promising therapeutic strategy to stably eradicate cancer.105, 106

CD95 and CD95 signaling have been connected to normal stem cells.45, 107 CD95 was, in fact, previously identified as a candidate stem cell marker (along with well-established stem cell markers such as Lin28, Oct4, Nanog, and Sox2, among others) in a serial analysis of gene expression profiling of human embryonic stem cells.108 Functional evidence of a prosurvival function of CD95 and CD95L signaling in normal stem cells came from experiments that showed that the stimulation of CD95 signaling in neuronal stem cells did not cause death, but rather increased the survival of neuronal stem cells via a Src/PI3K/AKT/mTOR signaling pathway, while, conversely, deletion of CD95 resulted in reduced neurogenesis.107 Because normal stem cells are often the origin of CSCs, these data were suggestive that CD95 may also have a nonapoptotic function in CSCs.

In the context of cancer, CD95 expression and CD95 signaling have been connected with the differentiation of cells. We reported this based on an analysis of the NCI-60 panel of cancer cells, which could be divided in two super-clusters with distinct differentiation stages that responded differently to CD95 stimulation.109 Interestingly, expression of CD95 inversely correlated with expression of the stem cell-inhibiting members of the let-7 family of micro RNAs (miRNAs), and stimulation of CD95 caused a reduction in let-7 expression.110 Moreover, and related to this, CD95 has been shown to be capable of inducing the epithelial-to-mesenchymal transition (EMT) differentiation program in gastrointestinal cancer111, 112 (Figure 2-5, EMT and CSC maintenance). In these studies, the authors demonstrated that CD95 signaling inactivates GSK3β by ERK/mitogen-activated protein kinase signaling resulting in increased nuclear import and interaction between AP-1 and NFAT4. This increases their transcriptional activity leading to nuclear accumulation of Snail and β-catenin and miR-23a expression, and subsequently, downregulation of E-cadherin and upregulation of MMP9 and vimentin in vivo and in vitro.111, 113 EMT has been previously connected with the generation of cells with the properties of CSCs.114

We recently demonstrated that CD95 is required for the survival of CSCs and allows new CSCs to emerge115 (Figure 2-5, EMT and CSC maintenance). Stimulation of CD95 on multiple tumor cells induced a conversion from non-CSCs to CSCs. This reprogramming activity of CD95 was independent of its apoptosis-inducing function, as it was not blocked by the pan-caspase inhibitor zVAD-fmk; rather, it represents a mechanism of retro-differentiation. Strikingly, CSCs from highly apoptosis-sensitive HeyA8 ovarian cancer cells enriched in tumor spheres were found to be almost completely resistant to CD95-mediated apoptosis. For breast cancer, we could connect this novel function of CD95/CD95L to the activity of miR-200, a miRNA previously linked to both EMT and CSCs.114, 116, 117 miR-200c expression increased the sensitivity of cancer cells to CD95-mediated apoptosis.118 Stimulation of CD95 not only increased the number of cancer cells with stem cell traits but also prevented differentiation of CSCs, suggesting that CD95 expression on cancer cells maintains the CSC pool.115 A connection between CD95 and CSCs was recently also reported for PDAC.83 CD95 expression strongly correlated with stemness and EMT markers and blocking CD95L reduced tumor growth and metastasis in vivo.

Death induced by CD95R/L elimination

Following up on our finding that CD95 contributes to the proliferation of cancer cells,44 we recently reported that the elimination of either CD95 or CD95L kills cancer cells (in vitro and in vivo) in a process we termed DICE (death induced by CD95 or CD95L elimination)119 (Figure 2-6, DICE). This activity of CD95 as a survival factor seems to be mostly relevant to cancer cells, as none of the normal tissues during embryonic development in either CD95 or CD95L knockout mice showed a growth defect or signs of cell death.6, 7, 8 Consistently, we found increased sensitivity to DICE in ovarian surface epithelial cells after they were immortalized by expression of hTERT.119

We found that all cancer cells tested (~40 lines tested to date) substantially die by DICE when either CD95 or CD95L is knocked down. We used 15 different non-overlapping si/shRNAs against either of the two genes, and all induce DICE. We generated Tet-inducible vectors (pTIP) to express the shRNAs. They kill all cancer cells when doxycycline is added. In two ovarian cancer mouse models and one mouse model of chemically induced liver cancer, tumor formation was severely reduced in the absence of CD95.44, 119 In fact, a reanalysis of the tumor samples revealed that not a single cancer cell could be detected in any of the models that had deleted both alleles of CD95.119 We reported that DICE has the following properties:119

  1. 1

    DICE represents a necrotic form of mitotic catastrophe with signs of apoptosis,119 autophagy, and senescence (unpublished data).

  2. 2

    DICE is characterized by cell swelling and production of reactive oxygen species followed by DNA damage and activation of caspase-2, resulting in mitochondrial outer membrane permeabilization. Cells eventually die by a RIP1/mixed lineage-like kinase-independent mechanism. Although multiple cell death pathways are activated, RIPK-dependent necroptosis does not seem to be critical, suggesting that DICE induction may not cause inflammation.

  3. 3

    DICE could not be inhibited by any of 1200 tested drugs or by knockdown of any single gene in a genome-wide shRNA screen,119 suggesting that it is a robust cell death mechanism that is difficult to block.

We recently postulated that DICE is a fail-safe mechanism, a dead man’s switch, that prevents the survival of cancer cells that are devoid of CD95, and, hence, would not be eliminated by the immune system through CD95L/CD95-mediated apoptosis.120 Thus, DICE is a naturally occurring antitumor defense mechanism. The observation that in tumor cells both alleles of CD95 are rarely if ever mutated or deleted (reviewed in Peter et al.41) is consistent with this interpretation. Our recent data show that all cancer cells autonomously produce a small amount of CD95L, suggesting that the loss of either CD95 or CD95L induces DICE, which is consistent with our observation that cancer cells never delete both alleles of CD95.119

Our study of CSCs revealed a crucial role for CD95 signaling in regulating cancer differentiation, and indicated that the two death mechanisms, DICE and canonical CD95-mediated apoptosis, have opposing roles in eliminating CSCs and non-CSCs. Conversion of non-CSCs to CSCs resulted in a loss of sensitivity to CD95-mediated apoptosis and a concomitant increase in the sensitivity of the cells to DICE.115 In fact, we found that DICE preferentially targets CSCs.115 When DICE was induced in multiple cancer cell lines or primary breast cancer cells, they became depleted of CSCs. Cells lost typical CSC surface markers, formed spheres less efficiently, and lost expression of endogenous CSC markers while becoming enriched in the stem cell-controlling miRNA miR-200c.

Targeting CD95L to Kill Cancer Cells

The data summarized above suggest that CD95 and CD95L act as oncogenes once cancer cells have become resistant to the apoptosis-inducing activity of CD95. The data further seem to suggest that the reason that cancer cells die after removal of either CD95 or CD95L is that they are addicted to their oncogenic activities. However, for the following reasons, we would argue that DICE is not the result of a broken oncogene addiction: (1) CD95 and CD95L intrinsically have tumor-suppressive activities in the context of the immune system (see above). (2) Elimination of CD95 or CD95L can kill any cancer cell we have tested, not just cells that overexpress CD95 or CD95L. In fact, CD95L expression in most cancer cells is barely detectable, yet elimination of CD95L induces DICE more effectively in cells that express less CD95L, perhaps because CD95L becomes rate limiting more easily. CD95 and CD95L may be the first identified tumor-suppressive genes that are so important that their loss (which could occur as neoplastically transformed cells continue to acquire mutations) triggers a fail-safe program to kill such cells. An interesting aspect of this model is that, by definition, the DICE mechanism has not been triggered in any cancer cell found in a cancer patient, the implication being that cancer cells do not become resistant to DICE, but they become resistant to apoptosis and may evade DICE by retaining expression of CD95 and CD95L.

Because neither CD95 nor CD95L knockout mice are known to exhibit any defects in the proliferation of any tissue and exhibit no defects in stem cell compartments,6, 7, 8 it is possible that CD95 or CD95L could be safely targeted for therapeutic purposes. Targeting CD95L systemically would block all the tumorigenic activities summarized in Figure 2.

Inducing DICE in Combination with Standard Chemotherapy

Although induction of DICE alone may be effective in killing cancer cells, the combination of induction of DICE with existing therapies and concepts may be beneficial in improving outcomes of cancer therapy. During our analysis of the role of CD95 in CSCs, we identified a strong synergy between DICE and CD95-mediated apoptosis in eradicating cancer.115 The synergy is a direct consequence of the differential sensitivities of CSCs and non-CSCs to the two death mechanisms. Thus, a therapy that combined the two death mechanisms could be beneficial to cancer treatment by targeting two differentiation stages of cancer development. It has been reported multiple times that many forms of chemotherapy act by inducing, at least in part, apoptosis in cancer cells, sometimes through upregulation of CD95L.121, 122 It is also established that cancer patients who become refractory to therapy have an increased CSC population,123, 124 which we recently showed to be more sensitive to DICE than non-CSCs. Thus, a combination of low-dose chemotherapy coupled with targeting CD95 may be beneficial as it should target both non-CSCs and CSCs. Targeting of CD95L could also be a beneficial addition to chemotherapy because chemotherapy-induced upregulation of CD95L has been suggested to not only drive cancer cells into apoptosis but to promote growth of drug resistant tumor cells.125

Inducing DICE in Combination with Inhibition of Immune Checkpoint Receptors

An effective mechanism to treat certain cancers involves the mobilization of the immune system. Cancer cells have found ways to suppress the antitumor response mounted by the immune system, but recent successes of therapies that are aimed toward de-repressing the tumor-imposed block on the immune system are evidence of the power of these mechanisms. Anti-PDL1 and anti-PD-1 clinical trials have shown promising effects in melanoma, renal, colorectal, and non-small cell lung cancer patients, and, for the first time ever in the development of immune therapy, a sizeable fraction of patients were observed who had a durable response that increased their life span.126, 127, 128, 129 Based on these early data, one can predict that success in cancer therapy will come from harnessing natural mechanisms that control cancer in general (e.g., an antitumor immune response) rather than from cancer-specific strategies. Empowering the immune system by targeting immune check point signaling and simultaneously attacking the cancer cells by inducing DICE may represent a viable combination of therapies both of which activate preexisting antitumor mechanisms.

Conclusions and Perspectives

Using CD95L for cancer therapy was never a viable option to treat cancer because of its devastating effects on the liver. Accumulating evidence now suggests that cancer cells can never lose CD95 or CD95L and if they do, they die. This provides an opportunity to use targeting either CD95 or CD95L to treat cancer. However, there are many open questions that need to be addressed first. Although excess of CD95L secreted by tumor cells may drive EMT and stemness and render tumor cells more motile and invasive, it is not clear whether targeting this secreted protein will be enough to block the tumor-promoting activities of CD95 and CD95L. In our hands, blocking the activity of extracellular CD95L has had no effect on cell viability.119 In addition, recent data suggest that both CD95L and CD95 are located in intracellular stores,119 hence they may exert their activity to protect cancer cells from DICE from within the cell. It may therefore be necessary to target the intracellular proteins or the mRNAs of CD95 and CD95L. This will require development of more efficient ways to deliver siRNAs to cells or the development of new technologies to eliminate genes from cells all together (i.e., using the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system).