Abstract
Retinoids and their naturally metabolized and synthetic products (e.g., all-trans retinoic acid, 13-cis retinoic acid, bexarotene) induce differentiation in various cell types. Retinoids exert their actions mainly through binding to the nuclear retinoic acid receptors (α, β, γ), which are transcriptional and homeostatic regulators with functions that are often compromised early in neoplastic transformation. The retinoids have been investigated extensively for their use in cancer prevention and treatment. Success has been achieved with their use in the treatment of subtypes of leukemia harboring chromosomal translocations. Promising results have been observed in the breast cancer prevention setting, where fenretinide prevention trials have provided a strong rationale for further investigation in young women at high risk for breast cancer. Ongoing phase III randomized trials investigating retinoids in combination with chemotherapy in non–small cell lung cancer aim to definitively characterize the role of retinoids in this tumor type. The limited treatment success observed to date in the prevention and treatment of solid tumors may relate to the frequent epigenetic silencing of RARβ. Robust evaluation of RARβ and downstream genes may permit optimized use of retinoids in the solid tumor arena. Clin Cancer Res; 19(7); 1651–9. ©2013 AACR.
Background
Vitamin A is derived from animal and plant food sources and has critical functions in many aspects of human biology. Its natural derivatives and metabolized products (retinoids) such as β-carotene, retinol, retinal, isotetrinoin, all-trans retinoic acid (ATRA), 9-cis retinoic acid, and 13-cis retinoic acid have important roles in cell differentiation, growth, and apoptosis (1). Synthetic retinoids are also available and include bexarotene and fenretinide. In clinical practice, retinoids have a wide range of dermatologic indications including for psoriasis, acneiform, and keratinization disorders (2). Systemic retinoids are approved by the U.S. Food and Drug Administration (FDA) for the treatment of cutaneous T-cell lymphoma (3) and acute promyelocytic leukemia (APL; refs. 4, 5). However, the chemopreventive and therapeutic effects of retinoids in solid tumors remain controversial. Therefore, an overview of the research to date and future directions in this area is the focus of this review.
Retinoic acid and the retinoic acid receptor pathway
Retinoic acids (RA) exert their functions through their specific receptors. The 2 distinct classes of receptors are retinoic acid receptors (RAR) and retinoic X receptors (RXR). Each class contains 3 different subtypes—α, β, and γ (6). ATRA and fenretinide can bind specifically to RARS, 13-cis RA and bexarotene only to RXRS, and 9-cis RA to RARS or RXRS (refs. 1, 5; Table 1). The expression of these receptors is regulated by the receptors themselves, other nuclear receptors such as ERα, or by other subtypes in the same family (5, 7). Upon the binding of ligands, RARs and RXRs form heterodimers and function as ligand-dependent transcription factors to activate their downstream effectors by binding to the retinoic acid response elements (RARE) located in the 5′-region of RA downstream genes (5). The above model of RAR or RXR function via binding to RARE is considered the RA classical or genomic pathway. Activation of the classical pathway will trigger cell differentiation, cell arrest, and eventual apoptosis (8).
Select clinical trials evaluating retinoids in solid tumors
| Retinoid | Other names | Target | Clinical trial setting | Dose and schedule (ref.) | Study outcome | Biomarker evaluation |
|---|---|---|---|---|---|---|
| ATRA | Tretinoin | RAR | Advanced NSCLC Phase II randomized (n = 107) | 20 mg/m2/d commencing 1 wk pre-paclitaxel/cisplatin every 3 wk (50) | RR (55.8% vs. 25.4%) and median PFS (8.9 vs. 6 months) favored the ATRA arm Phase III study pending | No significant association between RAR-β2 expression and response rate detected (n = 60) |
| Metastatic breast cancerPhase II single arm (n = 17) | 45 mg/m2/d for 4 d commencing 2 d preweekly paclitaxel (PMID 20596747) | Clinical benefit rate of 76.4% Note that the majority of patients had not received prior paclitaxel | ||||
| 13-cis RA | IsotretinoinRoaccutaneAccutane | RXR | Primary prevention: H+N cancer | Induction phase: high dose (1.5 mg/kg) for 3 mo; maintenance phase: low dose (0.5 mg/kg/d) vs. β-carotene (30 mg/d) for 9 mo (30) | Induction phase (n = 66): RR 55%; maintenance phase (n = 53): response or stable disease 92% (n = 22, isotretinoin) vs. 45% (n = 13, β-carotene) | |
| Advanced solid tumorsPhase I (n = 13) | 1 mg/kg twice daily 3 wk of 4 with MS-275 (69) | Antitumor activity observed and recommended phase II dose determined | ||||
| Metastatic breast cancer Phase II randomized (n = 99) | 1 mg/kg/d + tamoxifen 20 mg/m2 vs. tamoxifen alone vs. tamoxifen + IFN-α-2a 3 MU 3 times weekly IM (62) | No significant difference in RR or overall survival between the 3 arms | ||||
| 9-cis RA | Alitretinoin | RAR RXR | Metastatic breast cancerPhase I (n = 12) | 70 mg/m2/d + 20 mg/d tamoxifen (PMID 11352969) | Antitumor activity observed and recommended phase II dose determined | |
| Fenretinide | 4-OH Phenylretinamide | RAR | Primary prevention: women at high risk of breast cancer Randomized double-blind 2 × 2 design (n = 235) | Tamoxifen 5 mg/d vs. fenretinide 200 mg/d vs. the combination vs. placebo (58) | Low-dose tamoxifen plus fenretinide did not reduce breast cancer events vs. placebo; numerical reduction in annual odds of breast cancer observed with both single-agent tamoxifen and fenretinide | |
| Secondary prevention: early breast cancerPhase III randomized (n = 2,867) | 200 mg/d oral for 5 y with 3 d off every month vs. observation (55) | No difference in rates of breast cancer in overall population, but 35% reduction in events in premenopausal women in unplanned exploratory analysis | Baseline IGF-I/mammographic density, as well as change in mammographic density did not predict breast cancer events | |||
| Bexarotene | RXR | Chemotherapy-naïve advanced NSCLCPhase III randomized (n = 623) | 400 mg/m2/d every 4 wk with cisplatin/vinorelbine (52) | No survival benefit for addition of bexarotene to chemotherapy | Grade 3/4 hypertriglyceridemia associated with longer median survival in an unplanned subgroup analysis | |
| Metastatic breast cancer Phase II single arm (n = 148) | 200 mg/m2 oral daily (61) | Clinical benefit rate (complete response, partial response, and stable disease >6 mo) of ∼20% |
| Retinoid | Other names | Target | Clinical trial setting | Dose and schedule (ref.) | Study outcome | Biomarker evaluation |
|---|---|---|---|---|---|---|
| ATRA | Tretinoin | RAR | Advanced NSCLC Phase II randomized (n = 107) | 20 mg/m2/d commencing 1 wk pre-paclitaxel/cisplatin every 3 wk (50) | RR (55.8% vs. 25.4%) and median PFS (8.9 vs. 6 months) favored the ATRA arm Phase III study pending | No significant association between RAR-β2 expression and response rate detected (n = 60) |
| Metastatic breast cancerPhase II single arm (n = 17) | 45 mg/m2/d for 4 d commencing 2 d preweekly paclitaxel (PMID 20596747) | Clinical benefit rate of 76.4% Note that the majority of patients had not received prior paclitaxel | ||||
| 13-cis RA | IsotretinoinRoaccutaneAccutane | RXR | Primary prevention: H+N cancer | Induction phase: high dose (1.5 mg/kg) for 3 mo; maintenance phase: low dose (0.5 mg/kg/d) vs. β-carotene (30 mg/d) for 9 mo (30) | Induction phase (n = 66): RR 55%; maintenance phase (n = 53): response or stable disease 92% (n = 22, isotretinoin) vs. 45% (n = 13, β-carotene) | |
| Advanced solid tumorsPhase I (n = 13) | 1 mg/kg twice daily 3 wk of 4 with MS-275 (69) | Antitumor activity observed and recommended phase II dose determined | ||||
| Metastatic breast cancer Phase II randomized (n = 99) | 1 mg/kg/d + tamoxifen 20 mg/m2 vs. tamoxifen alone vs. tamoxifen + IFN-α-2a 3 MU 3 times weekly IM (62) | No significant difference in RR or overall survival between the 3 arms | ||||
| 9-cis RA | Alitretinoin | RAR RXR | Metastatic breast cancerPhase I (n = 12) | 70 mg/m2/d + 20 mg/d tamoxifen (PMID 11352969) | Antitumor activity observed and recommended phase II dose determined | |
| Fenretinide | 4-OH Phenylretinamide | RAR | Primary prevention: women at high risk of breast cancer Randomized double-blind 2 × 2 design (n = 235) | Tamoxifen 5 mg/d vs. fenretinide 200 mg/d vs. the combination vs. placebo (58) | Low-dose tamoxifen plus fenretinide did not reduce breast cancer events vs. placebo; numerical reduction in annual odds of breast cancer observed with both single-agent tamoxifen and fenretinide | |
| Secondary prevention: early breast cancerPhase III randomized (n = 2,867) | 200 mg/d oral for 5 y with 3 d off every month vs. observation (55) | No difference in rates of breast cancer in overall population, but 35% reduction in events in premenopausal women in unplanned exploratory analysis | Baseline IGF-I/mammographic density, as well as change in mammographic density did not predict breast cancer events | |||
| Bexarotene | RXR | Chemotherapy-naïve advanced NSCLCPhase III randomized (n = 623) | 400 mg/m2/d every 4 wk with cisplatin/vinorelbine (52) | No survival benefit for addition of bexarotene to chemotherapy | Grade 3/4 hypertriglyceridemia associated with longer median survival in an unplanned subgroup analysis | |
| Metastatic breast cancer Phase II single arm (n = 148) | 200 mg/m2 oral daily (61) | Clinical benefit rate (complete response, partial response, and stable disease >6 mo) of ∼20% |
Abbreviations: H+N, head and neck; IM, intramuscular; MS-275, entinostat; MU, million units; PFS, progression-free survival; RR, response rate.
The function of RA and its receptors involves not only the classical pathway but also multiple other important pathways. RAs have been shown to regulate NF-κB (9), IFN-γ (10), TGF-β (11), VEGF (12), mitogen-activated protein kinase (MAPK; ref. 13), and chromatin remodeling (14). Furthermore, RARs and RXRs can form heterodimers with other types of receptors, including the estrogen receptor-α (ERα; refs. 7, 15), AP-1 receptor (16), peroxisome proliferator-activated receptor (PPAR; ref. 17), liver X receptors (LXR; refs. 18, 19), and vitamin D receptor (VDR; ref. 20; Fig. 1). When RARs/RXRs heterodimerize with these receptors, they are involved in regulating their partner receptor's pathways, referred to as nonclassical or nongenomic pathways (5). Interestingly, these pathways often regulate processes that have functions opposite to the classical pathway. For example, a study has shown that RA activation of the PPARβ/δ pathway resulted in upregulation of prosurvival genes (17), contrary to the known differentiation function of RARs and RXRs in response to RA. The function of RAs, which involves nongenomic pathways, may provide opportunities for cancer cells to develop resistance to RA treatment, discussed later in this review. Another important function of RARA is the regulation of stem cell differentiation (11). RAs target stem cells via both genomic and nongenomic pathways such as the Notch pathway and inflammation (10, 11). In summary, RAs and their receptors play important roles as regulators of critical processes in cells.
The RARs and their action. In a series of enzymatic steps, vitamin A (retinol) is metabolized through the oxidizing action of retinaldehyde (RDH) to retinal, and by retinaldehyde dehydrogenase (RALDH), to RA. RA has 3 different isomers: all-trans, 9-cis, and 13-cis RA. RA is transported to the nucleus by the protein cellular RA–binding protein (CRABP) and delivered to the RARα. RARα heterodimerizes with and binds to RARE present most often in gene promoters. In the classical pathway of RA action, RA binds to dimers of RARα and RXRs (α, β, or γ) to induce expression of its downstream target genes, including RARβ. Upon activation, RARβ can regulate its own expression and that of its downstream genes, the function of which is mainly to inhibit cell growth. Alternatively, RA can be bound and transported to the nucleus by other factors such as FABP5. This delivers RA to other nonclassical receptors such as PPARβ/δ and ERα which activate nongenomic pathways such as PDK-1/Akt or the ERα pathway. Contrary to the differentiation functions attributed to the classical pathway, the nongenomic pathways exert strong antiapoptotic and proliferative effects on cancer cells. It is believed that the classical and nongenomic pathways are controlled by the relative abundance of their own ligands. RA has a stronger affinity for RARs than for the other receptors, and the classical pathway plays a dominant role over the nongenomic pathways. Thus, if RA is present with other ligands such as estrogen, signaling through the classical pathway is preferred to result in cell differentiation and growth inhibition.
The RARs and their action. In a series of enzymatic steps, vitamin A (retinol) is metabolized through the oxidizing action of retinaldehyde (RDH) to retinal, and by retinaldehyde dehydrogenase (RALDH), to RA. RA has 3 different isomers: all-trans, 9-cis, and 13-cis RA. RA is transported to the nucleus by the protein cellular RA–binding protein (CRABP) and delivered to the RARα. RARα heterodimerizes with and binds to RARE present most often in gene promoters. In the classical pathway of RA action, RA binds to dimers of RARα and RXRs (α, β, or γ) to induce expression of its downstream target genes, including RARβ. Upon activation, RARβ can regulate its own expression and that of its downstream genes, the function of which is mainly to inhibit cell growth. Alternatively, RA can be bound and transported to the nucleus by other factors such as FABP5. This delivers RA to other nonclassical receptors such as PPARβ/δ and ERα which activate nongenomic pathways such as PDK-1/Akt or the ERα pathway. Contrary to the differentiation functions attributed to the classical pathway, the nongenomic pathways exert strong antiapoptotic and proliferative effects on cancer cells. It is believed that the classical and nongenomic pathways are controlled by the relative abundance of their own ligands. RA has a stronger affinity for RARs than for the other receptors, and the classical pathway plays a dominant role over the nongenomic pathways. Thus, if RA is present with other ligands such as estrogen, signaling through the classical pathway is preferred to result in cell differentiation and growth inhibition.
Retinoids and cancer
The retinoids have been investigated extensively for the prevention and treatment of cancer, predominantly because of their ability to induce cellular differentiation and arrest proliferation. RA-regulated tumor suppressor genes, when expressed, can inhibit tumor growth (21). Among the 3 RARs, RARβ has been well known for its tumor-suppressive effects in epithelial cells (5, 8, 22). Exogenous expression of the RARβ gene can cause RA-dependent and -independent apoptosis and growth arrest (23). RARβ-induced growth arrest and apoptosis is mediated through RARα (24). As RA ligand-bound RARα binds to the RARE on the RARβ promoter, multiple activator proteins assemble at the site and result in the upregulation of the RARβ gene (5). The expression of RARβ results in the transactivation and expression of a number of its target genes that mediate cell differentiation and death (5, 6, 8). The ability of ATRA to initiate differentiation of promyelocytic leukemic cells to granulocytes is the basis of the dramatic success of retinoic acid therapy for acute promyelocytic leukemia harboring the RAR/PML translocation (4) and confirms the important role of RARβ in tumor growth inhibition. It is also becoming increasingly clear that RARβ expression is lost early in carcinogenesis or is epigenetically silenced (25) in many solid tumors, providing an opportunity for novel treatment strategies to be investigated using retinoids together with epigenetic modifiers that promote reexpression of silenced genes, described further below.
Clinical–Translational Advances
The retinoids have an established role in the treatment of certain hematologic malignancies, with FDA approval for use in cutaneous T-cell lymphoma and APL. Bexarotene (an RXR-selective retinoid or rexinoid) is associated with an overall response rate of approximately 50% in patients with refractory advanced-stage mycosis fungoides, a cutaneous T-cell lymphoma (3). ATRA, a synthetic retinoid, exhibited improvements in disease-free and overall survival when compared with chemotherapy alone in APL, with long-term remissions occurring in almost 70% of cases (4). The success of retinoids in treating this disease relates to the underlying chromosomal translocation and production of the PML/RARα fusion protein and the ability of retinoids to induce differentiation and inhibition of cell growth in this setting (26, 27). Clinical trials investigating the role of retinoids in the prevention and treatment of solid tumors will now be outlined with a focus on cancers of the upper aerodigestive tract (oropharyngeal and lung) and breast (Table 1).
Head and neck cancer
Premalignant oropharyngeal lesions have been shown to express low levels of RARβ, and it has been hypothesized that restoration of expression could reinstate normal growth and differentiation patterns. Indeed, RARβ mRNA expression was induced with retinoid therapy in specimens of oral mucosa available before and after 13-cis RA (n = 39). The levels of RARβ mRNA increased in the specimens from 18 of the 22 patients who had responses to 13-cis RA and in 8 of the 17 specimens from the patients without responses (P = 0.04), suggesting RARβ mRNA as a biomarker of response to therapy (28). An early randomized trial compared the use of 13-cis RA with placebo in patients with premalignant oral leukoplakia, with a dramatic decrease in the size of the lesions observed in 67% and 10% of patients, respectively. Unfortunately, relapse occurred in the majority of patients within a few months (29). In a follow-up trial, a lower dose of 13-cis RA was significantly more active against leukoplakia than β-carotene and was well tolerated (30). A Cochrane review has subsequently concluded, however, that there is not sufficient evidence currently to support the use of any agent to prevent the progression of oral leukoplakia to oropharyngeal cancer (31). The retinoids have also been evaluated in patients with a diagnosis of localized head and neck cancer after completion of surgery or radiation therapy with little promise overall (32). Because evidence supporting RA's nongenomic action, such as through inhibiting jun N-terminal kinase (JNK) phosphorylation or inhibiting the transactivation potential of NF-κB has been reported in head and neck cancer, this mode of action could have contributed to its limited success in treatment of this type of cancer (9, 33, 34).
Lung cancer
A number of studies have also investigated the role of single-agent retinoids in lung cancer prevention in patients at high risk for lung cancer (primary chemoprevention; refs. 35–39), in those with existing premalignant changes in bronchial epithelium or sputum (secondary chemoprevention; refs. 40–42), and in those with a history of lung cancer (tertiary chemoprevention; refs. 43–45). These studies have not indicated a benefit with use of retinoids in these settings, and indeed an increased risk of lung cancer was observed in smokers in some studies (36, 38). Interestingly, one study was designed to investigate whether either of 2 retinoid-based regimens could reverse RARβ expression loss in former smokers. A statistically significant restoration of RARβ expression and reduction of metaplasia were found in the 9-cis RA group when compared with placebo (46). A recent study revealed a dual growth-promoting and repressive role for RARβ2 in lung cancer cells, which may help explain the inconsistent results observed in clinical trials (47).
On the basis of preclinical observations of the ability of retinoids to enhance chemotherapy-induced cytotoxicity (48, 49), clinical studies have combined retinoids with chemotherapy in the treatment of lung cancer. A randomized phase II study of paclitaxel and cisplatin with or without ATRA was conducted in patients with advanced non–small cell lung cancer (NSCLC, n = 107; ref. 50). Both response rate (55.8% vs. 25.4%) and median progression-free survival (8.9 vs. 6 months) favored the arm incorporating ATRA. An association between RAR-β2 expression and response rate was investigated, but no significant association was identified, perhaps due to the small numbers of tumor samples that expressed the gene (10%, n = 6; ref. 50). On the basis of the promising clinical results, a phase III trial is now in the planning stages and aims to evaluate the benefit of RARβ2 and RARα expression as a response biomarker.
In contrast with these results, a phase III trial of bexarotene in combination with chemotherapy yielded disappointing results despite promising single-agent and phase II data (51). Cisplatin and vinorelbine with or without bexarotene were administered to 623 patients with chemotherapy-naïve advanced NSCLCs. There was no difference in survival (the primary study endpoint) between the arms (52).
Breast cancer
Fenretinide has been extensively studied in breast cancer prevention trials. Supportive preclinical studies revealed its inhibition of mammary carcinogenesis in animal models (53), and the selective accumulation of fenretinide in human breast tissue has been documented (54). The role of fenretinide in reducing contralateral or second ipsilateral breast cancer in patients with early breast cancer (n = 2,867) revealed no significant difference in these endpoints at 8-year follow-up (55). However, an unplanned exploratory analysis indicated a 35% reduction in events in premenopausal women, with a trend toward a detrimental effect being observed in postmenopausal women. These results have prompted a phase III primary prevention trial in premenopausal women at high risk for breast cancer (56).
Efforts to improve on these results have also included a biomarker trial of fenretinide and low-dose tamoxifen in premenopausal women at high risk of breast cancer. Tamoxifen is an approved agent for breast cancer prevention in high-risk individuals. Despite promising preclinical data supporting the combination (57) and its favorable effects on plasma insulin-like growth factor (IGF)-I levels and mammographic density in this clinical trial, the combination of low-dose tamoxifen plus fenretinide did not reduce breast cancer events compared with placebo. A numerical reduction in the annual odds of breast cancer was observed with both single-agent tamoxifen and fenretinide, supporting ongoing investigation of fenretinide in the breast cancer prevention setting (58).
Clinical trials investigating the retinoids as a single agent in metastatic breast cancer have been disappointing. In a phase II trial investigating single-agent 13-cis RA in metastatic breast cancer that was refractory to treatment, no objective responses were observed (59). ATRA administration as a single agent yielded a clinical benefit rate of 26.8% (60). A phase II trial of oral bexarotene (n = 148) in metastatic breast cancer reported a clinical benefit rate of approximately 20% with minimal toxicity observed (61).
Binding of RARs throughout the genome is highly coincident with ERα binding in an ER-dependent manner at ER-binding sites, potentially by maintaining ER–cofactor interactions. These findings suggest that RARs, acting in a nongenomic manner, can cooperate with ERα for effective transcriptional activity in breast cancer cells (7, 15). On the basis of the known interaction between the RARs and the ERα pathways, a clinical trial was conducted in patients with hormone-responsive metastatic breast cancer that investigated the addition of hormonal therapy to retinoids. No benefit to the combination therapy was observed at 8-year follow-up (62). Finally, a phase II single-arm trial of ATRA plus paclitaxel was conducted in patients with metastatic breast cancer (n = 17). Partial response was observed in 3 patients (17.6%) and stable disease in 10 patients (58.8%), with a clinical benefit rate of 76.4%. Although these results appear promising, they are comparable with historical reports with paclitaxel alone (63).
Potential Mechanisms of Resistance
Although pharmacologic doses of retinoids have proved effective in the treatment of hematologic malignancies (64), clinical trials in the prevention and treatment setting in a number of solid tumors, including lung cancer and breast cancer, have failed to show significant benefit to date (51, 63). The lack of a robust biomarker of response to therapy is one reason for this failure. In addition, a number of potential mechanisms of resistance to these therapies have been proposed.
Epigenetics
In solid tumors, RARβ gene expression is frequently lost in primary tumors and their metastasis compared with adjacent noncancerous tissues (65, 66). This provides a possible explanation as to why treatment using RAs in solid tumors such as breast cancer have previously failed. Our laboratory and others have provided extensive evidence that RARβ is silenced in breast cancer by epigenetic modification including both methylation at the promoter region of the gene and a compacted chromatin structure (25, 67). Epigenetically silenced RARβ has been shown to be reexpressed in the presence of DNA methyltransferase inhibitors (DNMT) and histone deacetylase (HDAC) inhibitors in RARβ2-silent breast cancer cells (67). Treatment with an HDAC inhibitor combined with 9-cis RA resulted in regression of prostate and breast cancer xenografts (67, 68). It is possible that the addition of epigenetic modifiers to RA-based therapy will be needed to reactivate RARβ in RARβ-silent tumors to accomplish significant growth inhibition (Fig. 2). With this in mind, a number of clinical trials have incorporated epigenetic modifiers with retinoids in an attempt to improve the outcomes observed with single-agent retinoids, and thereby potentially overcome resistance. Entinostat (MS-275), a HDAC inhibitor, has been combined with 13-cis RA in a phase I trial in patients with advanced solid tumors and lymphomas. The combination was reasonably well = tolerated and a recommended phase II dose was identified for future studies (69). A single-arm phase II study has also been reported that investigated the efficacy of 5-azacitidine (DNMT inhibitor), valproic acid (HDAC inhibitor), and ATRA in patients with hematologic malignancies (70). The best responses to this combination of agents included 14 complete responses and 3 partial responses to therapy. It is important to note that in the clinical trials described to date, the RARβ status of the tumors was not assessed before therapy. Tumors that do not express the RARβ receptor are unlikely to respond to RA treatment. The efficacy of retinoids may be further enhanced with the addition of cytotoxic agents to the combination of retinoids and HDAC inhibitors, perhaps by debilitating several critical interacting pathways that the cancer cell depends on for continued growth and proliferation. We have shown that the combination of the HDAC inhibitor entinostat ATRA and low-dose chemotherapy yielded the greatest inhibition of tumor cell growth in vitro and in human tumor breast cancer xenografts (71).
Mechanism of activation of RARβ, an important downstream effector of the RA pathway, in cancer growth inhibition. Under conditions where RARα is functional and the RARβ promoter is not epigenetically silenced, physiologic levels of RA can activate RARβ expression. A small number of solid tumors display this phenotype. Under less ideal conditions in which the RARβ promoter is hypoacetylated, pharmacologic doses of RA are needed to activate RARβ. In the majority of solid tumor types, the RARβ promoter is methylated and/or the histones are significantly deacetylated. In this case, treatment with pharmacologic doses of RA is not sufficient to overcome the repressive effect of epigenetic silencing. Epigenetic-modifying drugs such as DNA methytransferases or HDAC inhibitors are needed to release the epigenetic stress and activate the RARβ gene. NCOR, nuclear receptor corepressor 1; SMRT, silencing mediator for retinoid and thyroid receptors.
Mechanism of activation of RARβ, an important downstream effector of the RA pathway, in cancer growth inhibition. Under conditions where RARα is functional and the RARβ promoter is not epigenetically silenced, physiologic levels of RA can activate RARβ expression. A small number of solid tumors display this phenotype. Under less ideal conditions in which the RARβ promoter is hypoacetylated, pharmacologic doses of RA are needed to activate RARβ. In the majority of solid tumor types, the RARβ promoter is methylated and/or the histones are significantly deacetylated. In this case, treatment with pharmacologic doses of RA is not sufficient to overcome the repressive effect of epigenetic silencing. Epigenetic-modifying drugs such as DNA methytransferases or HDAC inhibitors are needed to release the epigenetic stress and activate the RARβ gene. NCOR, nuclear receptor corepressor 1; SMRT, silencing mediator for retinoid and thyroid receptors.
Cancer stem cells
Another potential mechanism of resistance to retinoids in solid malignancies is the presence of cancer stem cells. Many studies have attempted to target cancer stem cells with differentiation treatments including RAs (72, 73). Our laboratory has found that treatment of tumors using the HDAC inhibitor ATRA and low-dose doxorubicin not only results in striking tumor regression but also significantly reduces the number of cancer stem cells (unpublished data). Therefore, HDAC treatment may induce differentiation in the stem-like tumor cells, which may circumvent resistance to standard chemotherapy or ATRA treatment alone. Further studies are needed to delineate the role of RA in targeting these cells that are generally deemed treatment resistant.
Other potential mechanisms of resistance
Cancer cells may silence or repress RARβ by mechanisms other than epigenetic modulation to initiate and promote their growth and resist treatment with RA. A number of alternative mechanisms have been proposed, including the loss of coactivators (74), increased RA metabolism (75), decreased RA availability (76), and impaired RARα signaling (77). For example, studies have shown that AF2 co-activators of the RAR–thyroid hormone receptor complex are often lost in human lung cancer (74). The loss of AF-2 cofactors results in low levels of transcribed RARβ, suggesting an important function of these cofactors in mediating RARβ expression. Another study showed that impaired RARα function failed to facilitate changes in the chromatin structure of RARβ necessary for RARβ activation, implicating a critical role for RARα in controlling RARβ expression.
Potential mechanisms of resistance that are independent of RARβ have also been suggested. Aberrant p53 expression, for example, has been associated with 13-cis RA resistance in the clinic (78); RA, it appears, can promote intrinsic transactivation of p53 (79). It is also possible that cross-talk between the RAR and ER in breast cancer can create opportunities for cancer cells to bypass pathways inhibited by targeted therapies such as RA or hormonal therapies (7, 19).
Conclusions and Future Directions
In summary, retinoids have been investigated extensively for their use in solid tumor cancer prevention and treatment. Promising results have been observed in the breast cancer prevention setting, where fenretinide prevention trials have provided a strong rationale for a new trial in young women at high risk for breast cancer. Clinically relevant outcomes have also been observed with the use of retinoids combined with chemotherapy in NSCLC, prompting the development of confirmatory phase III randomized trials. Further delineation of the mechanisms of action and resistance of retinoids in solid tumors may provide the rationale for future studies and result in clinical benefit for patients. Ongoing and future studies that combine retinoids with epigenetic modifiers, such as the HDAC inhibitors, as well as standard cytotoxic agents, tyrosine kinase inhibitors, and other novel agents are more likely to yield clinically relevant outcomes than observed with single-agent therapy. Novel RA metabolism blocking agents (RAMBA) are also undergoing investigation at this time (80). Finally, clinical trialists should be encouraged to incorporate correlative endpoints in their studies to identify accurate biomarkers of response to retinoid therapy.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Authors' Contributions
Conception and design: R. Connolly, N.K. Nguyen, S. Sukumar
Development of methodology: N.K. Nguyen
Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): N.K. Nguyen
Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): R. Connolly, N.K. Nguyen
Writing, review, and/or revision of the manuscript: R. Connolly, N.K. Nguyen, S. Sukumar
Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): R. Connolly, N.K. Nguyen
Study supervision: R. Connolly, S. Sukumar
Acknowledgments
The authors thank Dr. Antonio Wolff for reviewing this article and providing helpful advice.
Grant Support
This study was supported by the Sidney Kimmel Comprehensive Cancer Center (SKCCC) Core grant P30 CA006973 and DOD COE W81XWH-04–1-0595.
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