Anti-tumor effects of retinoids combined with trastuzumab or tamoxifen in breast cancer cells: induction of apoptosis by retinoid/trastuzumab combinations

Introduction HER2 and estrogen receptor (ER) are important in breast cancer and are therapeutic targets of trastuzumab (Herceptin) and tamoxifen, respectively. Retinoids inhibit breast cancer growth, and modulate signaling by HER2 and ER. We hypothesized that treatment with retinoids and simultaneous targeting of HER2 and/or ER may have enhanced anti-tumor effects. Methods The effects of retinoids combined with trastuzumab or tamoxifen were examined in two human breast cancer cell lines in culture, BT474 and SKBR3. Assays of proliferation, apoptosis, differentiation, cell cycle distribution, and receptor signaling were performed. Results In HER2-overexpressing/ER-positive BT474 cells, combining all-trans retinoic acid (atRA) with tamoxifen or trastuzumab synergistically inhibited cell growth, and altered cell differentiation and cell cycle. Only atRA/trastuzumab-containing combinations induced apoptosis. BT474 and HER2-overexpressing/ER-negative SKBR3 cells were treated with a panel of retinoids (atRA, 9-cis-retinoic acid, 13-cis-retinoic acid, or N-(4-hydroxyphenyl) retinamide (fenretinide) (4-HPR)) combined with trastuzumab. In BT474 cells, none of the single agents except 4-HPR induced apoptosis, but again combinations of each retinoid with trastuzumab did induce apoptosis. In contrast, the single retinoid agents did cause apoptosis in SKBR3 cells; this was only modestly enhanced by addition of trastuzumab. The retinoid drug combinations altered signaling by HER2 and ER. Retinoids were inactive in trastuzumab-resistant BT474 cells. Conclusions Combining retinoids with trastuzumab maximally inhibits cell growth and induces apoptosis in trastuzumab-sensitive cells. Treatment with such combinations may have benefit for breast cancer patients.

Effects of retinoids on signaling by ER and HER2 have been reported. Inhibition of breast tumor cell growth by retinoids is greater for ER-positive cells than ER-negative cells [27], which may be partly related to alterations in retinoid metabolism [28]. Some studies have found that RA increased the amount of ER in MCF7 breast cancer cells [29], although others reported that RA downregulated ER in this cell line [30]. Regardless, activated RARs have been observed to exert anti-estrogenic effects by directly or indirectly impairing binding of ER to estrogen response elements (EREs) [31]. Conversely, the N-terminal region of ERα modulates the transcriptional activity of RAR [32].
Both RARα and RARβ have been implicated in the anti-proliferative effects of retinoids against breast cancer. RARα expression is correlated positively with ER and with RA sensitivity, and is inducible by estrogen [27]. RARβ has been ascribed tumor suppressor-type activity and is often down regulated in breast cancer; it is inducible by atRA, and inducibility correlates with atRA sensitivity [27]. In both ER-positive T47D cells and ER-negative SKBR3 cells, some evidence suggests that RARα is the receptor solely sufficient for the growth inhibition, cell cycle arrest, apoptosis, and modulation of RAR levels [33].
Inhibition of breast cancer cell growth by atRA and 4-HPR has been associated with downregulation of HER2; while atRA induced morphologic changes consistent with differentiation, 4-HPR induced those of apoptosis [34]. Another study demonstrated that RA can induce differentiation of cultured breast tumor cells, and this was again associated with reduction in cell surface HER2 [35]. atRA and 9-cis-RA caused decreases in HER2 and HER3, and inhibited SKBR3 cell growth with cell cycle arrest and induction of apoptosis [36], and the retinoids downregulated HER4 in T47D cells [37]; atRA also downregulated HER2 and HER3 in MCF cells [38]. 4-HPR was reported in another study to downregulate both HER2 and the epidermal growth factor receptor (EGFR, HER1) [39]. In contrast, stimulation of SKBR3 cells with epidermal growth factor or heregulin β1 (HRGβ1) upregulates RARα expression [40], yet resistance to atRA-induced growth inhibition has been reported for HER2-overexpressing breast cancer cells (either HER2-transfected MCF7 cells or naturally overexpressing BT474 or MDA-MB-453 cells); pretreatment for several days with trastuzumab could sensitize these latter two cell lines to inhibition by atRA [41,42]. Trastuzumab treatment increased RA response element binding activity measured by electrophoretic mobility shift assay in a HER2-overexpressing cell line [41]. Coamplification of RARα with HER2 has been reported in human breast tumors [43]. Finally, retinoids have been found to delay the onset of mammary tumors in HER2 transgenic mice [44,45], and a selective ER modulator/ rexinoid combination was synergistic in the prevention or treatment of such tumors, despite the ER-negative status of such tumors [46].
Given the known interactions of retinoids with ER and HER2, we hypothesize that treatment with retinoids and simultaneous targeting of HER2 and/or ER may be a fruitful approach to treating breast cancer. As models, we have used ER-positive (BT474) and ER-negative (SKBR3) HER2-overexpressing human breast cancer cell lines. In the present article we examine the effects of various retinoids (atRA, 9-cis-RA, 13-cis-RA, and 4-HPR), trastuzumab, tamoxifen, or combinations of these drugs on proliferation, cell cycle, differentiation, and apoptosis in BT474 and SKBR3 cells. Since retinoids, trastuzumab, and tamoxifen are individually agents that possess anti-tumor activity toward breast cancer, combinations of these drugs may translate into improved therapy for breast cancer patients. We report synergistic inhibition of cell proliferation for combinations of these drugs, but apoptosis-inducing activity only of the retinoid/trastuzumab combinations.

Materials and methods
Drugs Trastuzumab (Herceptin) was a gift from Genentech (South San Francisco, CA, USA), supplied as a stock solution in PBS. Tamoxifen citrate, atRA, 9-cis-RA, 13cis-RA, and 4-HPR were purchased from Sigma-Aldrich (St Louis, MO, USA); stock solutions of these drugs were prepared in 100% ethanol (EtOH) and were kept light-protected. HRGβ1 was purchased from R&D Systems (Minneapolis, MN, USA) and was reconstituted in PBS.
WST-1 colorimetric cell proliferation assay BT474 cells or SKBR3 cells were seeded in 96-well plates at 10,000 or 5,000 cells per well, respectively. On the following day, the cells were treated with vehicle (EtOH + PBS) or drug(s). In each independent experiment, eight replicate wells of cells were used for each treatment. On day 6 following treatment, the WST-1 proliferation assay was performed according to the protocol provided by the manufacturer (Roche Applied Science, Indianapolis IN, USA). Results are expressed as a percentage of control (vehicle-treated cells).

Analysis of drug interactions
Drug interaction results from the WST-1 proliferation assay were examined by the method of Chou and Talalay [51] using the commercially available software Cal-cuSyn [52,53]  where f a is the fraction affected, f u is the fraction unaffected, D is the dose, D m is the dose that produces a median effect (IC50), and m is the coefficient signifying the sigmoidicity of the curve (or the slope in a linear transformation).
For examining the effect of multiple drugs, a combination index (CI) is calculated based on the doses that have equivalent effects. The formula used for calculating the CI of two drugs is: and the formula used for determining the CI of three drugs is: where D1, D2, and D3 are the doses for each drug alone that inhibit a certain percentage, and D1c, D2c, and D3c are the doses for each drug in a combination that inhibit the same percentage. The CI is a quantitative measurement of the degree of interaction between two or more drugs: CI < 1 indicates synergism between the drugs, CI = 1 indicates additivity, and CI > 1 denotes antagonism.
Analysis of cell cycle and detection of apoptosis by determination of sub-G 1 DNA peak BT474 cells or SKBR3 cells were seeded in 25 cm 2 flasks at 1 × 10 6 or 0.5 × 10 6 cells per flask, respectively. On the following day, the cells were treated with vehicle (EtOH + PBS) or drug(s). At designated time points, floating cells in the growth media were collected, and adherent cells were trypsinized and collected. The pooled floating and adherent cells were washed twice with cold PBS. The washed cells were then resuspended in 2 ml of cold PBS, fixed by three stepwise additions of 2 ml each of cold 95% EtOH, and were stored at 4°C. For analysis of cell cycle and sub-G 1 DNA peak [54], the fixed cells were pelleted by centrifugation, incubated with 1 mg/ml ribonuclease A (Sigma-Aldrich) in PBS at 37°C for 30 minutes, and stained on ice with 50 μg/ml propidium iodide (Sigma-Aldrich) in PBS for 1 hour. The cell cycle distribution (percentages of cells in the G 0 /G 1 , S, and G 2 /M phases) and the percentage of cells in the sub-G 1 DNA peak were determined by flow cytometry. Detection of differentiation by Nile red staining of neutral lipids BT474 cells or SKBR3 cells were seeded in 25 cm 2 flasks at 1 × 10 6 or 0.5 × 10 6 cells per flask, respectively. On the following day, the cells were treated with vehicle (EtOH + PBS), or drug(s). On day 6 following treatment, the cells were collected by trypsinization, washed with PBS, and stained with 100 ng/ml Nile red fluorescent dye (Sigma-Aldrich) in PBS for 5 minutes at room temperature. The stained cells were then washed twice with PBS, resuspended in PBS, and analyzed by flow cytometry [55].

Measurement of estrogen receptor transcriptional activity by dual luciferase reporter assay
BT474 cells were seeded in six-well plates at 10 6 cells per well. On the following day, the cells were transiently co-transfected with Renilla luciferase reporter vector plasmid pRL-CMV (Promega, Madison, WI, USA) (a control to normalize for transfection efficiency) and a plasmid containing three consensus EREs fused to a firefly luciferase reporter vector (3 × ERE-TATA-Luc) described previously [56,57] using the TransFast transfection reagent (Promega) according to the manufacturer's protocol. Briefly, each well of cells was incubated at 37°C for 30 minutes in serum-free RPMI media containing 1,000 ng 3 × ERE-TATA-Luc reporter vector, 20 ng Renilla luciferase reporter vector pRL-CMV, and TransFast transfection reagent. Following incubation, the transfection mixture was removed, and normal growth media (including 10% FBS without charcoal stripping) was added to the cells, followed immediately by addition of experimental drugs. Drug treatment was with vehicle (EtOH + PBS), 1 μM Faslodex, 1 μM atRA, 1 μg/ml trastuzumab, 1 μM tamoxifen, or the various drug combinations at the same concentrations. Two days following transfection and treatment, the dual luciferase reporter assay was performed using the Promega Dual Luciferase Reporter Assay System according to the manufacturer's protocol. The firefly luciferase activities of the treated cells were normalized to their Renilla luciferase activities and are expressed as a percentage of activity of untreated cells.

Production of trastuzumab-resistant BT474 cells
Trastuzumab-resistant BT474 cells were selected as described [58] by long-term culture in media containing trastuzumab at 100 μg/ml. Cells were maintained in the same trastuzumab-containing media unless otherwise indicated.

Analysis of interactions between atRA, trastuzumab, and tamoxifen on cell proliferation
ER-positive/HER2-overexpressing BT474 cells were treated with atRA, trastuzumab or tamoxifen at a range of doses (0.2 to 10 μM for atRA and tamoxifen, and 0.2 to 10 μg/ml for trastuzumab) and with various combinations of the three drugs at fixed dose ratios. On day 6 following treatment, the effects of the single agents and drug combinations on BT474 cell growth were examined by the WST-1 proliferation assay. The results from WST-1 assays were expressed as a percentage of control growth, and drug interactions were analyzed by the Chou-Talalay method.

Analysis of cell cycle following drug treatment
The cell cycle distribution of BT474 cells was analyzed following treatment with single agents or various combinations of atRA, tamoxifen or trastuzumab. Each drug individually is known to cause G 1 cell cycle accumulation. Compared with untreated cells and vehicle-treated cells, single agents and various combinations of the three drugs led to an enhanced accumulation of cells in the G 0 /G 1 phase coupled with a reduction of cells in the S phase of cell cycle ( Figure 1). In general, the drug combinations produced lower percentage of cells in the S phase than did single agents.

Analysis of differentiation following drug treatment
Differentiation of BT474 cells was determined by Nile red fluorescent dye staining of neutral lipids on day 6 Doses of all-trans retinoic acid (atRA) and trastuzumab (Tzmab) in μM and μg/ ml, respectively. Each value is the mean of three independent experiments (with eight replicate wells for each treatment) ± standard error.
following treatment with HRGβ1 (positive control), single agents, or various combinations of atRA, tamoxifen or trastuzumab. Compared with untreated and vehicletreated cells, HRGβ1, single agents, or various drug combinations led to an increase in neutral lipid production ( Figure 2). Treatment with the atRA/trastuzumab, trastuzumab/tamoxifen, and atRA/trastuzumab/tamoxifen combinations resulted in greater neutral lipid production than the respective single agents alone (P < 0.05) ( Figure 2). The triple combination induced the greatest degree of differentiation (P < 0.05) ( Figure 2).

Analysis of apoptosis following drug treatment
To detect apoptotic cells, both floating and adherent BT474 cells were examined by annexin V staining and sub-G 1 DNA peak analysis following treatment with single agents or various combinations of the three drugs. Both annexin V staining and sub-G 1 DNA peak analysis demonstrate that only the atRA/trastuzumab and atRA/ trastuzumab/tamoxifen combinations induced apoptosis ( Figure 3). The atRA/trastuzumab combination resulted in 3%, 9%, and 16% annexin V-positive cells (Figure 3a), and in 3%, 13%, and 26% cells in the sub-G 1 DNA peak ( Figure 3b) on days 2, 4, and 6, respectively. The atRA/ trastuzumab/tamoxifen combination induced the greatest percentage of apoptotic cells (3%, 11%, and 25% annexin V-positive cells, and 3%, 13%, and 36% cells in the sub-G 1 DNA peak on days 2, 4, and 6, respectively) ( Figure 3). Therefore, while neither the single agents nor the atRA/tamoxifen or trastuzumab/tamoxifen combinations induced apoptosis, the atRA/trastuzumab and atRA/trastuzumab/tamoxifen combinations did result in apoptosis.
Given the unique ability of the atRA/trastuzumab combination to induce apoptosis in ER-positive BT474 cells, and the known interaction between retinoids and ER signaling discussed above, it was of interest to extend these experiments to ER-negative/HER2-overexpressing cells. In addition, it was of interest to compare the effects of other retinoids to those of atRA.
Effect of other retinoids with trastuzumab on cell proliferation of ER-positive and ER-negative cells HER2-overexpressing ER-positive/BT474 cells or ERnegative/SKBR3 cells were treated with each of the following single agents: 1 μg/ml trastuzumab, 1 μM atRA, 1 μM 9-cis-RA, 1 μM 13-cis-RA, 1 μM 4-HPR, 2.5 μM 4-HPR, or 5 μM 4-HPR -or with trastuzumab/retinoid combinations. On day 6 following treatment, the effects of the single agents and drug combinations on BT474 or SKBR3 cell growth were examined by the WST-1 proliferation assay. The results from WST-1 assays were expressed as a percentage of untreated cells. The combinations of trastuzumab with the various retinoids showed greater growth inhibition than the single agents alone in both BT474 cells ( Figure 4a) and SKBR3 cells ( Figure 4b) -with the exception of 4-HPR, which showed minimal ability to enhance trastuzumabmediated growth inhibition in both cell lines.

Effect of other retinoids with trastuzumab on cell cycle of ER-positive and ER-negative cells
The cell cycle distribution of BT474 cells or SKBR3 cells was analyzed on day 2 following treatment with single agents or combinations of the drugs. Each drug individually is known to cause G 1 cell cycle accumulation.
In BT474 cells, compared with untreated cells and vehicle-treated cells, the single agents trastuzumab and 4-HPR, and the combinations of trastuzumab with the various retinoids, led to an enhanced accumulation of cells in the G 0 /G 1 phase coupled with a reduction of cells in the S phase of the cell cycle ( Figure 5). In general, the drug combinations produced a lower percentage of cells in the S phase than did single agents alone in BT474 cells ( Figure 5).
In SKBR3 cells, all single agents resulted in a reduced S phase (4-HPR required higher concentrations), although there was only a modest effect of adding trastuzumab to 4-HPR and no additional effect of adding  trastuzumab to the other retinoids ( Figure 5). Unlike the BT474 cells, none of the drugs or their combinations had a significant impact on the G 1 phase in SKBR3 cells; rather, the retinoids (excluding 4-HPR) caused an increase in the G 2 /M phase, which was just slightly enhanced by addition of trastuzumab to any of the retinoids ( Figure 6).

Effect of other retinoids with trastuzumab on differentiation of ER-positive and ER-negative cells
Differentiation of BT474 cells or SKBR3 cells was determined by Nile red fluorescent dye staining of neutral lipids on day 6 following treatment with HRGβ1 (positive control), single agents, or combinations of the drugs. In BT474 cells, trastuzumab and single retinoid agents only slightly increased neutral lipid production, although it was significantly enhanced by adding trastuzumab to retinoids (other than 4-HPR) (Figure 7). In SKBR3 cells, single-agent retinoids induced significantly greater neutral lipid production, with the exception of 4-HPR; addition of trastuzumab to retinoids, however, did not enhance the effect of the retinoids, except for 4-HPR ( Figure 7).

Effect of other retinoids with trastuzumab on apoptosis of ER-positive and ER-negative cells
To detect apoptotic cells, both floating and adherent BT474 or SKBR3 cells were examined by sub-G 1 DNA peak analysis following treatment for 6 days with single agents or combinations of the drugs. In BT474 cells, sub-G 1 DNA peak analysis demonstrated that only the combinations of trastuzumab with retinoids induced apoptosis; none of the single agents (except 4-HPR at higher concentrations) induced apoptosis; and 4-HPR alone induced apoptosis at concentrations higher than 1 μM (2.5 μm or 5 μm), and this was not enhanced by  trastuzumab ( Figure 8). In contrast, the single retinoid agents did cause apoptosis in SKBR3 cells, with 4-HPR having a much weaker effect compared with the other retinoids; the addition of trastuzumab to the retinoids produced a small enhancement in the induction of apoptosis ( Figure 8).

Effect of drugs on receptor signaling
The effect on receptor signaling of treatment of BT474 cells with atRA, trastuzumab, or both was examined ( Figure 9). Single-agent trastuzumab at 1 μg/ml resulted in a moderate decrease in total levels of HER2, and, as expected, a more significant decrease in HER2 activity as reflected by the level of HER2 autophosphorylation. Treatment with atRA at 1 μM had no effect on HER2 expression level or the degree of phosphorylation, and, when added to 1 μg/ml trastuzumab, did not have a significant effect on HER2 expression level or activity. Single-agent atRA also did not significantly affect AKT or MAPK expression or activity. Trastuzumab treatment resulted in partial inhibition of AKT and MAPK activity; while addition of atRA to trastuzumab had no further effect on AKT activity, the combination did appear to result in a small further decrement in MAPK activity.
Single-agent atRA at 1 μM caused a small decrease in the level of RARα (Figure 9). Trastuzumab at 1 μg/ml had a similar effect, and the combination resulted in the greatest decrease in expression level of this receptor. Treatment with atRA did not appear to affect levels of RARβ, RXRα or RXRβ; however, trastuzumab caused a small decrement in expression of RARβ that was enhanced when combined with atRA ( Figure 9). ER transcriptional activity was examined using an ERE assay in BT474 cells. Treatment with single-agent atRA caused a profound inhibition of ERE activity, comparable with the ER downregulator Faslodex ( Figure 10). Single-agent trastuzumab caused partial inhibition of ERE activity comparable with that of tamoxifen, implicating peptide growth factor signaling pathway-driven ER activation in these cells; however, adding trastuzumab to tamoxifen did not further inhibit ERE activity. Adding trastuzumab, tamoxifen or both to atRA could not further inhibit ERE activity beyond that of atRA treatment alone.

Effect of atRA on growth of trastuzumab-resistant BT474 cells
Given the ability of the atRA/trastuzumab combination to synergistically induce apoptosis in BT474 cells under conditions where neither agent alone could do so, it was of interest to examine the activity of atRA in trastuzumab-resistant BT474 cells. Resistance was induced in these cells by long-term culture in trastuzumab-containing media. As shown in Figure 11, atRA did not affect the growth of trastuzumab-resistant BT474 cells, whether used in the presence or absence of trastuzumab in the media. In fact, removal of trastuzumab from the media did not affect growth of these cells. Slight growth inhibition was observed when trastuzumab-resistant cells were treated with 10 μM of the epidermal growth factor receptor/HER2 dual tyrosine kinase inhibitor laptinib analog GW2974 in the presence of trastuzumabcontaining media, although this required doses much greater than those typically required to inhibit growth of trastuzumab-sensitive cells; the addition of atRA to GW2974 was not able to enhance growth inhibition observed with the latter agent alone.

Discussion
HER2 and ER play critical roles in breast cancer and are validated therapeutic targets in this disease. Retinoids have also been shown to inhibit breast cancer growth.
We have demonstrated that combining atRA with trastuzumab, tamoxifen, or both results in strong synergistic growth inhibition of BT474 human breast cancer cells.
To elucidate the molecular mechanisms underlying this synergistic growth inhibition, we examined the effects of single agents and various drug combinations on cell cycle, differentiation, and apoptosis. We found that treatment with the atRA/trastuzumab and atRA/trastuzumab/tamoxifen combinations caused induction of apoptosis, which was not observed for single drugs or the trastuzumab/tamoxifen or atRA/tamoxifen combinations.
Since we observed that combining atRA with trastuzumab uniquely resulted in apoptosis, we examined the effects of other retinoids with trastuzumab, in both ERpositive (BT474) and ER-negative (SKBR3) HER2-  overexpressing human breast cancer cells. We found in BT474 cells that, while none of the single agents (except 4-HPR) induce apoptosis, the combinations of various retinoids with trastuzumab also result in apoptosis. In contrast, the single-agent retinoids (other than 4-HPR) do induce apoptosis in SKBR3 cells (weakest for 4-HPR), and adding trastuzumab to the retinoids causes only a small enhancement of that effect. The pan-retinoid receptor agonists 9-cis-RA and 13-cis-RA hence behave similarly to atRA, while 4-HPR has a different activity profile. A recent study reported synergistic growth inhibition and induction of apoptosis for the combination of trastuzumab and 9-cis-RA in hepatocellular cells [59], suggesting application to a broader range of malignancies; that report demonstrated that trastuzumab inhibited phosphorylation of RXRα and enhanced 9-cis-RA-induced RA response element and retinoid X response element activity.
The single agents employed in our study have been reported previously to promote accumulation of cells in the G 0 /G 1 phase of the cell cycle [36,[60][61][62][63][64][65]. Our results confirm these findings. Compared with untreated cells and vehicle-treated cells, the combinations of trastuzumab with various retinoids lead to enhanced accumulation of BT474 cells in the G 0 /G 1 phase and SKBR3 cells in the G 2 /M phase, coupled with a reduction of cells in the S phase. We further demonstrate that the combinations generally lead to a greater reduction of cells in the S phase of the cell cycle than the respective single agents in BT474 cells.
Retinoids have also been demonstrated to modulate breast cancer cell growth through differentiation as well as apoptosis [34,35], and to cooperate with heregulin to induce morphologic differentiation (branching morphogenesis) in three-dimensional culture [66]. We have found that the single-agent retinoids, trastuzumab and tamoxifen, individually induce differentiation but not apoptosis in BT474 cells. The combinations of various retinoids and trastuzumab result in greater differentiation than respective single agents in BT474 cells. Compared with untreated cells and vehicle-treated cells, the single retinoid agents alone induce greater differentiation and greater apoptosis in SKBR3 cells than in BT474 cells. The combinations of various retinoids and trastuzumab result in greater apoptosis than, but similar differentiation as, respective single agents alone in SKBR3 cells.
The combinations of trastuzumab and various retinoids do induce apoptosis in both BT474 cells and SKBR3 cells. Our findings are consistent with previous studies that show sensitivity to atRA is decreased in HER2-overexpressing breast cancer cells [41,42]. Consequently, we find that targeting of HER2 by trastuzumab in the presence of retinoids induces apoptosis and greater differentiation in BT474 cells; the potentiation of retinoid-induced apoptosis by trastuzumab was modest in SKBR3 cells. The capacities of retinoids to induce differentiation and apoptosis are thus enhanced when trastuzumab inhibits signaling by HER2. Through the induction of apoptosis, greater differentiation, and effects on cell cycle, the combinations of trastuzumab and various retinoids resulted in greater growth inhibition than single agents alone in both BT474 cells and SKBR3 cells.
Numerous previous studies have suggested promise for the combination of retinoids with anti-estrogens.
Tamoxifen was found to potentiate the effect of atRA to inhibit estrogen-induced growth of MCF7 cells [31]. In a rat carcinogen-induced mammary tumor model, the rexinoid bexarotene (Targretin) was able to induce complete remission of the majority of established tumors, and its combination with tamoxifen was more effective than either alone [67]. In this model, there was also some evidence that adding the retinoid to tamoxifen after the development of tamoxifen resistance may restore some sensitivity to tamoxifen, since response rates were higher than when tamoxifen was discontinued and Targretin was used instead [68].
A number of clinical trials have explored the therapeutic potential of retinoids in breast cancer patients or as prevention agents. Fenretinide, Targretin, 9-cis-RA, 13-cis-RA and atRA have been examined in clinical trials. A small phase II trial of 13-cis-RA in 18 heavily pretreated (chemotherapy and endocrine therapy refractory) advanced breast cancer patients yielded no objective responses [69]. Fenretinide had relatively mild and reversible toxicity in a small phase II trial in patients with advanced disease but also showed no clinical activity [70]. A small phase II trial of atRA in patients with hormone refractory metastatic breast cancer showed it to be relatively well tolerated, but noted only one partial response among 14 evaluable patients -although there was marked interpatient variability in pharmacokinetics [71]. A large phase III secondary prevention trial using fenretinide for 5 years after surgical treatment for ductal carcinoma in situ or stage I breast cancer revealed no statistically significant effect on the prevention of second contralateral or ipsilateral breast malignancies in the group as a whole, or in distant metastases or survivalalthough intriguingly there was a reduction of contralateral and ipsilateral breast cancer among premenopausal patients in the study [72], which may suggest a specific interaction with estrogen signaling; at 15-year follow up, the study continued to show the same trend [73].
Given the disappointing results for retinoids as single agents in advanced disease, combinations with other agents become of interest. The combinations of tamoxifen with atRA, retinyl acetate, 9-cis-RA, fenretinide, Targretin and retinyl palmitate have been studied in clinical trials.
A pilot breast cancer chemoprevention trial using fenretinide in combination with tamoxifen is being conducted [74]. This agent is also being tested as secondary chemoprevention (of contralateral breast cancer) as a single agent by the Milan group. In patients with advanced disease in a small phase I trial of the 9-cis-RA/tamoxifen combination, the dose-limiting toxicities were headache, hypercalcemia and noncardiogenic pulmonary edema; and of nine assessable patients, there was one partial response and one complete response,    Figure 11 Effect of atRA on growth of trastuzumab-resistant BT474 cells. WST-1 proliferation assay of trastuzumab-resistant BT474 cells treated for 6 days with 10 μM GW2974, 1 μM all-trans retinoic acid (atRA), or their combination, in the presence or absence of trastuzumab (Tzmab) in the culture media. Each bar is normalized to the growth of control (C, vehicle-treated) cells in trastuzumab-containing media. Each value is the mean of three independent experiments (with three replicate wells for each treatment) ± standard error. both in patients who had ER-positive tumors and previous tamoxifen therapy [75]. A phase I/II trial of tamoxifen with or without fenretinide in ER-positive or PR-positive, previously untreated metastatic breast cancer revealed no significant toxicity and improvement or stabilization of disease in 12 of 15 patients [76]. In a phase I/II trial of the atRA/tamoxifen combination in patients with potentially hormone-responsive advanced disease, the dose-limiting toxicity was headache and dermatologic toxicity; and two out of seven patients with measurable disease responded, while seven out of 18 patients with nonmeasurable but evaluable disease had stable disease [77].
Targretin has been tested in patients with metastatic breast cancer, as monotherapy and in combination with tamoxifen for tamoxifen-resistant patients; however, response rates were low -on the order of 3 to 6%, although up to 20% of patients had some clinical benefit [78]. In a phase II study of tamoxifen plus high-dose retinyl acetate in postmenopausal patients with advanced breast cancer, toxicity was generally mild and an overall response rate of 38.5% was reported [79].
In a phase II trial of the fenretinide/tamoxifen combination specifically in advanced disease patients with ER-negative tumors or patients with ER-positive tumors previously treated with tamoxifen, no objective responses were observed although three patients had prolonged stable disease [80]. A recent report with a 2 × 2 trial design found that either low-dose tamoxifen or fenretinide could lower risk of breast neoplasms compared with placebo, but curiously their combination could not, suggesting potential antagonism, although the study was underpowered to detect true differences [81]. A pilot phase II study of IFNβ/retinyl palmitate/tamoxifen in patients with advanced disease showed a clinical response rate of 55% [82]. No reported studies have evaluated the therapeutic effects of a retinoid/trastuzumab combination in a clinical trial, and our results suggest such a strategy could be of benefit.

Conclusions
In summary, the combinations of various retinoids with trastuzumab, tamoxifen, or both shows strong synergistic inhibition of proliferation accompanied by cell-cycle delay, differentiation, and, for retinoid/trastuzumab combinations, apoptosis in both ER-positive and ERnegative human breast cancer cells. The retinoid/trastuzumab combination resulted in enhanced inhibition of MAPK signaling and downregulation of RARα and RARβ. Treatment with a retinoid and simultaneous inhibition of HER2 and/or ER signaling may thus hold promise as therapy for breast cancer patients.