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. Author manuscript; available in PMC: 2022 Sep 1.
Published in final edited form as: Cancer Chemother Pharmacol. 2021 May 27;88(3):415–425. doi: 10.1007/s00280-021-04298-y

A triple combination gemcitabine+romidepsin+cisplatin to effectively control triple-negative breast cancer tumor development, recurrence, and metastasis

Pawat Pattarawat 1, Jessica T Hunt 2, Jacob Poloway 1, Collin J Archibald 1, Hwa-Chain Robert Wang 1,*
PMCID: PMC8489834  NIHMSID: NIHMS1743532  PMID: 34043046

Abstract

Purpose

Triple-negative breast cancer (TNBC) is an aggressive, lethal, heterogeneous type of breast cancer (BC). TNBC tends to have a lower response rate to chemotherapy and a lower fiveyear survival rate than other types of BC due to recurrence and metastasis. Our previous study revealed that a combination of gemcitabine, romidepsin, and cisplatin was efficacious in controlling TNBC tumor development. In this study, we extended our investigation of gemcitabine+romidepsin+cisplatin in controlling TNBC tumor recurrence and metastasis.

Methods

We investigated the ability of gemcitabine+romidepsin+cisplatin to control cell survival and invasiveness using cell viability, soft agar colony formation, and transwell invasion assays. We determined the efficacy of gemcitabine+romidepsin+cisplatin in controlling tumor recurrence and metastasis using cell-derived xenograft animal models. We used immunoblotting to study signaling modulators regulated by gemcitabine+romidepsin+cisplatin in TNBC cells and tumor tissues.

Results

Treatment with gemcitabine+romidepsin+cisplatin reduced the TNBC MDA-MB231 and MDA-MB468 cell survival to ~50% and ~15%, as well as invasiveness to ~31% and ~13%, respectively. Gemcitabine+romidepsin+cisplatin suppressed modulators involved in epithelialmesenchymal transition in an ROS-dependent manner. Controlling tumor recurrence, the Gem plus Rom+Cis regimen (~112%) was more efficacious than the Gem plus Cis regimen (~21%) in tumor growth inhibition. The Gem plus Rom+Cis regimen efficaciously reduced the development of metastatic nodules to 20% in animals.

Conclusion

The gemcitabine plus romidepsin+cisplatin regimen was highly efficacious in controlling TNBC tumor development, recurrence, and metastasis in animals. The combination regimen should be poised for efficient translation into clinical trials for controlling the recurrence and metastasis, ultimately contributing to reducing mortality and improving TNBC patients’ quality of life.

Keywords: triple-negative breast cancer, reactive oxygen species, metastasis, recurrence, combination regimens

Introduction

Triple-negative breast cancer (TNBC) is an aggressive, lethal, and heterogeneous type of breast cancer (BC). TNBC is defined by its lack of estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2 [1–3]. BC is the second leading cause of cancer deaths among women in North America and Europe; about 1 in 8 women in the United States (US) develop BC during their lifetime [4]. In 2020, more than 276,000 new cases of BC will emerge, resulting in >42,000 deaths in the US [5]. TNBC accounts for 15 – 20% of BCs and has lower 5year survival rates (~77%) than other BC types (84 – 94%), due to cancer recurrence and metastasis [3, 6].

In conventional chemotherapies, DNA damaging agents (cisplatin, carboplatin, cyclophosphamide, etc.), DNA synthesis inhibitors (gemcitabine, fluorouracil, etc.), topoisomerase inhibitors (doxorubicin, epirubicin, etc.), and microtubule inhibitors (taxane, vinorelbine, eribulin, etc.) are approved by the Food and Drug Administration (FDA) to treat TNBC [2, 3]. In general, TNBC is initially responsive to the conventional agents; however, >50% of cases are recurrent, and the 5-year survival rate of these TNBC patients is less than 65% [7]. Molecular analysis revealed heterogeneous subtypes of TNBC, and the TNBC heterogeneity conceivably accounts for diverse biological behaviors and its differential responses to conventional chemotherapies [1–3]. Compared to single agent treatments, combination regimens, such as capecitabine+docetaxel and Gem+paclitaxel, have shown improved survival rates for metastatic TNBC patients [8, 9]. In addition, immunotherapeutic regimens, such as atezolizumab plus nab-paclitaxel, are recently available to the control of metastatic TNBC; however, these regimens are efficacious in controlling PD-L1-positive TNBC [10, 11]. Thus, advanced combination therapeutic regimens are needed to address the problem of TNBC’s heterogeneity, recurrence, and metastasis in order to further increase patients’ survival. We have been investigating anticancer agents to identify combination regimens effective in controlling TNBC cells in vitro and cell-derived xenograft (CDX) tumors in animals. Our studies [12–19] have revealed that the histone deacetylase inhibitor romidepsin (Rom, also FK228) preferentially induces apoptosis and reduces clonogenic survival/drug resistance particularly in Ras-ERK-activated cancer cells. Rom is FDA-approved to treat T-cell lymphoma; however, its value in treating solid tumors has yet to be determined [2, 20, 21]. We identified that a combination of Rom and cisplatin (Rom+Cis) synergistically induces apoptosis and reduces the clonogenic resistance of BC, urothelial carcinoma, and colorectal cancer cells [12, 20]. Cis is a platinum-based DNA-damaging agent that is highly effective in treating BRCA- and PARP-deficient TNBC [23–25]. The Rom+Cis regimen is currently undergoing clinical trials for treating metastatic TNBC [2, 26]. Our studies furthered identified that a triple combination of gemcitabine (Gem) and Rom+Cis (Gem+Rom+Cis) not only synergistically induces BC cell death and reduces clonogenic resistance, but this combination also shows higher efficacy than Rom+Cis to control TNBC cells in vitro and CDX tumor development in animals [27]. Gem+Rom+Cis is able to induce the elevation of reactive oxygen species (ROS), the activation of caspase 3/7, and apoptotic cell death in the TNBC MDA-MB231 cells [27]. Blockage of ROS significantly suppresses Gem+Rom+Cis-induced caspase activation and apoptosis, indicating that ROS elevation plays an essential role in Gem+Rom+Cis-induced caspases and apoptosis [27]. Gem is a DNA synthesis-inhibiting agent [2, 28]. The Gem+Cis regimen shows an overall response rate at ~30% in controlling metastatic TNBC pretreated with taxane and anthracycline [29, 30]. A phase I trial reported a partial response of BC tumors to Rom+Gem with additive hematologic toxicities [31]. Results from clinical studies of Rom, in combination with other agents such as paclitaxel for BCs, were not conclusive [32, 33]. Our study demonstrated formulation of a safe regimen and protocol for Gem+Rom+Cis, resulting in the Gem plus Rom+Cis regimen, by adjusting treatment intervals and reducing drug doses to alleviate drug-related dose-limiting adverse effects in mice [27]. The Gem plus Rom+Cis regimen is highly efficacious in controlling MDA-MB231 CDX tumor development in mice. Accordingly, the value of Gem plus Rom+Cis regimen should be further determined in the control of TNBC recurrence and metastasis.

In this communication, we initially extended our investigation of the triple combination Gem+Rom+Cis in controlling survivability and invasion of TNBC cells in vitro. We subsequently demonstrated the efficacy of Gem plus Rom+Cis regimen in controlling the development and recurrence of CDXs and the metastasis of TNBC cells in animals.

Materials and Methods

Cell cultures and Reagents

The human TNBC MDA-MB231 and MDA-MB468 cells (American Type Culture Collection [ATCC], Rockville, MD) were maintained in DMEM with 10% heat-inactivated fetal bovine serum, 100 U/mL penicillin, and 100 μg/mL streptomycin [14]. Cultures were maintained in 5% CO2 at 37 °C and subcultured every 2–3 days. Stock solutions of Gem, Rom, and Cis (Medkoo, Chapel Hill, NC, USA) were prepared in DMSO. Stock solution of N-acetyl-Lcysteine (NAC) (Alexis, San Diego, CA, USA) was prepared in distilled water. Stock solutions were diluted in culture medium for assays [17].

Cell viability

Cultured cells were treated with anticancer agents for 48 h. A Methyl Thiazolyl Tetrazolium (MTT) assay kit (Travigen, Gaithersburg, MD, USA) was used to quantify cell viability with an enzyme-linked immunosorbent assay plate reader (Bio-Tek, Winooski, VT, USA) at 570 nm [16, 17]. Relative values of cell viability in treated cultures were normalized by the value determined in untreated counterpart cells, set as 100%.

Soft agar colony formation assay

1 × 104 cells were mixed with 0.4% low-melting agarose (Sigma-Aldrich) in a mixture (1:1) of medium and plated on top of a 2% low-melting agarose base layer in 60-mm culture dishes. Cultures were maintained for 14 days to develop 3-dimensional (3D) cell colonies.

Transwell invasion assay

The cell invasion assay was performed using 24-well transwell insert chambers with a polycarbonate filter with a pore size of 8.0 μm (Costar, Corning, NY). Two × 104 cells in serumfree medium were seeded on top of a Matrigel-coated filter (BD Biosciences, Franklin Lakes, NJ) in each insert chamber. Then, the insert chambers were placed into wells on top of culture medium containing 10% fetal bovine serum as a chemoattractant. The invasive ability of cells was determined by the number of cells translocated to the lower side of filters.

Immunoblotting

Cell lysates were prepared as shown previously [19], and protein concentrations were measured using the BCA assay (Thermo, Rockford, IL, USA). Equal amounts of cellular proteins were resolved by electrophoresis in 10% SDS–polyacrylamide gels and transferred to nitrocellulose filters for immunoblotting, using specific antibodies to detect Matrix metallopeptidase 9 (MMP9), E-cadherin (ECAD), vimentin (VIM), Thioredoxin-1 (TRX-1) (Cell Signaling Technology, Danvers, MA, USA), vascular endothelial growth factor (VEGF), and βactin (Santa Cruz, Santa Cruz, CA, USA). Antigen-antibody complexes on filters were detected by the SuperSignal West Dura kit (Thermo).

Tumor recurrence animal model

5 to 6 weeks old, female immuno-deficient athymic nu/nu (nude) mice (Envigo, Indianapolis, IN, USA) were used to develop CDXs. In brief, 5 × 106 MDA-MB231 cells were mixed with Matrigel basement membrane matrix (BD) and inoculated subcutaneously into mammalian fat pad area of each mice. Each cohort contained 3 mice calculated for power analysis (at a power of 80%) to detect a difference in tumor size of 100 ± 40 mm3 in the study. Tumor volume was determined with a formula (length × width2 × ½) [34].

Tumor metastasis animal model

5 × 105 MDA-MB231 cells suspended in 100 μL of PBS were injected intravenously into 5 to 6 weeks old female nude mice (Envigo) through the tail vein. Each cohort contained 4 mice calculated for power analysis (at a power of 80%) to detect a difference in metastatic nodules of 200 ± 100 in the study.

Animals

Mice were housed in sterile cages in a temperature-controlled room with 12 h light-dark cycle at the University of Tennessee Laboratory Animal Facility. Mice were provided with irradiated diet and water ad libitum. Isoflurane (3–5 %) (Zoetis, NJ, USA) was used as an anesthesia by inhalation during inoculation. Animals were euthanized by CO2 exposure followed by cervical dislocation. All animal procedures were approved by the University of Tennessee Animal Care and Use Committee and were following the NIH Guide for the Care and Use of Laboratory.

Histological examination

Tissues were isolated, fixed in neutral-buffered formalin, and embedded in paraffin, followed by hematoxylin and eosin staining of tissue sections for histopathological examination.

Statistical analysis

A Student t-test was used to analyze statistical significance. A multiple comparison analysis was evaluated using one-way ANOVA followed by post-hoc Tukey’s multiple comparison test. Mann-Whitney U-test was used for Nonparametric test. Statistical analysis was performed using SPSS software (IBM, Armonk, NY, USA). Statistical significance was indicated by *p < 0.05, **p < 0.01, ***p < 0.001; a p value < 0.05 was considered significant. Combination indices analysis was performed using the method by Chou and Talalay [35] via the CompuSyn software suite (Paramus, NJ, USA). Combination indices <1, =1, and >1 indicate synergistic, additive, and antagonistic effects, respectively.

Results

1. Gem+Rom+Cis controlled cancerous properties of TNBC cells in vitro

Continuing to determine the ability of Gem+Rom+Cis to control TNBC, we investigated the effectiveness of Gem+Rom+Cis in controlling the cancerous properties of cellular invasion, survival, and anchorage-independent growth, as well as related signaling modulators in MDAMB231 and MDA-MB468 cells. As shown in Fig. 1a & 1b, a combination of Gem, Rom, and Cis synergistically reduced viability of MDA-MB231 and MDA-MB468 cells [27]. We used the results of Gem+Rom+Cis-reduced cell viability to normalize the results of colony formation and cell invasion.

Figure 1. Gem+Rom+Cis controlled cancerous properties of TNBC cells in vitro.

Figure 1.

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(a) MDA-MB231 and MDA-MB468 cells were treated with Gem, Rom, Cis, or a combination of Gem+Rom+Cis at their IC10 doses for 48 h. MTT assay kit was used to determine cell viability. Relative cell viability was normalized by the value determined in untreated counterpart cells set as 100%. (b) The combination indices, < 1, were determined to reveal synergistic effects of Gem+Rom+Cis on reducing cell viability (a). (c, d, & e) MDA-MB231 and MDA-MB468 cells were treated with Gem+Rom+Cis (G+R+C) at IC10 doses for 48 h, followed by soft agar colony formation assay to determine the clonogenic survival of cells. Cell colonies ≥ 0.1 mm diameter were counted. Numbers of colonies were normalized with cell viability (a), and then relative colony formation was normalized by the value determined in untreated counterpart cultures, set as 100%. (d) Representative images of the wells in colony formation assay. (f & g) MDAMB231 and MDA-MB468 cells were treated with G+R+C at IC10 doses for 24 h, followed by transwell invasion assay to determine relative invasive activities of cells by counting the numbers of cells translocated through a matrigel-coated filter in 10 arbitrary visual fields. Numbers of invaded cells were normalized by cell viability (a), then normalized by the value determined in untreated counterpart cultures, set as 100%. Columns, mean of triplicates; bars, SD. Statistical significance were determined using student’s t-test (a, e, & g), indicated by *p < 0.05, **p < 0.01, ***p < 0.001. (h) Cells were treated with G+R+C in the presence or absence of 5 μM NAC for 48 h. Cell lysates were prepared and analyzed by immunoblotting using specific antibodies to detect E-cadherin (ECAD), vimentin (VIM), matrix metallopeptidase 9 (MMP9), vascular endothelial growth factor (VEGF), with β-actin as a control. The levels of these modulators were quantified by densitometry and normalized with the level of β-actin set in control cells as 1 (X, arbitrary unit). All results are representative of three independent experiments.

Cellular adhesion to the extracellular matrix is required for normal epithelial cell survival. In contrast, cancerous cells acquire the ability of anchorage-independent growth (AIG) to increase survivability. The AIG is also associated with self-renewal ability of cancer cells [36]. Using the soft agar colony formation assay, we studied the ability of Gem+Rom+Cis to control clonogenic survival as an index for cancer recurrence [37] of TNBC cells. Our results revealed that Gem+Rom+Cis was able to reduce the clonogenic survival of MDA-MB231 and MDA-MB468 cells to ~50% and ~15%, respectively (Fig. 1c, 1d, & 1e), after normalization with the results of cell viability. The results indicated that Gem+Rom+Cis was effective in controlling clonogenic survival/recurrence of these TNBC cells.

Invasiveness is an important hallmark of cancer cells for their malignancy [38]. Our studies revealed that Gem+Rom+Cis was able to reduce the invasive ability of MDA-MB231 and MDA-MB468 cells to ~31% and ~13%, respectively (Fig. 1f & 1g), after normalization with the results of cell viability (Fig. 1a). The result of our research indicated that Gem+Rom+Cis was effective in controlling invasiveness of these TNBC cells.

To understand signaling modulators involved in Gem+Rom+Cis-reduced clonogenic survival and invasiveness, we studied the epithelial-mesenchymal transition (EMT) program associated with cancer cell malignancy and metastasis [39]. During EMT, decreased E-cadherin (ECAD) is associated with a loss in cell-cell adhesion, and increased Vimentin (VIM) plays a role in filament formation and cell motility [39]. The Gem+Rom+Cis treatment resulted in an elevation of ECAD in MDA-MB231, but not in MDA-MB468; however, Gem+Rom+Cis treatment reduced VIM in both MDA-MB231 and MDA-MB468 cells (Fig. 1h). On the other hand, Matrix metallopeptidase 9 (MMP9) is known to enhance invasion and metastasis by degrading the extracellular matrix [40], while vascular endothelial growth factor (VEGF) promotes angiogenesis and supports metastasis [41]. We detected that Gem+Rom+Cis treatment suppressed MMP9 and VEGF in MDA-MB231 and MDA-MB468 cells (Fig. 1h). These results indicated that Gem+Rom+Cis treatment may suppress recurrence and metastasis of TNBC cells by modulating the EMT program.

Our studies showed that induction of reactive oxygen species (ROS) is essential for the anticancer activity of Gem+Rom+Cis [27, 42]. Using the ROS inhibitor N-acetyl cysteine (NAC), we detected that blockage of ROS abrogated the Gem+Rom+Cis-induced ECAD and alleviated Gem+Rom+Cis-reduced VIM, MMP9, and VEGF (Fig. 1h). These results indicated that Gem+Rom+Cis treatment modulated the EMT program in an ROS-dependent manner.

2. Gem plus Rom+Cis completely suppressed tumor recurrence.

Our previous reports showed that the Gem plus Rom+Cis regimen is effective in controlling CDX tumor development of TNBC and urothelial carcinomas [27, 42]. To determine the ability of the Gem plus Rom+Cis regimen to control tumor recurrence, we implanted MDAMB231 subcutaneously into the mammalian fat pad area of nude mice. Mice developing CDX tumors reaching ~18 mm3 were admitted into the treatment study (Fig. 2a). Mice were administered (i.p.) with PBS as a control vehicle (V), Gem plus Cis regimen (G plus C), or Gem plus Rom+Cis regimen (G plus R+C) for 5 cycles. Body weight loss was used to detect adverse side effects [43]. We observed that mice administered with combination regimens induced a reversible body weight loss after the first 2 consecutive cycles of treatment, and 1 or 2 days of interval between the rest of treatment cycles helped mice regain body weight (Fig. 2b). Thus, the determined regimen-administering schedule and doses were tolerable and safe to mice. In order to study the efficacy of combination regimens in controlling CDX development of MDA-MB231 cells, we measured tumor volume and performed histo-pathological examination during necropsy 8 days after the last treatment (day 22). We detected that both the Gem plus Cis and the Gem plus Rom+Cis regimen were highly efficacious in controlling CDX development throughout treatment cycles; however, tumors recurred in animals after treatments with the Gem plus Cis regimen (Fig. 2c). In contrast, the Gem plus Rom+Cis regimen completely suppressed tumor recurrence after completing the 5 cycles of treatment (Fig. 2c). Analysis of the final tumor volumes revealed a growth of CDXs in 22 days to ~560% of their original volume in mice treated with vehicle, and tumor growth was suppressed but recurred to ~460% of their original volume in mice treated with Gem plus Cis (Fig. 2d). In contrast, treatment with the Gem plus Rom+Cis regimen efficaciously suppressed tumor growth and recurrence and reduced tumor size to ~40% of their original volume. Comparing tumor weights (final T/C: 0.1/1) verified the efficacy of Gem plus Rom+Cis in controlling CDX development and recurrence (Fig. 2d). Calculation of the tumor growth inhibition rates (TGI, %) furthered revealed that the Gem plus Rom+Cis regimen (112%) was more efficacious than the Gem plus Cis regimen (21%) in controlling TNBC CDX recurrence.

Figure 2. Gem plus Rom+Cis suppressed CDX tumor recurrence in mice.

Figure 2.

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5 × 106 MDAMB231 cells were mixed with Matrigel and inoculated into mammalian fat pad areas of nude mice. Tumor volume was measured with a caliper and determined with a formula (length × width2 × ½). Mice developing CDXs reaching ~18 mm3 were entered into the treatment study (a to c, day 1). Nude mice, 3 per group, were injected (i.p.) with PBS (V, Control), 20 mg/kg Gem followed by 5 mg/kg Cis (Gem plus Cis, G plus C), or 20 mg/kg Gem followed by 1 mg/kg Rom mixed with 5 mg/kg Cis (Gem plus Rom+Cis, G plus R+C) for 5 treatment cycles at indicated (a). (b) Body weight was measured every 2 days to reveal adverse side effects of a regimen on animals. (c) Tumor volume was measured at indicated days. Mice were histo-pathologically examined during necropsy at day 22. (d) Tumor volume at days 1 and 22 were presented in mean ± SD. Changes of tumor volume (%) were calculated by T22 (tumor volume determined at day 22)/T1 (tumor volume determined at day 1). The average weight of tumors isolated at day 22 was measured, mean ± SD. Final tumor/control ratios (T/C) were calculated by T (mean tumor weight of treatment group)/C (mean tumor weight of control group) of tumors isolated from mice at day 22. Tumor growth inhibition rate (TGI, %) was calculated by 1-(T22/T1 / C22/C1).

Statistical significance was determined by Tukey’s multiple comparison test. Statistical significance, indicated by *p < 0.05, **p < 0.01. (e) Representative CDX tumors were shown. (f) Histological features of tumors isolated from mice treated with V, G plus C, or G plus R+C were shown. White arrows indicate mitotic cells, and black arrows indicate necrosis area (irreversible damage). Images were taken at 400x; scale bar, 50 μm. (g) Lysates were prepared from MDAMB231 cultured cells, CDX tumor tissues isolated from mice treated with V, G plus C, or G plus R+C. Cell and tumor lysates were then analyzed by immunoblotting using specific antibodies to detect levels of TRX-1, ECAD, VIM, MMP9, VEGF, with β-actin. Levels of these modulators were quantified by densitometry, normalized with the level of β-actin, and the level set in MDAMB231 cultured cells as 1 (X, arbitrary unit).

Histological examination of isolated tumors (Fig. 2e) revealed that necrosis area and mitotic figure were detectably higher and lower in tumors, respectively, isolated from mice treated with Gem plus Rom+Cis than in mice treated with vehicle or Gem+Cis (Fig. 2f). The results indicated that the Gem plus Rom+Cis regimen inhibited proliferation and induced death of tumor cells more efficaciously than Gem plus Cis regimen.

Thioredoxin-1 (TRX-1) is an antioxidant protein, plays an important role in regulation of ROS, and is positively associated with drug resistance and cancer recurrence [44]. We detected the TRX-1 level was lower in tumors isolated from mice treated with Gem plus Rom+Cis than mice treated with Gem plus Cis or control vehicle (Fig. 2g). We also detected EMT markers modulated by Gem plus Cis and Gem plus Rom+Cis regimens in isolated tumors. Interestingly, the EMT marker ECAD was highly increased in tumors than in cultured parental MDA-MB231 cells, and ECAD level was reduced or increased in tumors isolated from mice treated with Gem plus Cis and Gem plus Rom+Cis, respectively (Fig. 2g). VIM and MMP9 were reduced in tumors isolated from mice treated with Gem plus Cis and Gem plus Rom+Cis, and Gem plus Rom+Cis treatment reduced VIM and MMP9 more than Gem plus Cis. In addition, we detected that VEGF increased in tumors more than in cultured parental MDA-MB231 cells and was reduced in tumors isolated from mice treated with Gem plus Cis and Gem plus Rom+Cis. Gem plus Rom+Cis reduced VEGF more than Gem plus Cis (Fig. 2g). These results indicated the Gem plus Rom+Cis regimen was effective in controlling signaling modulators involved in drug resistance, EMT, and angiogenesis that are known to support tumor recurrence and metastasis.

3. Gem plus Rom+Cis controlled metastasis in animals.

To study the efficacy of Gem plus Rom+Cis regimen in controlling tumor metastasis, we injected MDA-MB231 cells intravenously via the tail vein into nude mice [45]. After 30 days of injection, we administered mice 5 cycles of the Gem plus Rom+Cis regimen (Fig. 3a). During necropsy on day 38, 24 days after the last cycle of treatment, histo-pathological examination of animals revealed that tumor nodules were visually detectable in the lungs, lymph nodes, ovaries, and abdominal wall in all the vehicle-treated, MDA-MB231-injected mice (Fig. 3b). Histological examination identified tumor tissues in these organs (Fig. 3c). The number of nodules detected in the lungs, lymph nodes, ovaries, and abdominal wall of the control group were 166, 15, 6, and 20, respectively (Fig. 3d). Forty-eight tumor nodules were detected in the lung of only 1 out of 4 mice treated with Gem plus Rom+Cis (Fig. 3d). The total numbers of tumor nodules detected in mice treated with control vehicle and Gem plus Rom+Cis regimen were 207 vs. 48, respectively (Fig. 3d). The result indicated that the Gem plus Rom+Cis regimen efficaciously suppressed tumor metastasis of the TNBC MDA-MB231 cells to 20% in animals.

Figure 3. Gem plus Rom+Cis controlled metastasis in animals.

Figure 3.

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(a) Nude mice, 4 per group, were injected (i.v.) through tail-veins with 5 × 105 MDA-MB231 cells/mouse. Thirty days after cell injection, mice were injected (i.p.) with vehicle (V) or G plus R+C at indicated days. Histopathological examination was performed during necropsy at day 38, and metastatic tumor nodules were counted. (b) Representative tumor nodules were detected on various organs of mice treated with V or G plus R+C. (c) Histological features of representative metastatic tumors isolated from lungs, lymph nodes, ovaries, and abdominal walls of control (V) and mice treated with G plus R+C were shown. Black arrows indicate tumor areas, while white arrows indicate non-tumor areas. Images were taken at 100x; scale bar, 50 μm. (d) Tumor nodules visually detected in each group (4 mice) were counted. The total number is the combined number of metastatic nodules from the lung, lymph nodes, ovary, and abdominal wall examined in each group. Efficacy (%) in inhibition of metastatic tumors was normalized by the number of metastatic nodules determined in the vehicle-treated group, set as 100%. Statistical significance was determined by Mann-Whitney U-test, indicated by *p < 0.05.

Discussion

In this communication, we demonstrated, for the first time, that the triple combination Gem plus Rom+Cis regimen was highly efficacious and more efficacious than the double combination Gem plus Cis regimen in controlling CDX development and recurrence of the TNBC MDA-MB231 cells in mice. Histological examination supported that Gem plus Rom+Cis was more effective than Gem plus Cis in inducing growth inhibition and individual cell death of tumor tissues in animals. The Gem plus Rom+Cis regimen was also highly efficacious in controlling the tumor metastasis of MDA-MB231 cells in mice. Our results of animal studies strongly suggested that the Gem plus Rom+Cis regimen should be seriously considered to control TNBC tumor development, recurrence, and metastasis.

Our in vitro studies indicated that Gem+Rom+Cis was able to synergistically reduce cell viability and effectively reduce survivability (3D colony formation) and invasive ability of the TNBC MDA-MB231 and MDA-MB468 cells. Our in vitro studies also revealed that Gem+Rom+Cis treatment suppressed the EMT program, including increased E-cadherin and reduced vimentin, suppressed the invasive modulator MMP9, and suppressed the angiogenesis modulator VEGF in TNBC cells in an ROS-dependent manner. These results were consistent with the results of these modulators regulated in tumor tissues of MDA-MB231 CDXs isolated from mice treated with control vehicle, Gem plus Cis, and Gem plus Rom+Cis. In addition, the antioxidant modulator TRX-1, involved in drug resistance and cancer recurrence, was substantially suppressed in tumor tissues of mice treated with the Gem plus Rom+Cis regimen, as compared to mice treated with control vehicle or the Gem plus Cis regimen. Gem and Cis are known to induce drug resistance through induction of PARP-involved DNA repair and ROS elevation for cell survival [46]. Our published report [27] revealed the ability of Gem+Rom+Cis to induce proteolysis of PARP and lethal levels of ROS, thereby suppressing the Gem+Cisinduced, PARP-involved DNA repair and survival-related ROS. These data taken together lead us to suggest that the Gem plus Rom+Cis regimen was complementary to effectively induce tumor cell death and suppress tumor cell proliferation, invasion, angiogenesis, drug resistance, and the EMT program, leading to efficaciously control tumor development, recurrence, and metastasis. However, our study revealed that Gem+Rom+Cis did not induce E-cadherin level in MDA-MB468 cells as induced in MDA-MB231 cells. Studies showed the methylation CpG islands distributed differently in MDA-MB231 and MDA-MB468 cells [47]. Promoter of the Ecadherin gene is regulated by methylation of CpG island in MDA-MB231 but not in MDAMB468 cells [48, 49]. Histone deacetylase inhibitor treatment has been shown to reactivate genes that are suppressed by CpG methylation [50–52]. Accordingly, it is conceivable that Gem+Rom+Cis, in which Rom is a histone deacetylase inhibitor, may regulate expression of Ecadherin, via activation of CpG in MDA-MB231 but not in MDA-MB468 cells. Although Ecadherin level was not modulated by Gem+Rom+Cis in MDA-MB468 cells, MDA-MB468 cells appeared to be more susceptible than MDA-MB231 cells to Gem+Rom+Cis for suppressing clonogenic survival and invasiveness. Whether E-cadherin played a minor role in the EMT program, clonogenic survival, or invasiveness modulated by Gem+Rom+Cis in MDA-MB468 cells remained to be clarified.

Conclusion

The overall survival rate of TNBC has not been significantly improved by recent chemotherapies for 20 years [53]. A recent clinical study reported a similar overall response rate of controlling recurrent TNBC with the double combination Gem plus Cis regimen to the Gem plus paclitaxel regimen [54]. Our studies demonstrated that the tolerable triple combination Gem plus Rom+Cis regimen was not only highly efficacious in controlling TNBC metastasis but also more efficacious than the double Gem plus Cis regimen in the control of TNBC CDX development and recurrence in animals. Gem, Rom, and Cis are already FDA-approved anticancer agents. Thus, the regimen should be poised for efficient translation into clinical trials for controlling the recurrence and metastasis, ultimately contributing to reducing mortality and improving TNBC patients’ quality of life.

Acknowledgments

We are grateful to Dr. Robert Donnell for his support in histological analysis.

Declarations Funding

This study was supported by the National Institutes of Health [CA177834 to H-C.R. W.] and the University of Tennessee, Center of Excellence in Livestock Diseases and Human Health [HC.R.W.].

Abbreviations:

3D

3-dimensional

AIG

Anchorage-independent growth

ATCC

American Type Culture Collection

BC

Breast Cancer

CDX

Cell-derived xenograft

Cis

Cisplatin

DMEM

Dulbecco’s Modified Eagle Medium

ECAD

E-cadherin

EMT

Epithelial to Mesenchymal transition

FDA

Food and Drug Administration

Gem

Gemcitabine

IC

Inhibitory concentration

IP

Intraperitoneal

IV

Intravenous

MDA-MB

MD Anderson-Metastatic Breast

MMP9

Matrix metallopeptidase 9

MTT

Methyl thiazolyl tetrazolium

NAC

N-acetyl-L-cysteine

NIH

National institutes of health

PBS

Phosphate-buffered saline

Rom

Romidepsin

ROS

Reactive oxygen species

TNBC

Triple-negative breast cancer

TRX-1

Thioredoxin-1

VEGF

Vascular endothelial growth factor

VIM

Vimentin

Footnotes

Conflicts of interest/Competing interests The authors declare no conflicts of interest.

Ethics approval

All animal procedures were approved by the University of Tennessee Animal Care and Use Committee and were in accordance with the NIH Guide for the Care and Use of Laboratory Animals.

Availability of data and material

All the data related to this study are included in this article.

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