Evaluation of the Mammalian Aquaporin Inhibitors Auphen and Z433927330 in Treating Breast Cancer

Abstract

AQPs contribute to breast cancer progression and metastasis. We previously found that genetic inhibition of Aqp7 reduces primary tumor burden and metastasis in breast cancer. In this study, we utilized two AQP inhibitors, Auphen and Z433927330, to evaluate the efficacy of therapeutic inhibition of AQPs in breast cancer treatment. The inhibitors were evaluated in breast cancer for both cytotoxicity and metabolic stability assays across both murine and human breast cancer cell lines. Both AQP inhibitors also affected the expression of other AQP transcripts and proteins, which demonstrates compensatory regulation between AQP family members. As a single agent, Auphen treatment in vivo extended overall survival but did not impact primary or metastatic tumor burden. However, Auphen treatment made cells more responsive to chemotherapy (doxorubicin) or endocrine treatment (tamoxifen, fulvestrant). In fact, treatment with Tamoxifen reduced overall AQP7 protein expression. RNA-seq of breast cancer cells treated with Auphen identified mitochondrial metabolism genes as impacted by Auphen and may contribute to reducing mammary tumor progression, lung metastasis, and increased therapeutic efficacy of endocrine therapy in breast cancer. Interestingly, we found that Auphen and tamoxifen cooperate to reduce breast cancer cell viability, which suggests that Auphen treatment makes the cells more susceptible to Tamoxifen. Together, this study highlights AQPs as therapeutic vulnerabilities of breast cancer metastasis that are promising and should be exploited. However, the pharmacologic results suggest additional chemical refinements and optimization of AQP inhibition are needed to make these AQP inhibitors appropriate to use for therapeutic benefit in overcoming endocrine therapy resistance.

Keywords: Auphen; Z433927330; aquaporin; breast cancer; inhibitor.

Scheme 1

Synthesis of Z433927330 and Auphen.

Figure 1

Cytotoxicity and stability of aquaporin inhibitors in vitro. (A) Structure of aquaporin inhibitors used in this study. (B,C) Viability of murine (B) and human (C) breast cancer cell lines after treatment with Auphen or Z433927330 for 3 days. Average of n = 3 independent experiments with at least technical triplicates. (D) Viability of HepG2 cells treated with Auphen and Z433927330. Representative image of two independent experiments with technical quadruplicates. (E) Metabolic stability of Z433927330 in rat and human liver microsomes. Each dot with error bars represents the average of three independent experiments +/− S.E.M.

Figure 2

AQP redundancy and off-target effects of aquaporin inhibitors in vitro. (A) RT-qPCR analysis of AQP expression in cell lines treated with Auphen or Z433927330 (IC50 concentration) after 3 days. Representative image of three independent experiments, each with technical triplicates +/− S.D. *, p < 0.05, **, p < 0.01, ***, p < 0.001, unpaired t-test. (B,C) Western blot analysis of breast cancer cell lines following three days of treatment with Auphen (B) and Z433927330 (C). Representative image of two independent experiments.

Figure 3

Auphen treatment of metastatic breast cancer cells increases overall survival in vivo. (A) Schematic of orthotopic injection of 4T1 cells in NOD SCID mice treated with either vehicle (n = 15) or Auphen (n = 15). (B) Growth of tumors in (A). (C) Kaplan–Meier plot showing overall survival (OS). p-values for OS were determined using the log-rank (Mantel–Cox) test. (D) Quantification of median lung metastasis using Welch’s t-test. (E) Schematic of orthotopic injection of 4T1 cells in BALB/cJ mice treated with either vehicle (n = 14) or Auphen (n = 14). (F) Tumor burden of (E). (G) Kaplan–Meier plot showing overall survival (OS) of (E). p-values for OS were determined using the log-rank (Mantel–Cox) test. (H) Quantification of median lung metastasis was analyzed using Goodman and Kruskal’s gamma test.

Figure 4

Z433927330 treatment of metastatic mammary tumors in vivo. (A) Schematic of orthotopic injection of 4T1 cells in syngeneic BALB/cJ mice treated with either vehicle (n = 4, weekly and biweekly) or Z433927330 (n = 4, weekly and biweekly). (B) Tumor growth curves for biweekly treatment. (C) Quantification of lung metastasis using Fisher’s exact test. (D) Kaplan–Meier plot showing overall survival for biweekly treatment. p-values for OS were determined using the log-rank (Mantel–Cox) test. (E) Tumor growth curves for weekly treatment. (F) Quantification of lung metastasis using Goodman and Kruskal’s gamma. (G) Kaplan–Meier plot showing overall survival for weekly treatment. p-values for OS were determined using the log-rank (Mantel–Cox) test.

Figure 5

AQP inhibition increases the antitumorigenic effects of breast cancer therapy in vitro. (A) Viability of MCF-7 and PyMT cells after treatment with Auphen and Doxorubicin alone or in combination after 3 days using CellTiter-Glo. (B) Viability of MCF-7 and PyMT after treatment with Auphen and tamoxifen as either single agents or in combination with the indicated endocrine therapy after 3 days using CellTiter-Glo. (C) Viability of MCF-7 and PyMT cells after treatment with Auphen and fulvestrant alone or in combination after 3 days using CellTiter-Glo. (A–C) Representative images of three independent experiments completed with technical quadruplicates +/− S.E.M. *, p < 0.05, **, p < 0.01, ***, p < 0.001, unpaired t-test.

Figure 6

Combination treatment of Auphen and tamoxifen in metastatic breast cancer cells inhibits primary tumor growth and metastasis but increases overall survival in vivo. (A) Schematic of orthotopic injection of PyMT cells in FVB mice treated with either vehicle, Auphen, or tamoxifen alone or in combination (n = 15 per cohort). (B) Tumor growth curves for tumors generated. (C) Kaplan–Meier plot showing overall survival (OS). p-values for OS (right panels) were determined using the log-rank test (Mantel–Cox) test. (D) Quantification of lung metastasis. Tables with detailed statistics corresponding to figures with statistically significant results are highlighted in red (B–D).

Figure 7

Proliferation and apoptosis do not change in response to single or combination treatment with Auphen and tamoxifen. (A) Immunohistochemistry of Ki67 and cleaved caspase-3 in tumors from FVB mice treated with either vehicle, Auphen, or tamoxifen alone or in combination. (B) Analysis of IHC staining of Ki67 and cleaved caspase-3. The data are shown as mean percent positive cells +/− S.E.M. and are representative of vehicle: n = 6; Auphen: n = 6; Tamoxifen: n = 6; Auphen + Tamoxifen: n = 6; and Tamoxifen + Auphen: n = 6. p values were calculated using two-way ANOVA.

Figure 8

Transcriptomic analysis of Auphen-treated cells. RNA-seq analysis of PyMT and MCF-7 cells treated with vehicle or Auphen for 3 days. (A) Volcano plot of differentially expressed genes in MCF-7 cells (FDR < 0.1 and p-value < 0.05). Genes that are significantly upregulated are represented as orange dots and significantly downregulated genes are shown in blue. (B) Top 20 GO terms from DAVID functional GO analysis for biological processes that resulted in downregulated and upregulated DEGs in MCF-7 cells. (C) Volcano plot of differentially expressed genes in PyMT cells (FDR < 0.1 and p-value < 0.05). Genes that are significantly upregulated are represented as orange dots and significantly downregulated genes are shown in blue. (D) Top 20 GO terms from DAVID functional GO analysis for biological processes that resulted in downregulated and upregulated DEGs in PyMT cells. (E) RT-qPCR analysis of PyMT cells +/− Auphen for selected genes of interest for RNA-seq confirmation. Average of n = 3 independent experiments (mean +/− S.E.M). *, p < 0.05, **, p < 0.01, ***, p < 0.001, unpaired t-test.