Suberoylanilide hydroxamic acid blocks self-renewal and homotypic aggregation of inflammatory breast cancer spheroids

Abstract

Background: Inflammatory breast cancer (IBC) is the most aggressive form of locally advanced breast cancer (LABC). Patients with IBC commonly present with skin metastasis, which are observed microscopically as tumor emboli within dermal lymphatics. These metastatic tumor cells aberrantly overexpress E-cadherin and exhibit the ability to undergo self-renewal and are highly invasive. There are no therapeutics yet identified that target the structure and functions of IBC tumor emboli. The present studies evaluated the effects of the pan-histone deacetylase (HDAC) inhibitor suberoylanilide hydroxamic acid (SAHA) using IBC tumor spheroids derived from established IBC cell lines and tumor spheroids derived from pleural effusion (PE) aspirates of patients with IBC and LABC, designated as PE-IBC and PE-LABC.

Methods: Methods used are as follows: culture of IBC cells from clonal density single cells in low adherence culture conditions that promote formation of IBC tumor spheroids; clonogenic assays; cell fractionation and Western blotting; confocal microscropy; and modified Boyden chamber invasion assays.

Results: SAHA inhibited self-renewal of IBC tumor spheroids from established IBC cell lines and PE-IBC and PE-LABC, as assessed by decreased clonogenic growth. SAHA blocked homotypic aggregation of the cells that comprised the IBC tumor spheroids leading to loss of their 3-dimensional (3D) structure, which was associated with a change in location of E-cadherin protein from the plasma membrane in untreated IBC tumor spheroids to the cytoplasm of cells within IBC tumor spheroids with SAHA treatment. In addition, SAHA blocked the robust invasion exhibited by IBC tumor spheroids of established cell lines as well as by tumor spheroids derived from PE-IBC and PE-LABC.

Conclusions: SAHA targets the integrity and biological activities of IBC tumor spheroids and may be a promising agent to evaluate for its effectiveness in treatment of IBC.

Copyright 2010 American Cancer Society.

Figures

Figure 1. A and B. SAHA inhibited the homotypic aggregation and 3D integrity of IBC tumor spheroids

Phase contrast micrographs revealed that SAHA induced a dose dependent inhibition of the homotypic aggregation of cells within SUM149 (Figure 1 A) and SUM190 tumor spheroids (Figure 1 B) with a loss of their 3D integrity of both SUM149 and SUM190 tumor spheroids (Figure 1 A and B).

Figure 2. A–E. SAHA induced translocation of E-cadherin protein into the cytoplasm of IBC tumor spheroids

Figure 2 A and B. Western blotting combined with densitometric analysis revealed that SUM149 cells produced approximately 2-fold greater amounts of E-cadherin protein when cultured as 2D adherent cells compared to levels of E-cadherin protein produced by SUM149 cells cultured as 3D tumor spheroids. This appears to be due to the presence of a glycosylated protein product in 2D adherent SUM149 cells. The amount of protein produced by SUM190 cells was equivalent in 2D adherent compared to 3D tumor spheroids. Figure 2 C and D. SAHA treatment did not alter the total amount of E-cadherin protein produced by either SUM149 or SUM190 tumor spheroids. Figure 2 E and F. Cell fractionation combined with Western blotting and densitrometry analysis revealed that SAHA altered the location of E-cadherin protein from the plasma membrane to the cytoplasm without altering the total amount of E-cadherin protein.

Figure 3. A–F. SAHA altered the location of E-cadherin protein and blocked homotypic aggregation of IBC tumor spheroids

Figure 3 A–C. E-cadherin protein (red fluorescence) was visualized at the adherens junctions of SUM190 cells that comprise the IBC tumor spheroids. Blue fluorescence is associated with nuclear DNA stained with TO-PRO-3. Figure 3 C is the merged image showing E-cadherin protein localization at the plasma membrane with nuclear staining shown in blue. Figure 3 D–F. Following exposure to 5 mM SAHA for 6 hrs, E-cadherin protein was diminished at the adherens junctions at the plasma membrane, with visible E-cadherin protein within the cytoplasm. The decreased E-cadherin protein staining at the cell surface was associated with the loss of homotypic aggregation of cells within the tumor spheroid, leading to disintegration of the tumor spheroid structure.

Figure 4. SAHA blocks invasion of IBC tumor spheroids from established IBC cells lines and from PE-IBC and PE-LABC

SAHA significantly inhibited invasion of IBC tumor spheroids derived from established cell lines and from pleural effusion cells isolated from a patient with IBC (PE-IBC) and patient with LABC (PE-LABC).