Targeting of Rac GTPases blocks the spread of intact human breast cancer

Abstract

High expression of Rac small GTPases in invasive breast ductal carcinoma is associated with poor prognosis, but its therapeutic value in human cancers is not clear. The aim of the current study was to determine the response of human primary breast cancers to Rac-based drug treatments ex vivo. Three-dimensional organotypic cultures were used to assess candidate therapeutic avenues in invasive breast cancers. Uniquely, in these primary cultures, the tumour is not disaggregated, with both epithelial and mesenchymal components maintained within a 3-dimensional matrix of type I collagen. EHT 1864, a small molecule inhibitor of Rac GTPases, prevents spread of breast cancers in this setting, and also reduces proliferation at the invading edge. Rac1+ epithelial cells in breast tumours also contain high levels of the phosphorylated form of the transcription factor STAT3. The small molecule Stattic inhibits activation of STAT3 and induces effects similar to those seen with EHT 1864. Pan-Rac inhibition of proliferation precedes down-regulation of STAT3 activity, defining it as the last step in Rac activation during human breast cancer invasion. Our data highlights the potential use of Rac and STAT3 inhibition in treatment of invasive human breast cancer and the benefit of studying novel cancer treatments using 3-dimensional primary tumour tissue explant cultures.

Figure 1. RAC1 is more highly expressed in breast cancer and is associated with poor prognosis

(A) Box plots demonstrating that invasive ductal carcinomas (IDC) have higher RAC1 expression than normal breast ducts, but range is similar. (B-C) RAC1 is not associated with estrogen receptor alpha (ER) status or histological grade of primary breast tumours. (D) Kaplan-Meier survival curves for all breast cancer patients with available data in the meta-dataset show that high levels of RAC1 expression are associated with poorer prognosis (n=1654). RAC1 expression levels were divided into low and high groups at the median. (E) An example of invasion ex vivo: 3D reconstruction of optical projection tomography of a representative ER+ HER2- tumour. The original tumour material is in blue (autofluorescence) and invading epithelial tumour cells are in green (cytokeratin labelled). (F) Immunohistochemistry showing that cells invading ex vivo express Rac1 protein (upper panel) and its short isoform Rac1b (lower panel). Rac1 and Rac1b are expressed in both tumour epithelial (arrowheads) and mesenchymal (arrows) invading cells. Images are from a representative ER- HER2- tumours. Lower magnification images are in Figure S2. Bars, 50 μm. The dotted lines show border between original tumour explant (OT) and surrounding collagen (SC). Brown staining indicates cells positive for Rac1 and Rac1b staining.

Figure 2. Rac inhibition ex vivo blocks tumour invasion

(A) Tumour invasion into surrounding collagen is readily detectable using H&E staining (upper panel). Continuous Rac inhibition with EHT1864 for 14d (bottom panel) results in blockage of invasion beyond the original tumour material (black outline) and extensive cell death: Bars, 50 μm. Outgrowth of tumour cultures were categorised following treatment with EHT 1864 or vehicle control, as follows: (B) continuous exposure from start of culture, or (C) exposure of existing outgrowths, 10d after start of culture. All inhibitor treatments lasted 14d. Results were obtained from light microscopy examination and subsequently confirmed by H&E staining. Tumour biomarkers shown were determined by a pathologist as part of clinical practice.

Figure 3. Rac inhibition with of tumour outgrowth ex vivo results in block of proliferation and cell death

(A) The effect of treatment with EHT 1864 or vehicle control on outgrowth of tumour cultures was obtained from light microscopy examination and subsequently confirmed by H&E staining. Shown are reperentative results from ER- HER2- tumour explant culture lasting 14d (n=4). Note that continuous EHT1854 treatment reduces viable outgrowth (regimen #4) in comparison to short-treatment (#2) or vehicle control (#1). Rac inhibition of tumour invasion is accompanied with induction of apoptosis as detected by immunohistochemistry for cleaved caspase-3 (B) and block of proliferation, detected by Ki67 staining (C). (D) E-cadherin levels are unchanged ex vivo. Shown here representative images from a single ER- HER2- tumour, (left panel) without or (right panel) with EHT 1864. The dotted lines show border between original tumour explant (OT) and surrounding collagen (SC). Bars, 50 μm.

Figure 4. STAT3 signaling is downstream of Rac activity during invasion of human breast cancer

(A) Immunohistochemistry shows that Rac1 protein expression (left panel) and STAT3 phosphorylation on Ser727 (right panel) frequently coincide in human breast cancer. Epithelial cells which are Rac1+ pSTAT3- are also present. (B) STAT3 phosphorylation is associated with invasion ex vivo and is down regulated by Rac inhibition (right panels; inhibition as in Figure 2). Identical patterns are seen with the STAT3 transcriptional target Survivin, ex vivo (C). Even after exposure to EHT 1864, a few cells are still positive for phospho-STAT3 and Survivin (arrows in B and C). Images are shown from a representative ER- HER2- tumour. Lower magnification images are in Figure S6. The dotted lines show border between original tumour explant (OT) and surrounding collagen (SC). Bars, 50 μm.

Figure 5. The early cellular response to Rac inhibition ex vivo

(A) Tumour explants were grown ex vivo for 14 days prior to inhibitor treatment in order to define events induced by a single post-growth treatment. Immunohistochemistry suggest that STAT3 phosphorylation (vehicle, left panels) is down regulated by EHT 1864 (middle panels) or Stattic (right panels) within 72 h of treatment. (B) Wide spread apoptosis is detected in Stattic treated cultures (right panel) and to lesser extent in EHT 1864 treated cultures (middle panel). Effects on proliferation are shown by staining with the marker Ki67 and (C) the cell cycle protein cyclin D1 (D): reduced in EHT 1864 (middle panel) or Stattic (right panel) exposed cultures). The dotted lines show border between original tumour explant (OT) and surrounding collagen (SC). Bars, 50 μm. Quantification is shown for percent of positive invading cells from two preparations per treatment, all originating from the same tumour.

Figure 6. STAT3 inhibition blocks tumour outgrowth ex vivo

Outgrowth of tumour cultures were categorised following treatment with Stattic or vehicle control, as follows: (A) continuous exposure from start of ex vivo culture, or (B) exposure of existing outgrowths, 10d after start of culture. All inhibitor treatments lasted 14d. Results were obtained from light microscopy examination and subsequently confirmed by H&E staining. Tumour biomarkers shown were determined by a pathologist as part of clinical practice.