Cellular Adhesion Promotes Prostate Cancer Cells Escape from Dormancy

<div><p>Dissemination of prostate cancer (PCa) cells to the bone marrow is an early event in the disease process. In some patients, disseminated tumor cells (DTC) proliferate to form active metastases after a prolonged period of undetectable disease known as tumor dormancy. Identifying mechanisms of PCa dormancy and reactivation remain a challenge partly due to the lack of <i>in vitro</i> models. Here, we characterized <i>in vitro</i> PCa dormancy-reactivation by inducing cells from three patient-derived xenograft (PDX) lines to proliferate through tumor cell contact with each other and with bone marrow stroma. Proliferating PCa cells demonstrated tumor cell-cell contact and integrin clustering by immunofluorescence. Global gene expression analyses on proliferating cells cultured on bone marrow stroma revealed a downregulation of TGFB2 in all of the three proliferating PCa PDX lines when compared to their non-proliferating counterparts. Furthermore, constitutive activation of myosin light chain kinase (MLCK), a downstream effector of integrin-beta1 and TGF-beta2, in non-proliferating cells promoted cell proliferation. This cell proliferation was associated with an upregulation of CDK6 and a downregulation of E2F4. Taken together, our data provide the first clinically relevant <i>in vitro</i> model to support cellular adhesion and downregulation of TGFB2 as a potential mechanism by which PCa cells may escape from dormancy. Targeting the TGF-beta2-associated mechanism could provide novel opportunities to prevent lethal PCa metastasis.</p></div

Constitutive activation of MLCK promotes proliferation of LuCaP PDX cells via upregulation of CDK6.

<p>A) LuCaP cells were infected with lentivirus containing A-tMK (constitutively activate MLCK) showed positive Ki67 staining, whereas cells transduced with an empty vector did not. B) In LuCaP 86.2, 92, and 93, ectopic expression of A-tMK induced an upregulation of CDK6 and a concurrent downregulation of E2F4 when compared to that of the empty vector-transduced cells. Inhibition of MLCK with the MLCK inhibitor ML-7 suppressed proliferation by C) abolishing Ki67 expression, D) decreasing cell viability assessed by WST-1 assay and E) downregulating CDK6 expression. E2F4 expression was not altered by the ML-7. Green, EpCAM; Red, Ki67; Blue, DAPI. Magnification: 200x. Scale bar: 20 μm. **p< 0.01 as compared to the DMSO control. CDK6: cyclin-dependent kinase 6; E2F4: E2F transcription factor 4.</p

CDK6 overexpression induced proliferation of LuCaP PDX cells <i>in vitro</i>.

<p>LuCaP 86.2, 92 and 93 cells were lentivirally transduced to overexpress CDK6 and cultured <i>in vitro</i> to assess proliferation. Positive Ki67 indicated that CDK6 overexpression facilitated proliferation in these cells. Green, EpCAM; Red, Ki67; Blue, DAPI. Magnification: 200x. Scale bar: 20 μm.</p

Genes associated with cellular movement were downregulated in proliferating LuCaP cells.

<p>A) Heat map of hierarchically clustered differential gene expression in NG and G LuCaP PDX cells. Green, downregulated; red, upregulated. B) Ingenuity pathway analysis showing cellular movement was the top molecular and cellular function altered between NG and G cells. C) List of eight genes that were involved in the decreased activation of cellular movement in G when compared to NG cells. D) EDN1 was predicted to be the top regulator that affected the cell movement via downregulation of FN1, CDC42, and FOSL1. E) Quantitative real-time PCR showed a downregulation of FN1, CDC42 and TGFb2 in growing LuCaP lines. Data were normalized to the levels of housekeeping gene RPS15. NG: not growing; G: growing.</p