Proprotein convertase inhibition results in decreased skin cell proliferation, tumorigenesis, and metastasis

Abstract

PACE4 is a proprotein convertase (PC) responsible for cleaving and activating proteins that contribute to enhance tumor progression. PACE4 overexpression significantly increased the susceptibility to carcinogenesis, leading to enhanced tumor cell proliferation and premature degradation of the basement membrane. In the present study, we sought to evaluate a novel approach to retard skin tumor progression based on the inhibition of PACE4. We used decanoyl-RVKR-chloromethylketone (CMK), a small-molecule PC inhibitor, for in vitro and in vivo experiments. We found that CMK-dependent blockage of PACE4 activity in skin squamous cell carcinoma cell lines resulted in impaired insulin-like growth factor 1 receptor maturation, diminished its intrinsic tyrosine kinase activity, and decreased tumor cell proliferation. Two-stage skin chemical carcinogenesis experiments, together with topical applications of CMK, demonstrated that this PC inhibitor markedly reduced tumor incidence, tumor multiplicity, and metastasis, pointing to a significant delay in tumor progression in wild-type and PACE4 transgenic mice. These results identify PACE4, together with other PCs, as suitable targets to slow down or block tumor progression, suggesting that PC inhibition is a potential approach for therapy for solid tumors.

Figure 1

PACE4 expression and activity in murine skin cell lines. NK and 308, normal murine keratinocytes; MT1/2, papilloma cell line; all others, SCC cell lines. DNAse-treated messenger RNA was analyzed for PACE4 expression by reverse transcription-polymerase chain reaction (A) and quantified by real-time quantitative reverse transcription-polymerase chain reaction (B). The conditioned media from these cells were tested for PC proteolytic activity and evaluated as production of the fluorescent product AMC (C). Note that PACE4 expression directly correlates with enzymatic activity. GAPDH indicates glyceraldehyde-3-phosphate dehydrogenase.

Figure 2

(A) Small interference RNA-mediated silencing of PACE4 expression resulted in decreased proteolytic activity in the conditioned medium extracted form the highly PACE4-expressing cells JWF-2 and CH72: P < .0001, P < .05, respectively. (B) CMK-dependent inhibition of PC activity. Conditionedmediumfromthe JWF-2 and CH72 cell lines were collected and assayed for PC proteolytic activity in the presence of increasing amounts of CMK. The enzymatic activity was evaluated as the production of the fluorescent product AMC: P < .002.

Figure 3

CMK inhibits IGF-1Rmaturation and phosphorylation. Cells were incubated in the absence or presence of different concentrations of CMK. Cells were plated and incubated with CMK and serum-containingmedia. After 24 hours, themediumwas replaced with serum-free medium containing the same CMK concentrations. On the third day, cells were induced with 50 nM IGF-1 for different incubation periods as indicated. pIGF-1R and the extent of processing of IGF-1R (A) or ERK1/2 phosphorylation (B) were evaluated by Western blot. Note that CMK abolished almost completely IGF-1R phosphorylation even at the lowest CMK concentration.

Figure 4

Inhibition of IGF-1R processing and phosphorylation results in decreased in vitro proliferation rates. Cells were subsequently treated with the indicated CMK concentrations for 24 hours, starved an additional 16 hours, and incubated in the presence of serum for 4 hours. Cells (A) JWF-2 and (B) CH72 were labeled with [³H]-methyl thymidine for 4 hours. DNA was extracted, and the levels of radioactivity associated with the newly synthesized DNA were measured in a scintillation counter. Note the dramatic decreased in proliferation in CMK-treated cells (P < .0001).

Figure 5

FITC-CMK skin penetration. CMK was labeled with FITC, purified, and analyzed by HPLC/MS. FITC-CMK-treated skin sections were analyzed with the Metamorph software using pseudocolor to assess the relative amount of fluorescence in the epidermis (Epi), dermis (Der), and skin surface (Sur). Sham (A), 100 µM(B), and 1mMFITC-CMK (C) treatments, respectively. The epidermal fluorescence was subtracted from background levels and plotted in a bar diagram (D). Exposure time: 1 second, (P = .001).

Figure 6

CMK-dependent inhibition of TPA-induced epidermal proliferation. The skin of K5-PACE4 transgenic mice was treated with vehicle (A and C) or CMK (300 µm; B and D) for 2 days before TPA treatment. Tissues were collected and fixed, and the relative expression of total and phosphorylated IGF-1R was assessed by immunohistochemistry. Note the decrease in IGF-1R phosphorylation in the hair follicles in the treated epidermis despite the relative constant IGF-1R expression. pIGF-1R immunohistochemistry and hematoxylin counterstain.

Figure 7

Skin from wild-type (A and B) or Tg (K5-PACE4; C and D) mice were treated with TPA (twice weekly) to increase the low basal level of cell proliferation and then treated either with vehicle alone (A and C) or with CMK 100 µM (B and D) daily for 2 weeks. Fragments of dorsal skin were fixed and embedded in paraffin; sections were stained with anti-Ki-67 antibody. The number of labeled basal epidermal keratinocytes wasmeasured per length of interfollicular epidermal basement membrane and expressed as positive cells per millimeter of basement membrane (E) P < .001. Original magnification, x100.

Figure 8

CMK inhibitory effect on tumor multiplicity. Wild-type and transgenic (Tg; K5-PACE4) mice were initiated with DMBA and treated with TPA twice a week for 30 weeks in the presence or absence of CMK(100 µM, starting atweek 2). The numbers of papillomas per mouse in wild-type (A) and K-PACE4 mice (B) and SCC per mouse in wild-type (C; P < .05) or K5-PACE4 animals (D; P < .01) were determined weekly.

Figure 9

Analysis of SCC growth rate patterns in CMK-treated (100 µM) and untreated mice. Tumor multiplicity curves were adjusted to fit linear functions, and the slopes, intercept, and their SDs were calculated by regression analysis. The confidence interval of the slope and the P value to reject the null hypothesis were determined using a t distribution. (A) Linear approximation of tumor multiplicity curves versus time for K5-PACE animals. (B) Mean slopes with their SDs.

Figure 10

CMK inhibitory effect on tumor volume andmetastatic potential. Mice were initiated with 100 nmol ofDMBA and treated twice a week., (A) SCC volume wasmeasured at 30 weeks of TPA treatment, (P<.05)., (B) Metastases to lymph nodes, (LN) and lungs weremeasured as percentage of animals with metastasis, (*P = .055, **P = .045).