In vivo targeting of ADAM9 gene expression using lentivirus-delivered shRNA suppresses prostate cancer growth by regulating REG4 dependent cell cycle progression

Abstract

Cancer cells respond to stress by activating a variety of survival signaling pathways. A disintegrin and metalloproteinase (ADAM) 9 is upregulated during cancer progression and hormone therapy, functioning in part through an increase in reactive oxygen species. Here, we present in vitro and in vivo evidence that therapeutic targeting of ADAM9 gene expression by lentivirus-delivered small hairpin RNA (shRNA) significantly inhibited proliferation of human prostate cancer cell lines and blocked tumor growth in a murine model of prostate cancer bone metastasis. Cell cycle studies confirmed an increase in the G1-phase and decrease in the S-phase population of cancer cells under starvation stress conditions, which correlated with elevated intracellular superoxide levels. Microarray data showed significantly decreased levels of regenerating islet-derived family member 4 (REG4) expression in prostate cancer cells with knockdown of ADAM9 gene expression. This REG4 downregulation also resulted in induction of expression of p21(Cip1/WAF1), which negatively regulates cyclin D1 and blocks the G1/S transition. Our data reveal a novel molecular mechanism of ADAM9 in the regulation of prostate cancer cell proliferation, and suggests a combined modality of ADAM9 shRNA gene therapy and cytotoxic agents for hormone refractory and bone metastatic prostate cancer.

Figure 1. Knockdown of ADAM9 expression decreased prostate cancer cell proliferation.

Cell proliferation decreased starting 2 days after seeding (1×10⁴ cells/well) of ADAM9-knockdown PC3 (a) and LNCaP (b) (* Student’s t-test, p≤0.05). (c) Clonogenic cell proliferation assays of PC3, PC3_shGFP, PC3_pLKO and PC3_shADAM9-46978 revealed decreased colonies in PC3_shADAM9-46978. (d) Quatitative analyses of clonogenic studies demonstrated that the number of cell colonies was lower in lentiviral knockdown of ADAM9 expression compared to wild-type, mock infected, and non-target controls (** Student’s t-test, p≤0.001).

Figure 2. ADAM9 knockdown reduces PC3-induced tumor growth and osteolytic activity.

(a) PC3_shGFP and PC3_shADAM9 were injected into the left and right tibia of the same mouse (n = 10). (b) Bioluminescence imaging at 40 days post-inoculation. Black arrows show the only signal detected in a PC3_shADAM9 bone lesion. (c) Computerized radiographic scanning of lesion area. Upper image: Bone destruction is extensive in the left leg compared to the right leg; Lower image: mouse with a destructive bone lesion in the left leg and no lesion in the right leg. (d) Histological imaging showing that both trabecular and cortical bone was replaced by the PC3_shGFP tumor. In contrast, trabecular and cortical bone was still intact in the PC3_shADAM9 tumor (H & E). Masson’s trichrome staining indicated only traces of bone left in PC3_shGFP compared to PC3_shADAM9 tumors (Trichrome). (e) TRAcP analysis of osteoclast activity. Arrow indicates osteoclasts observed in a PC3_shGFP tumor. Red highlight indicates loci of 40× enlarge.

Figure 3. ADAM9 reduction therapy decreased tumor volume by inhibiting cancer cell proliferation.

(a) Schematic of ADAM9 knockdown therapy experiment. Mice were injected subcutaneously with PC3 cells in both flanks, and tumor size was measured twice a week until the size reached approximately 200 mm³. Mice then received injections of either PBS into both tumor sites (n = 5) or shGFP lentivirus into the left side tumor and shADAM9 into the right side (n = 10). Viruses were injected and tumors measured weekly for 6 consecutive weeks. (b) After shADAM9 therapy, tumor volumes did not increase compared to shGFP therapy or PBS controls (** p≤0.01, Student’s t test). (c) Immunoblotting assay of ADAM9 expression confirms decreased ADAM9 expression after shADAM9 therapy. EF-1α was used as a loading control (* p≤0.05; ** p≤0.01, Student’s t test). (d) Statistical analysis of ki67 staining. shADAM9 therapy significantly decreased cell proliferation activities in PC3 tumors. PBS and shGFP therapy showed no difference in the proliferation index (** p≤0.001, One Way ANOVA). (e) Statistical analysis of TUNEL staining showed no significant (N.S.) difference between therapies (p≥0.05, One Way ANOVA).

Figure 4. ADAM9 silencing induced G₁ arrest.

Starvation reduced the S-phase cell population in ADAM9 knockdown cancer cells compared to that of controls in PC3 (a), LNCaP (b). (c) Expression levels of p21^Cip1/WAF1, p27^Kip1, and cyclin D1 increased in shADAM9 cells under starvation stress conditions in PC3 cells. (d) Expression levels of p21^Cip1/WAF1 and p27^Kip1 increased in LNCaP_shADAM9 under both normal and starvation conditions.

Figure 5. Knockdown ADAM9 expression enhances endogenous superoxide levels under stress condition.

(a) PC3_shGFP and PC3_shADAM9 cells were cultured in either 5% FBS or under serum starvation for 24 hours, followed by treatment with 0.5 µM MitoSOX fluorescence. Equal intensity of fluorescence was used in each image capture for quantification of superoxide levels. (b) Elevation of endogenous superoxide concentrations level can be detected during serum starvation in both PC3_shGFP and PC3_shADAM9. (c) Endogenous hydrogen peroxide levels did not differ between cells cultured in 5% FBS and under serum starvation.

Figure 6. Decreased REG4 and increased p21^Cip1/WAF1, p27^Kip1, and cyclin D1 expression in ADAM9 knockdown prostate cancer cells.

(a) Quantitative PCR confirmed the decrease of REG4 mRNA and increase of CD33 mRNA. (b) Overexpression of REG4 decreased p21^Cip1/WAF1 expression under FBS starvation conditions in PC3_shADAM9. (c) The starvation-induced increase in superoxide levels was reversed by overexpression of REG4 in PC3_shADAM9. (d) The strongest inhibition of REG4 expression by shREG4 lentiviral vectorwas observed in PC3_shREG4-225663 by RT-PCR. (e) PC3_shREG4-225663 reduced the S-phase and increased the G1-phase populations under starvation conditions.

Figure 7. Scheme demonstrated the role of ADAM9 in the regulation of REG4 and p21^Cip1/WAF1/p27^Kip1 activities in prostate cancer cells during environmental stresses, such as radiation therapy or chemotherapy.