Mitochondrial ASncmtRNA-1 and ASncmtRNA-2 as potent targets to inhibit tumor growth and metastasis in the RenCa murine renal adenocarcinoma model

Abstract

Knockdown of antisense noncoding mitochondrial RNAs (ASncmtRNAs) induces apoptosis in several human and mouse tumor cell lines, but not normal cells, suggesting this approach for a selective therapy against different types of cancer. Here we show that in vitro knockdown of murine ASncmtRNAs induces apoptotic death of mouse renal adenocarcinoma RenCa cells, but not normal murine kidney epithelial cells. In a syngeneic subcutaneous RenCa model, treatment delayed and even reversed tumor growth. Since the subcutaneous model does not reflect the natural microenviroment of renal cancer, we used an orthotopic model of RenCa cells inoculated under the renal capsule. These studies showed inhibition of tumor growth and metastasis. Direct metastasis assessment by tail vein injection of RenCa cells also showed a drastic reduction in lung metastatic nodules. In vivo treatment reduces survivin, N-cadherin and P-cadherin levels, providing a molecular basis for metastasis inhibition. In consequence, the treatment significantly enhanced mouse survival in these models. Our results suggest that the ASncmtRNAs could be potent and selective targets for therapy against human renal cell carcinoma.

Keywords: antisense therapy; mitochondria; murine renal cancer; ncRNA.

Figure 1. Expression of the mSncmtRNA and the mASncmtRNAs in normal mouse kidney epithelial cells (mKEC) and RenCa cells

A. Scheme depicting the putative origin of the mouse ncmtRNAs. Segments generated from bidirectional transcription of the 16S region of the mouse mtDNA are processed to give rise to the SncmtRNA and the ASncmtRNAs. In blue, heavy-strand transcript; in red, light-strand transcript. B. Schematic representation of the mASncmtRNA-1 and -2, indicating the size of the loop, the length of the IR and position of ASO-1232S used in this study. C. FISH of mSncmtRNA and the mASncmtRNAs in RenCa and mKEC cells (Bars = 25 μm).

Figure 2. ASK induces inhibition of proliferation and death of RenCa cells

A. RenCa cells (100,000/ well) were transfected in triplicate with 100 nM of ASO-C, or ASO-1232S or left untreated (NT). At 24, 48 and 72 h post-transfection, total cell number was determined. At 72 h, ASO-1232S induced drastic inhibition of cell proliferation compared to controls (*p < 0.005). B. Cells were treated as in (A) for 48 h. ASK induced over 70% cell death evaluated by PI staining and cytometric analysis (*p < 0,05). C. ASK of normal mKEC for 48 h does not induce significant death, compared to controls. D. After a 48 h treatment, knockdown of the ASncmtRNAs was confirmed by RT-PCR amplification of mASncmtRNA-1 (648 bp amplicon) and mASncmtRNA-2 (209 bp amplicon), using 18S rRNA (180 bp amplicon) as control (M, 100-bp ladder).

Figure 3. ASK induces apoptosis in RenCa cells

A. ASK induces dissipation of Δψm. RenCa cells were transfected with 100 nM ASO-C or ASO-1232S or left untreated (NT). As positive control, a parallel culture was incubated with STP. At 24 h post-transfection, cells were harvested, stained with 20 nM TMRM for 15 min and analyzed by flow cytometry. Both ASK and STP induce dissipation of Δψm compared to controls. A triplicate analysis showed that ASK induced around 70% of depolarized cells and STP about 55%, compared to controls (*p < 0.001). B., C. ASK induces DNA fragmentation. RenCa cells treated as in (A) for at 48 h were fixed, subjected to fluorescent TUNEL assay and counterstained with DAPI (see Materials and Methods). As positive control, cells were treated with 1 μM STP. ASK and STP but not controls induced marked DNA fragmentation (Bars = 25 μm). A triplicate analysis shows that ASK and STP induce about 80% and 70% DNA fragmentation, respectively (*p < 0.05). D. ASK induces translocation of phosphatidylserine to the outer layer of the plasma membrane. RenCa cells treated as in (A) for 24 h were fixed and stained with Annexin-V-Alexa fluor 488 and analyzed by flow cytometry. ASK induced a drastic increase in Annexin V-positive cells, compared to controls (*p < 0.001).

Figure 4. ASK induces activation of pro-apopototic factors and downregulation of anti-apoptotic factors

A. RenCa cells were transfected with 100 nM ASO-C or ASO-1232S or left untreated (NT) for 48 h, harvested and processed for Western blot using calnexin as loading control. ASK induces processing of procaspase-3, procaspase-9 and PARP1. B. ASK induces downregulation of the anti-apoptotic factors Bcl-xL and Bcl-2 compared to controls; β-actin and calnexin were used as loading controls. C. RenCa cells treated as in (A) for 24 h displayed a drastic reduction in survivin, compared to controls, which was around 85% as evidenced by a triplicate densitometric analysis (D.; *p < 0.005). E. Western blot analysis of mKEC cells treated as in (A) for 48 h showed that ASK did not affect PARP1, procaspase-3 or procaspase-9, compared to controls. F. ASK did not induce downregulation of Bcl-2 or Survivin in mKEC cells. In E and F, calnexin and β-tubulin were used as loading controls.

Figure 5. ASK inhibits tumorogenic and metastatic potential of RenCa cells

A. RenCa cells were transfected with 100 nM ASO-C or ASO-1232S or left untreated (NT) for 48 h. After harvesting and counting, 2,000 Tb-negative cells were seeded in soft agar (see Materials and Methods) and colonies >50 μm in diameter were scored at 2-3 weeks. A triplicate analysis showed that ASK inhibited colony formation by about 90% (*p < 0.005). B. ASK downregulates proteins involved in metastasis. RenCa cells were transfected as in (A) for 24 h and protein extracts were analyzed by Western blot with antibodies against N-cadherin and P-cadherin, using β-tubulin as loading control. Both cadherins were strongly downregulated by ASK. C. ASK inhibits invasiveness of RenCa cells. Cells (100,000) treated as in (A) for 48 h were grown over Matrigel-coated inserts. After 24 h, inserts were fixed in cold methanol, stained with DAPI and mounted in Mowiol. At least 10 fields were evaluated by fluorescence microscopy (Bars = 25 μm). A triplicate analysis showed that ASK induced an inhibition of around 90% in invasiveness of RenCa cells (*p < 0,001).

Figure 6. ASK inhibits subcutaneous RenCa tumor growth

A. Experimental scheme. B. Twelve Balb/C mice were inoculated sc with 200,000 RenCa cells. At 10 days post-cell inoculation, mice harbored tumors of about 100 mm³. Animals were randomized into 3 groups of 4 mice, which received 10 ip injections of 200 μl saline alone or containing 200 μg ASO-C or ASO-1232S. At day 26 post-cell inoculation, the control groups reached tumors over 1000 mm³ and were sacrificed. The remaining mice reached different tumor size followed by progressive decrease of tumor volume. These four mice were healthy at day 150 post-cell inoculation. C. Kaplan-Meier survival curve. Saline and ASO-C control groups displayed 24 and 26-day mean survival, respectively, whereas all ASO-1232S-treated mice were alive and healthy at 150 days post-cell inoculation (p = 0.00051).

Figure 7. ASK in vivo induces downregulation of survivin and MMP9

Eight Balb/c mice were inoculated sc with 200,000 RenCa cells. Eleven days post-cell inoculation, mice contained tumors of about 200 mm³ and were randomized into 2 groups of 4 mice, which were treated by 6 ip injections on days 11, 13, 15, 17, 19 and 21 with 200 μg ASO-C or ASO-1232S in 200 μl saline. The day after the last injection, mice were euthanized and tumors were harvested and processed for Western blot. A. Scheme of the experiment. B. Growth curve of sc tumors, showing a drastic retardation in ASO-1232S treated mice, compared to controls. C. Representative blot of survivin in lysates of 3 tumors from each group. Tumors treated with ASO-1232S (T4, T5 and T6) exhibited downregulation of survivin, compared to ASO-C tumors (T1, T2 and T3). D. A triplicate analysis showing that ASK induced about 80% downregulation of survivin (*p < 0,001). E. Drastic downregulation of MMP-9 induced by ASK (*p < 0.0005).

Figure 8. Kinetics of orthotopic RenCa tumor growth and tumoral 1232S entry

A. Twenty one Balb/C mice were inoculated into the subcapsular structure of the kidney with 100,000 RenCa cells. Three mice each were sacrificed on days 4, 6, 8, 10, 12, 14 and 18 and kidneys were fixed. Four sections from each kidney were used to evaluate tumor growth. At day 4, 1-2 mm³ tumors were found (insert, magnification 4X). B. Incorporation of ASO-1232S into orthotopic tumors. RenCa cells (100,000) were injected into the subcapsular region of the left kidney of 2 Balb/C mice and sutured. On day 12 post-cell inoculation, one mouse was injected ip with 100 μg ASO-1232S conjugated to Alexa Fluor 488 and the other mouse was injected with saline. Two h after injection, mice were sacrificed, kidneys were collected, frozen at -70°C and sectioned with a cryostat. Sections were counterstained with DAPI and analyzed by fluorescence microscopy. Green fluorescence was found in the kidney parenchyma and in the tumor only of the mouse injected with Alexa 488-ASO-1232S (magnification 4X).

Figure 9. ASK in vivo inhibits primary tumor growth and lung metastasis in an orthotopic renal cancer model

A. Scheme of the orthotopic protocol. Ten female Balb/C mice were injected under the renal capsule with 100 μl saline containing 100,000 RenCa cells and sutured. At day 4 post-cell inoculation, mice were randomized into 2 groups of 5 mice each, receiving 8 ip injections on days 4, 6, 8, 10, 12, 14, 16 and 18 of 250 μl saline containing 200 μg of ASO-C or ASO-1232S. On day 19 post-cell inoculation, mice were sacrificed under anesthesia and both kidneys and lungs were resected and fixed. Kidneys were weighed before fixing. B. Macroscopic presence of kidney tumors comparing the left and the right (control) kidney (arrows) of each mouse. Only one mouse from the ASO-1232S group developed a small tumor, whereas all 5 mice form the ASO-C group displayed large tumors. C. Tumor weight of the left kidney, comparing ASO-C and ASO-1232S (**p < 0.005). D. Four sections from each fixed lung were stained with H&E and metastatic nodules (arrows) were microscopically scored. The figure shows representative images from the left kidney of each mouse (magnification 4X). E. ASK induces a low number of metastatic nodules as compared to the control ASO-C (**p < 0.005).

Figure 10. Tail vein RenCa metastasis assay

A. Scheme of the metastasis protocol. Fifteen female Balb/c mice were inoculated through the tail vein with 100 μl saline containing 100,000 RenCa cells and animals were randomized into 3 groups of 5 mice each, which received 7 ip injections on days 1, 3, 5, 7, 9, 11 and 13 of 250 μl saline alone (S) or containing 200 μg ASO-C or ASO-1232S. On day 15, mice were sacrificed and lungs were excised, fixed and sections stained with H&E. B. Saline and ASO-C-treated mice harbored abundant metastatic nodules (arrows) in the lungs, whereas the ASK group displayed considerably fewer nodules (magnification 4X). C. Metastatic nodule count depicts an important and statistically significant reduction in the number of metastatic nodules in the ASK group, compared to both controls (*p < 0.005). D. Metastasis survival assay. Ten mice were inoculated with 100,000 RenCa cells as described in (A). On day 1 post-cell inoculation, mice were randomized into 2 groups of 5 mice each, which were injected ip with 200 μg ASO-C or ASO-1232S in 250 μl saline, on days 1, 3, 5, 7, 9, 11 and 13. Tumor growth and survival was monitored until day 100 post-cell inoculation (p = 0.07).