Genome-wide expression analysis of therapy-resistant tumors reveals SPARC as a novel target for cancer therapy

Abstract

Overcoming resistance to chemotherapy and radiation therapy has been a difficult but important goal in the effort to cure cancer. We used gene-expression microarrays to identify differentially expressed genes involved in colorectal cancer resistance to chemotherapy and identified secreted protein, acidic and rich in cysteine (osteonectin) (SPARC) as a putative resistance-reversal gene by demonstrating low SPARC expression in refractory human MIP101 colon cancer cells. We were able to achieve restoration of their radiosensitivity and sensitivity to 5-fluorouracil and irinotecan by reexpression of SPARC in tumor xenografts. Moreover, treatment of mice with SPARC conferred increased sensitivity to chemotherapy and led to significant regression of xenografted tumors. The results show that modulation of SPARC expression affects colorectal cancer sensitivity to radiation and chemotherapy. SPARC-based gene or protein therapy may ameliorate the emergence of resistant clones and eradicate existing refractory clones and offers a novel approach to treating cancer.

Figure 1

Human SPARC mRNA and protein levels in cell lines sensitive and resistant to chemotherapy. (A) The oligonucleotide microarray result was confirmed by semiquantitative RT-PCR. MIP101/R, resistant MIP101 cells. (B) SPARC mRNA expression in cancer cell lines (paired sensitive and resistant uterine sarcoma MES-SA and MES-SA/DX5; breast cancer MDA435 and MCF-7; pancreatic MIA PaCa-2; normal colon CCD-112 CoN; mesothelioma JMN 1B; and colorectal HCT 116, RKO, SW620, and HT29). (C) Protein levels in sensitive MIP101 and resistant cells (MIP/5FU, MIP/CPT, MIP/ETO, MIP/CIS) and another pair of sensitive and resistant cells (MES-SA and MES-SA/DX5).

Figure 2

SPARC expression in human colonic epithelium. Normal colon shows SPARC expression in normal colonic epithelium (A and boxed area in B), and endothelial cells in the submucosa (A, inset). SPARC levels are lower in colorectal adenocarcinomas (arrows) at primary sites (B) and following metastasis to liver (*) (C). Lower expression was also observed in paired human colorectal cancers (patient A: D and E; patient B: F and G) following treatment with 5-FU/leucovorin (E and G) compared with the untreated primary tumor (D and F). Sections, 6 μm; scale bars: 20 μm.

Figure 3

SPARC alters the sensitivity of MIP101 colorectal cancer cells to chemotherapy. (A) TUNEL assay shows the effect of exogenous SPARC on apoptosis in MIP/5FU cells. (B) Levels of SPARC secreted by MIP/SP cells (clones c1, c2, and c3) compared with control MIP/Zeo cells and following concentration of the incubation media (conc, c2-conc). (C) Colony-forming assay of MIP/SP and control MIP/Zeo cell lines following exposure to incremental concentrations of 5-FU, CPT-11, or ETO. (D) Sensitivity of MIP/SP compared with MIP/Zeo cells to chemotherapy (FACS analysis of annexin V–labeled cells). *P < 0.05, Student’s t test; n = 3 different experiments. (E) Immunoblots of MIP/Zeo and MIP/SP cells exposed to 500 μM 5-FU for 12 hours and probed for caspase-3 and α-fodrin.

Figure 4

Effect of SPARC on cell cycle progression. There is a delay in the G₁/S phase in MIP/SP cells compared with control MIP/Zeo cells. See Table 1 for quantitative results.

Figure 5

Effect of chemotherapy and radiation therapy on tumor xenografts of MIP/SP cells. (A) Tumor regression in MIP/SP tumor xenografts exposed to 5-FU or CPT-11 compared with MIP/Zeo cells. To allow better visualization of the results provided in A, data from control animals only (tumors of MIP/Zeo and nontreated MIP/SP xenografts) are shown in B, while data from all MIP/SP xenografts treated with 5-FU or CPT-11 are represented in C. (D) Representative MIP/Zeo or MIP/SP tumor xenografts following treatment with 2 cycles of 5-FU or CPT-11. (E) Effect of radiation therapy on MIP/SP tumor xenografts (n = 10 animals/group; total dose of radiation, 100 Gy, single dose; P = 0.02 after 3 weeks of radiation; standard 2-sample t test). (F) SPARC levels in serum obtained from animals with xenografts of MIP/SP an MIP/Zeo 42 days after implantation.

Figure 6

Effect of combination therapy with SPARC(s) and 5-FU on tumor regression. Exposure of MIP101 tumor xenografts to combination therapy consisting of intraperitoneal (IP) SPARC(s) and 5-FU (n = 6; mean ± SE) (A and B) or subcutaneous (SC) SPARC(s) and 5-FU (results are representative of those obtained from 8 animals) (D). (C) Treatment of resistant MIP/5FU tumor xenografts with combination therapy with SPARC(s) (n = 6 animals; mean ± SE).

Figure 7

Effect of SPARC in combination with 5-FU on tumor regression and apoptosis. (A) Treatment of MIP101 tumor xenografts with purified recombinant SPARC in combination with 5-FU (n = 3; mean ± SE). (B) Tumors recovered from animal xenografts treated with SPARC(s) or 5-FU alone did not show increased numbers of apoptotic bodies (arrows) compared with tumors recovered from animals treated with a combination of SPARC(s) and 5-FU. Sections, 6 μm; scale bars: 15 μm.

Figure 8

Evaluation of cell proliferation and blood vessel formation in tumor xenografts following treatment with SPARC(s) and 5-FU. Immunoperoxidase staining shows no significant difference in the levels of SPARC and no effect on cell proliferation (staining with Ki-67). Decreased staining with CD34 shows fewer blood vessels in SPARC(s)-treated animals. Sections, 6 μm; scale bars: 20 μm.

Figure 9

Evaluation of cell proliferation and blood vessel formation in tumor xenografts following treatment with SPARC(s) and 5-FU. (A) No significant differences were noted in Ki-67–positive nuclei in the xenografts harvested from various treatments. (B) As indicated by the lower numbers of CD34-positive blood vessels, significantly fewer blood vessels were noted in xenografts from animals treated with 5-FU compared with controls, but more significant is the further decrease in animals treated with SPARC alone or in combination with 5-FU. *P < 0.05, Student’s t test. LPF, low-power field.

Figure 10

Assessment of the effect of SPARCs exposure on the ECM of tumor xenografts. Collagen IV is noted predominantly around blood vessels, and no significant differences in immunoperoxidase staining is seen in the ECMs between control and any of the treatment groups. Laminin expression in the ECM within the tumor xenograft is similarly unaffected in treatment groups, with its predominant expression noted within the fibrous capsule surrounding the graft. A thicker fibrous band (arrows) is noted surrounding xenografts of animals treated with SPARCs alone.