Simultaneous siRNA targeting of Src and downstream signaling molecules inhibit tumor formation and metastasis of a human model breast cancer cell line

Abstract

Background: Src and signaling molecules downstream of Src, including signal transducer and activator of transcription 3 (Stat3) and cMyc, have been implicated in the development, maintenance and/or progression of several types of human cancers, including breast cancer. Here we report the ability of siRNA-mediated Src knock-down alone, and simultaneous knock-down of Src and Stat3 and/or cMyc to inhibit the neoplastic phenotype of a highly metastatic human model breast cancer cell line, MDA-MB-435S, a widely used model for breast cancer research.

Methodology/results: Src and its downstream signaling partners were specifically targeted and knocked-down using siRNA. Changes in the growth properties of the cultured cancer cells/tumors were documented using assays that included anchorage-dependent and -independent (in soft agar) cell growth, apoptosis, and both primary and metastatic tumor growth in the mouse tumor model. siRNA-mediated Src knock-down alone, and simultaneous knock-down of Src and Stat3 and/or cMyc inhibited the neoplastic phenotype of a highly metastatic human model breast cancer cell line, MDA-MB-435S. This knock-down resulted in reduced growth in monolayer and soft agar cultures, and a reduced ability to form primary tumors in NOD/SCID mice. In addition, direct intra-tumoral injection of siRNAs targeting these signaling molecules resulted in a substantial inhibition of tumor metastases as well as of primary tumor growth. Simultaneous knock-down of Src and Stat3, and/or Myc exhibited the greatest effects resulting in substantial inhibition of primary tumor growth and metastasis.

Conclusions/significance: These findings demonstrate the effectiveness of simultaneous targeting of Src and the downstream signaling partners Stat3 and/or cMyc to inhibit the growth and oncogenic properties of a human cancer cell line. This knowledge may be very useful in the development of future therapeutic approaches involving targeting of specific genes products involved in tumor growth and metastasis.

Figure 1. Src and some of the signaling pathways downstream of Src.

Src regulates several downstream signaling pathways including the STAT3 pathway, the PI3K pathway, and the MAPK pathway.

Figure 2. Src siRNA causes reductions in Src protein and activity and inhibits cell growth.

MDA-MB-435S cells were left untreated or transfected with control or Src siRNA. 24 hours later, some cells were plated into soft agar or tissue culture dishes. After 48 hours, the remainder of the cells were processed for Src immunoblots or Src kinase activity. The results from the immunoblot and kinase assay were quantitated, the results are shown, and significant differences are indicated (* and **; p<0.05) (A). The cells in soft agar were grown for 14 days and colonies were quantitated (B) with significant differences indicated (*; p<0.05). The cells in monolayers were quantitated 5 days after plating and the significant differences indicated (*; p<0.01) (C). Each data point is the mean of duplicate (western and kinase) or triplicate (soft agar and monolayer) samples +/−1 S.D. and is representative of at least 3 independent experiments.

Figure 3. siRNA targeting various signaling molecules causes corresponding reductions in protein expression and inhibits cell growth.

MDA-MB-435S cells were transfected with siRNA, and 24 hrs post transfection, cells were plated into soft agar or into tissue culture dishes. Cells to be analyzed by immunoblotting were lysed 60 hrs post-transfection and duplicate samples of each cell extract were analyzed (A). The cells in soft agar were grown for 14 days (B) and colonies larger than approximately 50 cells were quantitated (C). The cells in monolayer culture were quantitated 4 days after plating (D). Each growth condition was assayed in triplicate and is representative of at least 3 independent experiments. In C and D, comparisons were made between the group treated with control siRNA and various single targeting siRNA, and statistically significant differences are noted (*; p<0.05). In C and D, comparisons between singly and multiply transfected cells are made, and significant differences are noted (**; p<0.05).

Figure 4. Src+STAT3 siRNA reduces tumor formation in NOD/SCID mice.

1×10⁶ cells transfected with control (mice 1–4) or Src+STAT3 siRNAs (mice 5–8) were implanted into the mammary fat pad of mice. After 66 days, the tumors were excised and weighed (significant differences are indicated (*; p<0.05)(A). Sections of the tumor were stained (B) or subjected to immunostaining with anti-GFP antibody (C). Malignant cells are indicated by an arrow. A portion of each tumor was homogenized with RIPA buffer and immunoblotted with anti-GFP antibodies (D).

Figure 5. Injection of siRNA/oligofectamine complexes into mouse tumors inhibits tumor growth.

MDA-MB-435S cells were implanted in the mammary fat pad of SCID/NOD mice. Two days post-implantation, the mice were divided into 3 groups (one group of 10 control siRNA-injected mice and two groups of 5 targeted siRNA mice) and the groups received twice weekly injections of either control, Src+STAT3, or Src+STAT3+Myc siRNA into the site of the cell implantation/tumor. 47 days post-implantation, 5 mice in the control group were switched to Src+STAT3 siRNA injections for the remainder of the experiment (Control, then Src+STAT3 group). Tumor growth was monitored and the results are shown (A), with each treatment group shown in a single panel, and individual mouse tumors represented by connected data points. 126 days post-implantation, the primary tumor(s) were excised (B) and weighed. The tumor weights for each mouse (data points) and the average tumor weight for each group (horizontal bar) is shown (C). Tumor weight data was analyzed by Analysis of Variance and Tukey's pairwise comparison, with significant differences between the control group and two of the treatment groups noted ( *p<0.05).

Figure 6. Injection of siRNA/oligofectamine complexes into mouse tumors inhibits tumor metastasis.

Mice treated in fig. 5 were examined grossly and microscopically for the presence of tumor metastasis and representative pictures (A) (tumors indicated with yellow outline and arrows) are shown. A summary of results from gross and microscopic examination was compiled (B) with the presence to metastatic tumor growth indicated (+).

Figure 7. Src siRNA causes apoptosis of MDA-MB-435S cells.

MDA-MB-435S cells were transfected with siRNA or treated with 1 uM staurosporine. 48 hours post-treatment, the cells were collected and assayed for apoptosis using the TUNEL assay. Results are expressed with log(fluorescence) on the horizontal axis and cell counts on the vertical axis and is representative of triplicate experiments.