August 12, 2017

The Inhibitory Protein That Plays an Important Role in Canine Mammary Tumors

The inhibitory protein known as SET plays an important role in canine mammary tumor progression.
By JoAnna Pendergrass, DVM
A research team has recently determined that the inhibitory protein SET plays an important role in canine mammary tumor progression. The team’s findings were published in Nature Scientific Reports.

Canine mammary tumor is the most common cancer type affecting female dogs and is gaining traction as a translational model for human breast cancer. It is often first treated with surgical resection; adjuvant chemotherapy or radiation therapy is recommended if metastasis is present. However, as with human breast cancer, chemotherapy is not always effective, highlighting the need for new targeted treatments.

SET inhibits PP2A, a tumor suppressor protein that inhibits tumor-promoting cell signaling pathways. Human studies have reported SET’s suppression of PP2A and increased expression in breast cancer and other cancers. Although SET has been identified in dogs, its role in canine mammary tumors has not yet been evaluated.

For the current study, the research team obtained tumor samples from 13 female pet dogs with mammary tumors that received surgical treatment. A series of tests were performed on the tumor samples, on normal canine mammary tissue, and on 2 canine mammary tumor cell lines to evaluate SET’s role in canine mammary tumors. The cell lines—CIP-p (primary origin) and CIP-m (metastatic origin)—had been generated previously.

SET Protein Levels
Immunoblotting, a protein detection technique, was used to analyze SET protein levels. Levels were significantly higher in the tumor tissue and cell lines than in the normal tissue, and higher in stage 2–4 tumors than stage 1 tumors. Levels were similar between cell lines.

Researchers infected the tumor cells with lentivirus to express small hairpin RNA that would silence the set gene (shSET). Immunoblotting revealed marked SET suppression by shSET in both cell lines.

SET KnockdownTumor Growth
SET knockdown (SET KD) with shSET effected tumor growth in several ways:
  • Cell proliferation inhibition, delayed cell doubling time, and decreased colony numbers of CIP-m, but not CIP-p, cells
  • Inhibition of anchorage-independent growth (marker of tumorigenicity) and in vivo mammary tumor growth of CIP-m cells
These findings indicated SET’s changing role during mammary tumor progression, particularly within metastatic cells. In addition, the differential effect of SET KD between the cell lines could be attributed to genetic mutations in CIP-m cells, noted the researchers.

SET KnockdownCell Signaling and PP2A Activity
Using immunoblotting, the researchers observed SET KD’s effects on levels of tumor-promoting signaling proteins in the CIP-m cells:
  • No effect: c-MYC, AKT/pAKT, ERK1/2/pERK1/2
  • Significant suppression: mTORC1/p70S6K, b-catenin, NFkB/pNFkB
In the CIP-p cells, SET KD had little effect on these protein levels. However, SET KD significantly increased PP2A activity in the CIP-m cells.

Such findings suggest that, in canine mammary tumors, SET increases activity of mTORC1/p70S6K, b-catenin, and NFKB/pNFKB signaling pathways by inhibiting PP2A.

SET KnockdownTumor Therapy
Researchers tested SET KD’s effect on several breast cancer chemotherapies in the CIP-m cells. Sensitivity to tamoxifen, carboplatin, and bortezomib remained unchanged with SET KD. Unexpectedly, though, SET KD produced doxorubicin resistance in CIP-m cells, suggesting a partial dependence of doxorubicin’s anti-tumor activity on SET.

SET KD had little effect on colony formation of CIP-m cells exposed to radiation therapy, suggesting that SET plays no role in mammary tumor cells’ response to radiation therapy.

Bringing It Together
The researchers concluded that the SET protein has enhanced activity in canine mammary tumors. The study’s findings, they wrote, “support further investigation into the therapeutic potential of SET as a target for anti-tumor drugs for canine mammary tumor/human breast cancer.”

Dr. JoAnna Pendergrass received her Doctor of Veterinary Medicine degree from the Virginia-Maryland College of Veterinary Medicine. Following veterinary school, she completed a postdoctoral fellowship at Emory University’s Yerkes National Primate Research Center. Dr. Pendergrass is the founder and owner of JPen Communications, a medical communications company.

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