Targeting CD44 With CRISPR Gene Editing May Improve Prognosis of Osteosarcoma

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CD44 protein, which is substantially unregulated in advanced osteosarcoma, is known to contribute to tumor metastasis and lead to worse prognosis. Unfortunately, there has not been an effective tool to target CD44 and explore its function in osteosarcoma.

Most studies of CD44 in osteosarcoma have relied on small interfering RNA—two synthetic RNA strands that specifically target a particular mRNA for degradation. This technique is temporary, incomplete and limited to certain organisms, and it sometimes results in off-target effects and toxicity.

The clustered regularly interspaced short palindromic repeats (CRISPR)–associated 9 (Cas9) nuclease system is gaining attention as a potential method of targeted gene regulation. It works at the DNA level, so it can knock out a gene permanently. Also, because it's an exogenous system, it does not compete with endogenous processes.

In collaboration with colleagues in China, the late Henry Mankin, MD, former chief of Orthopaedic Surgery at Massachusetts General Hospital, and Tang Liu, MD, PhD, postdoctoral researcher in the Department of Orthopaedics, have shown that it's possible to silence CD44 with CRISPR-Cas9. As the team reports in Cellular Physiology and Biochemistry, further experimentation showed that CD44 has a role in the proliferation, migration and invasion of osteosarcoma cells—and that it promotes tumor resistance to chemotherapy. Thus, targeting CD44 with CRISPR-Cas9 has potential as a therapeutic strategy in osteosarcoma.

Using immunohistochemistry, the researchers detected CD44 in 97 osteosarcoma samples from 56 patients (56 primary, 30 metastatic and 11 recurrent samples). As in previous research, high expression of CD44 was associated with metastasis and recurrence of osteosarcoma.

Of the 56 primary tumor samples, 53 were from patients who had received preoperative chemotherapy. As expected, higher CD44 levels predicted poorer response to chemotherapy and poorer survival.

The researchers then found they could use the CRISPR-Cas9 system to specifically silence CD44 in two drug-resistant osteosarcoma cell lines, KHOSR2 and U-2OSR2. Using an assay that measures cell metabolic activity, the researchers found that knockout of CD44 significantly restored cell sensitivity to doxorubicin.

Confirming that finding, drug uptake assays showed that knockout of CD44 by CRISPR-Cas9 significantly increased intracellular adsorption of doxorubicin in KHOSR2 and U-2OSR2 cells.

These results are particularly important because the relatively low survival rate of patients with aggressive or metastatic osteosarcoma is principally due to chemotherapeutic drug resistance.

In additional experiments, the researchers found that:

• In the two drug-resistant osteosarcoma cell lines, knockout of CD44 by CRISPR-Cas9 significantly inhibited expression of P-glycoprotein, an important marker of drug resistance. This suggests, the authors say, that CD44 may mediate tumor drug resistance in osteosarcoma via regulation of P-glycoprotein
• In three-dimensional cell culture, CD44 promoted the proliferation of KHOSR2 and U-2OSR2 cells
• Knockout of CD44 dramatically decreased the capabilities of KHOSR2 and U-2OSR2 cells for migration and invasion, which are preconditions for metastasis

The team concludes that the use of CRISPR-Cas9 to suppress CD44 expression has the potential to inhibit osteosarcoma migration, invasion and proliferation, and reduce resistance to chemotherapy.

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