Genomics: Insight

Introduction

An estimated 150,000 women die annually as a result of triple-negative breast cancer (TNBC).³ This figure represents about 1 out of every 3 deaths of any form of breast cancer.¹ TNBC is defined by the lack of expression of human epidermal growth factor receptor 2 (HER2), progesterone receptors, and estrogen receptors, hence the name triple negative.² TNBC is most common in women of African descent and in those carrying mutated BRCA, a tumor-suppressing gene.² Currently, surgery, chemotherapy, and radiotherapy exist as the sole treatments for TNBC patients. The scant treatment options, in conjunction with the highly metastatic tendency of TNBC, have resulted in the highest mortality rate in comparison to all subtypes of breast cancer.² However, new developments in CRISPR/Cas9 gene editing technology have suggested a more effective method of TNBC therapy: reducing expression of the gene Lipocalin-2 (Lcn2).

Current CRISPR knowledge

CRISPR is a revolutionary genome editing tool that can accurately, efficiently, and easily edit genes by using a guide RNA sequence to direct Cas9, a DNA-cutting enzyme, to mutated DNA. CRISPR’s ability to shut down specific genes serves as a potential cure for many genetic diseases, such as cancer. However, to date, CRISPR has mainly been studied in monogenic diseases, which are caused by variations in only one gene. While CRISPR therapies for monogenic diseases like hereditary tyrosinemia and cystic fibrosis have been successful², there are far fewer studies on multigenic diseases such as TNBC.

Lipocalin-2 (Lcn2) knockout effects on TNBC metastasis

While there are many genes associated with TNBC, the overexpression of Lipocalin-2 (Lcn2), a gene associated with breast cancer, has been shown to significantly contribute to the cancer’s metastasis and mortality; reduction of its expression decreases the severity of TNBC.² Lcn2 has been shown to be highly expressed in patients suffering from TNBC. In a study involving 178 TNBC patients², the expression level of Lcn2 was shown to be correlated with survival probability, in which patients with lower Lcn2 expression had a higher chance of survival than those with a high Lcn2 expression. In this study, a cell culture model for TNBC used two cell lines derived from TNBC patients and focused on key characteristics of cancer such as cell proliferation and cell migration. Lcn2 CRISPR knockout in the two TNBC cell lines, while having no effect on cell proliferation, reduced TNBC cell migration, a hallmark of cancer metastasis, by over 60% compared to controls. These findings suggest that Lcn2 CRISPR knockout could greatly decrease the probability of cancer metastasis in TNBC patients.

“…the expression level of Lcn2 was shown to be correlated with survival probability, in which patients with lower Lcn2 expression had a higher chance of survival than those with a high Lcn2 expression.”

Other Lcn2 knockout experiments that mitigate TNBC symptoms in patients

In the same study2, using the same model system where the Lcn2 gene was knocked out, the effects on the following cellular characteristics were also studied: cell speed, migration, length, height, area, and volume. When treated with Lcn2KO, TNBC cells migrated a shorter distance compared to cancerous cells. Lcn2KO gene editing therapy also impacted TNBC cell shape and behavior, which decreased cell motility and metastasis compared to WT cells. TNBC cell speed, migration, length, and volume were significantly decreased by Lcn2KO treatment in comparison to WT gene therapy controls. Cell height and area also decreased but not as significantly. Microscopic images of the cells taken over time demonstrate the decreased cell movement when treated with Lcn2KO CRISPR/Cas9 gene editing, indicating the restoration of characteristics typical in healthy-type cells. The expression of the proteins Fibronectin and E-Cadherin in treated TNBC cells was also decreased when treated with Lcn2KO CRISPR therapy. Since both of these protein expressions correlate with dangerous cell proliferation and malignancy levels, their decreased expression demonstrates the treatment’s effectiveness compared to WT cells. Together these results indicate that CRISPR-mediated Lcn2 knockout in TNBC patient cell lines reduces cancer symptoms by several measures relative to controls.

“Lcn2 CRISPR knockout could greatly decrease the probability of cancer metastasis in TNBC patients”

Effects of CRISPR-mediated lipocalin-2 knockout in TNBC

The paper by Guo et al. also used a tumor-bearing mouse model of TNBC wherein low Lcn2 expression by a CRISPR gene targeting strategy reduces symptoms of TNBC. TNBC cells were injected into mice on Day 0; CRISPR plasmid (Lcn2 knockout or control) was injected four subsequent times: on days 35, 42, 49, and 56. tNLG-Lcn2KO resulted in the greatest effect on tumor volume (mm3) compared to controls. The control caused the smallest decrease in volume compared to the CRISPR-Lcn2 knockout vector and other gene-targeting controls.² Lcn2 knockout relative to controls exhibits the largest decrease in tumor mass, with an average of under 50mg measured from seven mice. When treated with the Lcn2 knockout vector, the tumors display a sharp decrease in size in comparison to the control group. The study also demonstrates that the in vivo editing efficiency is high and that treatment with the Lcn2 knockout vector does not cause significant toxicity since no pathological changes occurred in the liver or spleen of the tumor-bearing mice after tNLG-Lcn2KO treatment. These results from an in vivo model indicate that the CRISPR gene targeting strategy attenuates many cancer characteristics, such as tumor volume and weight while maintaining relative safety.

“CRISPR gene targeting strategy attenuates many cancer characteristics such as tumor volume and weight while maintaining relative safety”

Conclusion

Reduced Lcn2 gene expression significantly increases patient survival probability; in a cell culture system, it rescues metastasis by 60% in TNBC cell lines², and in a mouse model system, it reduces tumor growth by 77%. Therefore, the application of CRISPR Lcn2 gene editing therapy to human TNBC patients could reduce cancer proliferation and metastasis and thus drastically reduce the disease’s lethality.

A personal statement

Before researching CRISPR treatments for TNBC, we both had close family friends with TNBC and witnessed the debilitating effects of the disease firsthand. After conducting research into the disease and new emerging treatments for TNBC, we are hopeful that CRISPR will revolutionize survival chances for patients and make the treatment process more painless and efficient.

References

1. Saraiva, D. P., Cabral, M. G., Jacinto, A., & Braga, S. (2017). How many diseases is triple negative breast cancer: the protagonism of the immune microenvironment. ESMO Open, 2(4). https://doi.org/10.1136/ esmoopen-2017-000208
2. Guo, P., Yang, J., Huang, J., Auguste, D. T., & Moses, M. A. (2019). Therapeutic genome editing of triple-negative breast tumors using a noncationic and deformable nanolipogel. Proceedings of the National Academy of Sciences of the United States of America, 116(37). https://doi.org/10.1073/ Pnas.1904697116
3. World Health Organization. Women and health: today’s evidence tomorrow’s agenda. 2009.