IIT Kanpur-led team discovers the molecular mechanism by which a protein (EZH2) is produced in excess leading to a reduction in the levels of two microRNAs. This causes an oncogene (SPINK1) to get overexpressed in the SPINK1-positive prostate cancer subtype.  Synthetic microRNAs and epigenetic drugs that increase the microRNA levels reduce tumour growth and metastasis.

The molecular mechanism and pathobiology of SPINK1-positive prostate cancer subtype, which is the second most recurrent and aggressive in nature, that affects about 15% of patients has been finally unravelled. The study was done by a multi-institutional team led by Prof. Bushra Ateeq from the Department of Biological Sciences and Bioengineering at Indian Institute of Technology (IIT) Kanpur. The SPINK1-positive prostate cancer subtype derives its name from the excess amount of SPINK1 oncogene found in the cancer cells.

Excess production of SPINK1 gene responsible for tumour and metastasis is not restricted to prostate cancer alone but also seen in colorectal, lung, pancreatic, breast and ovarian cancers. The insights gained in this study might therefore help in the treatment and disease management of several SPINK1-positive malignancies.

In addition to excess amount of the SPINK1 oncogene, the researchers found that most cancer cells belonging to this subtype also have more than normal amount of a particular protein called EZH2. Also, the levels of two microRNAs (miRNA-338-5p and miRNA-421) produced in SPINK1-positive cancer cells were much less.

Deciphering the molecular mechanism

“We found that the increased levels of EZH2 protein triggers the reduction in the synthesis of these two microRNAs in SPINK1-positive cancers. And the reduced levels of the two microRNAs in turn lead to over production of SPINK1,” says Prof. Ateeq who is an Intermediate Fellow of the Wellcome Trust/DBT India Alliance. “The EZH2 protein sits on the regulatory region of the two microRNAs and shuts down their synthesis leading to excess production of SPINK1.”

The researchers first discovered excess levels of SPINK1 protein and reduced amount of the two microRNAs on analysing the global data sets of prostate cancer patients. These findings were then corroborated in SPINK1-positive prostate cancer cell lines.

To validate the role of the two microRNAs in regulating the expression of the SPINK1 oncogene, the researchers introduced the microRNAs into the SPINK1-positive cancer cell lines. When the amount of microRNAs in the cell lines was increased, the SPINK1 level reduced and there were marked changes in the oncogenic properties — the cell proliferation and invasion reduced.

“The observations made using SPINK1-positive cell lines were validated in chicken embryos and mouse models,” says Anjali Yadav from the Department of Biological Sciences and Bioengineering at IIT Kanpur and one of the first authors of a paper published in Clinical Cancer Research.

Validating in mice and chicken embryos

In both the chicken embryo and mouse model experiments, the SPINK1-positive prostate cancer cells were modified by introducing the microRNAs and were tested for tumour growth and metastasis. “Tumour was found growing aggressively in mice of the control group but significantly reduced in size in the microRNA overexpressing group,” says Vipul Bhatia from IIT Kanpur and the other first author of the paper. “A significant reduction in tumour growth was also observed in chicken embryos that were implanted with microRNA-modified prostate cancer cells.”

Metastasis was also significantly reduced in both the lungs and bone marrow of mice implanted with microRNA-modified prostate cancer cells. But metastasis results were a little different in the case of chicken. While metastasis was less in the lungs, but both control and microRNA modified cancer cells failed to metastasise in the liver.

Testing epigenetic drugs

The researchers tested the effectiveness of epigenetic drugs to restore the levels of the microRNAs and reduce the expression of the SPINK1 gene using SPINK1-positive cancer cell lines that did not have the two microRNAs. “The epigenetic drugs could restore the expression of the two microRNAs leading to a reduction in the SPINK1 expression. We could abrogate the SPINK1-mediated oncogenicity in terms of cell-cycle progression, stemness and drug resistance,” says Prof. Ateeq. “We could see similar effects when we replenished the SPINK1-positive cancer cell lines with synthetic microRNAs.”

Future studies

“We will soon start studying the role of other RNA species which don’t code for proteins but play an important role in SPINK1 gene regulation. We are also looking at other genetic aberrations that play an oncogenic role in the SPINK1-positive subtype,” says Prof. Ateeq.

Published in The Hindu on January 5, 2019