An ‘off’ switch for aggressive breast cancer

Date: 8th June 2020

Article in brief:

There were an estimated 2.09 million cases of breast cancer worldwide in 2018, and 627,000 deaths. The most common cause of death for breast cancer patients is metastatic disease, which can affect vital organs. Now scientists have identified a gene that causes an aggressive form of breast cancer to rapidly grow and metastasise. By inhibiting the gene, TRAF3IP2, tumour formation was inhibited, preformed tumours regressed and metastasis was prevented.

Whilst genetic and epigenetic factors are the underlying cause of cancer, once established, the tumour microenvironment (TME) plays an important role in its growth and dissemination. The TME is composed of a cellular and acellular fraction, and signalling between the two fractions form the basis of pro-tumorigenic signalling, promoting further tumour growth and metastasis.

Now a team of scientists, from the Tulane University School of Medicine, and the University of Missouri School of Medicine and Harry S. Truman Veterans Memorial Hospital, US, have been investigating the role of the TME in tumour development and progression by studying two genes, TRAF3IP2 and Rab27a. Both are expressed in the TME and play roles in the secretion of substances that can cause tumour formation.

The team wanted to focus their research on triple negative breast cancer (TNBC) by inhibiting TRAF3IP2 and Rab27a. As TNBC is considered to be the most aggressive of breast cancers, and has a poor prognosis for treatment and survival, identifying potentially novel therapeutic targets would be hugely beneficial.

• To begin, expression levels of Rab27a and TRAF3IP2 were assessed in malignant (TNBC) and non-malignant breast cells and were shown to be significantly higher in the malignant cell line.
• To inhibit expression of the two genes the team used lentiviral delivery of specific short hairpin RNAs (shRNAs) and showed suppression of either Rab27a or TRAF3IP2 expression upon transduction of the shRNAs into TNBC cells.
• Suppression of either Rab27a or TRAF3IP2 expression significantly increased the population doubling times of the TNBC cells.
• Rab27a is known to play a role in exosome release - exosomes of non-malignant cells, such as mesenchymal stem cells (MSCs), play a crucial role in tumour progression and increased tumour cell migration. Exosomes originating from breast cancer cells can also transform MSCs into tumour-like cells, thus they play crucial roles in cancer progression. Here, the team showed that Rab27a knockdown resulted in a marked reduction in exosome-specific markers.
• In contrast, TRAF3IP2 is known to contribute to the inflammatory response by regulating multiple pro-inflammatory and pro-tumorigenic mediators. TRAF3IP2 knockdown, resulted in the differential expression of a panel of 12 inflammatory cytokines. One to note was the marked decrease of Interleukin 11 (IL11) expression, which is involved in migration and invasion of breast cancer cells.
• Silencing Rab27a or TRAF3IP2 also altered gene expression in TNBC cells and electron microscopy showed significant cell morphological changes in both cases.
• To examine the effects of supressing the genes in vivo TNBC cells were injected into mice and tumours were allowed to establish over time as either ‘wild type’ TNBC cells, or TNBC cells depleted of RAB27a or TRAF3IP2.
• The wild type TNBC cells produced tumours which had metastasised within 8 weeks, whereas no visible tumours could be detected in the mice injected with either knockdown TNBC cells at that time.
• After 15 weeks post injection very small tumours were detected in 6/6 mice injected with Rab27a depleted cells and only 3/6 mice injected with the TRAF3IP2 depleted cells showed very small tumours.
• Silencing Rab27a was shown to delay tumour development, and it significantly extended survival by more than 3-fold (30 weeks) compared with the wild type-injected mice. However, whilst macro-metastasis were supressed micrometastasis still occurred.
• Targeting TRAF3IP2 suppressed tumour growth, and the mice survived the length of the experiment (52 weeks). Importantly, macro- and micrometastasis were not observed.
• Finally, the team wanted to determine whether treating pre-existing tumours with lentiviral TRAF3IP2 shRNA could regresses their size. Indeed, a marked reduction in tumour size after 8 weeks post-treatment was seen, with only a small residual tumour detected and no metastases.

Conclusions and future applications:

The team have shown here that both Rab27a and TRAF3IP2 play a causal role in breast cancer growth and metastasis. TRAF3IP2 in particular has been identified here as a potentially novel therapeutic target for breast cancer. Silencing of TRAF3IP2 in TNBC cells suppressed tumour growth as well as macro- and micro-metastasis.

The really exciting part of the work, was that treatment with lentiviral TRAF3IP2 shRNA regressed pre-formed tumours and prevented metastasis. The animal study data was so compelling that the team is now working on FDA approval to begin clinical trials.

Several of the genes altered by silencing TRAF3IP2 are individually the subject of clinical trials for other types of cancer and disease. As such, this gene may be a potential target for other cancers and diseases too. In a collaborative effort the team are now also looking into targeting TRAF3IP2 for the treatment of glioblastoma, a brain cancer with limited treatment options.

However, whilst this preliminary data looks hopeful the team do acknowledge that in the immediate future the treatments will need to be extended to other patient-derived xenotransplants as only one was used here.

With a plethora of next generation tools now becoming available to treat cancer such as living biotherapeutics and nanoparticles able to delivery CRISPR machinery to edit cancerous cells, it is likely that this new target will receive much interest. With drug and therapeutic delivery systems evolving at a staggering rate, there is increasing hope for more efficient treatment for patients with such devastating and aggressive diseases.

For more information please see the press release from Tulane University

Alt, E. U., P. M. Wörner, A. Pfnür, J. E. Ochoa, D. J. Schächtele, Z. Barabadi, L. M. Lang, S. Srivastav, M. E. Burow, B. Chandrasekar and R. Izadpanah (2020). “Targeting TRAF3IP2, Compared to Rab27, is More Effective in Suppressing the Development and Metastasis of Breast Cancer.” Scientific Reports 10(1): 8834.

https://doi.org/10.1038/s41598-020-64781-z