RNA-seq analysis of wild-type and mutated TBPL1 gene in breast cancer cells lines through CRISPR/Cas9 approach reveals novel molecular signatures – Nature

Report on the Transcriptional Regulation Role of TBPL1 in Breast Cancer and its Alignment with Sustainable Development Goals

Executive Summary

Breast cancer remains a primary cause of mortality among women globally, presenting a significant challenge to achieving Sustainable Development Goal 3 (SDG 3), which aims to ensure good health and well-being. This report details an investigation into the role of the TATA-box binding protein-like 1 (TBPL1) gene, a transcriptional regulator whose function in breast cancer has been previously undisclosed. Utilizing advanced genomic technologies, in line with SDG 9 (Industry, Innovation, and Infrastructure), this study sought to determine the transcriptomic impact of TBPL1. Key findings indicate that TBPL1 is overexpressed in aggressive breast cancer cell lines and its knockout via CRISPR/Cas9 technology alters gene expression profiles related to cell proliferation, migration, and metastasis. These results identify TBPL1 as a potential biomarker and therapeutic target, contributing vital knowledge toward SDG Target 3.4, which seeks to reduce premature mortality from non-communicable diseases.

Introduction: Aligning Cancer Research with Global Health Goals

The Global Burden of Breast Cancer and SDG 3

Breast cancer is a leading non-communicable disease (NCD) and a major impediment to global health equity. The high mortality rate associated with the disease directly contravenes the objectives of SDG 3: Good Health and Well-being. Specifically, understanding the molecular mechanisms of breast cancer is fundamental to developing effective prevention and treatment strategies required to meet SDG Target 3.4: to reduce premature mortality from NCDs by one-third by 2030.

Genetic Basis of Cancer and the Focus on TBPL1

The progression of cancer is characterized by the dysregulation of genes controlling cellular processes. This study focuses on the TATA-box binding protein (TBP) family, which is central to gene transcription. While the role of TBP is well-documented, its paralog, TATA-box binding protein-like 1 (TBPL1), remains poorly understood in the context of breast cancer. Previous studies have implicated TBPL1 in other malignancies, such as colorectal and pulmonary carcinoid tumors. The objective of this investigation was to elucidate the transcriptomic function of TBPL1 in breast cancer cell lines to identify novel molecular signatures that could inform future therapeutic strategies and contribute to the knowledge base required to achieve global health targets.

Methodology: Leveraging Innovation for Health Research (SDG 9)

This research employed advanced scientific methodologies, reflecting the aims of SDG 9: Industry, Innovation, and Infrastructure, particularly Target 9.5, which encourages enhancing scientific research and upgrading technological capabilities.

Cell Line Analysis

A comparative analysis was conducted using a panel of human cell lines:

• Healthy Breast Cell Lines (Controls): MCF-12F, MCF-10A
• Breast Cancer Cell Lines: T47D, SKBR3, MDA-MB-231, HCC-1937, MCF-7

Genetic Manipulation and Analysis

The study utilized a multi-step approach to determine gene function:

1. Gene Expression Analysis: Initial mRNA expression levels of TBP family members (TBP, TBPL1, TBPL2) were quantified using RT-qPCR.
2. Gene Knockout: The CRISPR/Cas9 system was used to precisely knock out the TBPL1 gene in selected healthy and cancerous cell lines.
3. Transcriptome Profiling: Next-Generation Sequencing (RNA-seq) was performed to generate comprehensive gene expression profiles for both wild-type and TBPL1-knockout cell lines, allowing for a comparative analysis of downstream genetic regulation.

Key Findings: Uncovering Molecular Signatures of Breast Cancer

Differential Expression of TBP Family Genes

• A direct correlation was observed between the expression levels of TBP and TBPL1 and the aggressiveness of breast cancer cell lines. The most aggressive lines, SK-BR-3 and MDA-MB-231, exhibited significant overexpression of both genes compared to healthy controls.
• Expression of TBPL2, another TBP paralog, was not detected in any of the cell lines studied.
• These findings suggest that elevated TBPL1 expression could serve as a potential biomarker for aggressive breast cancer phenotypes, aiding in risk assessment and contributing to the goals of SDG 3.

Impact of TBPL1 Knockout on Gene Expression

• The successful knockout of the TBPL1 gene resulted in significant alterations to the transcriptome in all tested cell lines.
• RNA-seq data revealed distinct sets of upregulated and downregulated genes in TBPL1-mutated cells compared to their wild-type counterparts, indicating that TBPL1 is a key regulator of a broad network of genes.
• Notably, the analysis identified several novel long non-coding RNAs (lncRNAs) whose expression is dependent on TBPL1, opening new avenues for research into non-coding regulators in cancer.

Pathways Regulated by TBPL1

Functional analysis of the differentially expressed genes identified several critical pathways influenced by TBPL1, which are directly relevant to cancer progression and public health outcomes:

• Cell Proliferation, Migration, and Metastasis: TBPL1 was found to regulate genes involved in cell duplication, morphology, and growth, which are hallmarks of cancer development and spread.
• Apoptosis: The gene appears to play a role in anti-apoptotic pathways, which enable cancer cells to evade programmed cell death.
• Tumor Formation: In vivo data suggests that TBPL1 may contribute to tumor formation in aggressive cell lines like MDA-MB-231.

Discussion: Implications for Sustainable Development Goals

Contribution to SDG 3: Good Health and Well-being

The findings of this report have direct implications for advancing SDG 3 by addressing the global challenge of breast cancer.

• Therapeutic Target Identification: By establishing TBPL1 as a regulator of cancer-promoting pathways, this study identifies it as a promising therapeutic target. Developing inhibitors against TBPL1 or its downstream effectors could lead to novel treatments for aggressive breast cancer, supporting SDG Target 3.4.
• Biomarker Development: The correlation between high TBPL1 expression and cancer aggressiveness suggests its potential as a prognostic biomarker. This could improve patient stratification and treatment planning, contributing to better health outcomes.
• Foundation for New Research: The discovery of novel transcripts and lncRNAs regulated by TBPL1 provides a foundation for future research into the complex regulatory networks of breast cancer, fostering the continuous innovation needed to combat NCDs.

Advancing Scientific Innovation (SDG 9)

This research exemplifies the application of cutting-edge scientific innovation to address pressing global health issues. The use of CRISPR/Cas9 and RNA-seq technologies underscores the importance of SDG Target 9.5, which calls for enhancing scientific research and technological capacity. Such advancements are crucial for developing the sophisticated tools needed to understand and treat complex diseases like cancer.

Conclusion and Recommendations

This report concludes that the TBPL1 gene is a significant transcriptional regulator in breast cancer, with its expression level correlating with disease aggressiveness. Its demonstrated role in controlling pathways essential for cell proliferation, migration, and survival positions it as a key factor in breast cancer biology. This research provides a strong foundation for future studies aimed at developing targeted therapies against TBPL1, which would represent a significant step toward achieving the health targets outlined in SDG 3.

It is recommended that further research focuses on:

1. Investigating the precise molecular mechanisms by which TBPL1 regulates downstream cancer-related genes.
2. Validating the potential of TBPL1 as a prognostic biomarker in clinical patient cohorts.
3. Exploring the functional roles of the novel lncRNAs discovered in this study to determine their potential as therapeutic targets or diagnostic markers.

SDGs, Targets, and Indicators Analysis

1. Which SDGs are addressed or connected to the issues highlighted in the article?

The primary Sustainable Development Goal (SDG) addressed in the article is:

• SDG 3: Good Health and Well-being

  Explanation: The article directly addresses a major global health challenge by focusing on breast cancer, which it identifies as “the leading cause of death among women globally.” The research aims to understand the molecular mechanisms underlying this disease, specifically the role of the TBPL1 gene in the development and progression of breast cancer. This investigation into a non-communicable disease is central to the mission of SDG 3, which is to ensure healthy lives and promote well-being for all at all ages.

2. What specific targets under those SDGs can be identified based on the article’s content?

Based on the article’s focus, the following specific targets under SDG 3 can be identified:

1. Target 3.4: By 2030, reduce by one-third premature mortality from non-communicable diseases through prevention and treatment and promote mental health and well-being.

   Explanation: Breast cancer is a major non-communicable disease (NCD). The research presented in the article is a fundamental step toward developing more effective treatments. By exploring the role of the TBPL1 gene in “cell migration, proliferation, anti-apoptosis, and metastasis,” the study contributes to the body of knowledge required to create targeted therapies that could ultimately reduce premature mortality from breast cancer.

2. Target 3.b: Support the research and development of vaccines and medicines for the communicable and non-communicable diseases that primarily affect developing countries, provide access to affordable essential medicines and vaccines, in accordance with the Doha Declaration on the TRIPS Agreement and Public Health, which affirms the right of developing countries to use to the full the provisions in the Agreement on Trade-Related Aspects of Intellectual Property Rights regarding flexibilities to protect public health, and, in particular, provide access to medicines for all.

   Explanation: The article represents a direct contribution to the research and development (R&D) of potential new cancer therapies. By identifying the TBPL1 gene’s involvement in cancer aggressiveness and its potential as a therapeutic target (“underscores the significance of investigating this transcription factor as a key target in cancer biology”), the study supports the foundational scientific work necessary for the future development of medicines for a globally significant NCD.

3. Are there any indicators mentioned or implied in the article that can be used to measure progress towards the identified targets?

The article does not mention official SDG indicators directly, but it implies several scientific metrics that serve as proxies for measuring progress toward the identified targets:

• Implied Indicators for Target 3.4:
  • Understanding of molecular mechanisms of cancer: The study measures progress by analyzing “differential gene expression patterns” and identifying the roles of specific genes (like TBPL1) in cancer-related processes. The article states its aim was to “determine the transcriptome of the TBPL1 gene in breast cancer cell lines,” which serves as a measure of advancing scientific knowledge needed to combat the disease.
  • Assessment of cancer cell properties: The research uses metrics such as “cell duplication, morphology, and growth,” as well as “cell migration, invasion, metastasis,” to quantify the aggressiveness of cancer cells. The finding that knocking out the TBPL1 gene “reduces cell viability” in both healthy and cancerous cell lines is a quantifiable outcome that contributes to understanding the disease and potential treatment side effects.
• Implied Indicators for Target 3.b:
  • Identification of potential therapeutic targets: A key outcome of the research is the identification of the TBPL1 gene as a factor in cancer progression. The article concludes that its findings suggest a “potential role of the TBPL1 gene in cancer aggressiveness,” making it a candidate for future drug development. This identification is a direct measure of progress in the R&D pipeline.
  • Application of advanced research technologies: The use of cutting-edge methods like “CRISPR/Cas9 technology” and “RNA-seq analysis” demonstrates active and advanced research efforts. The successful application of these technologies to study cancer biology is an indicator of ongoing R&D activities supported by the scientific community.

4. Summary Table of SDGs, Targets, and Indicators

Source: nature.com