Comprehensive Notes on Circadian Rhythms and Triple Negative Breast Cancer Research

Introduction to the Speaker and Research Focus

• Speaker: Olaji Mokeo Ogulosi (Often goes by "Ola").
• Background: Fourth-year PhD student in the Department of Biology at Texas A&M University (TAMU).
• Primary Research Focus: Investigating how disruption to the circadian clock promotes the progression of Triple Negative Breast Cancer (TNBC), the most aggressive and difficult-to-treat subtype of breast cancer.
• Purpose: To provide a comprehensive overview of circadian biology, clock gene mechanisms, the health consequences of rhythm disruption, and specific laboratory findings regarding the molecular link between clock disruption and tumor aggressiveness.

Fundamental Concepts of the Circadian Clock

• Definition: The circadian clock is an internal, biological alarm system that regulates the timing of cellular and organ functions.
• Etymology: The word "circadian" is derived from the Latin phrase "circa diem," meaning "about a day."
• Cycle Duration: Most biological rhythms controlled by this clock occur over a period of approximately $24$ hours.
• Hierarchical Structure:
  • Master Clock: Located in the suprachiasmatic nucleus (SCN), a very tiny region within the brain. It acts as the central coordinator for all other rhythms in the body.
  • Peripheral Organs: Organs such as the liver, lungs, and heart possess their own internal clocks, which are synchronized by the master clock in the SCN.
• Genetic Regulation: Approximately $10\%$ of all human genes are under direct circadian control, including genes responsible for immunity and cell division.

The Molecular Mechanism: Transcription-Translation Feedback Loop (TTFL)

• Positive Arm of the Clock:
  • The primary positive regulators are the genes CLOCK and BMAL1 (Brain and Muscle ARNT-Like $1$).
  • These two genes must team up (forming a dimer) to become active. Once active, they initiate the expression of other clock genes.
• Negative Arm of the Clock:
  • The positive arm triggers the expression of genes called PER (Period) and CRY (Cryptochrome).
  • As the day progresses into evening, the expression levels of PER and CRY proteins peak.
  • Upon reaching high concentrations, these proteins enter the cell nucleus and bind to the CLOCK-BMAL1 complex, effectively blocking their activity.
• Cycle Maintenance: The clock stays active in a cycle; PER and CRY eventually degrade over time, which releases the inhibition on CLOCK and BMAL1, allowing the sequence to restart approximately every $24$ hours.

Physiological Processes Regulated by the Clock

• Sleep-Wake Cycle:
  • Melatonin: A hormone responsible for inducing sleep. It typically begins to rise between $8\text{--}10\text{PM}$.
  • Cortisol: A hormone responsible for waking the body. It peaks in the morning between $6\text{--}8\text{AM}$.
• Cell Division and Growth: The clock regulates the timing of the cell cycle, including mitosis (prophase, metaphase, anaphase, and telophase) and DNA repair mechanisms.
• Immune System Function: The secretion and patrolling activity of immune cells, such as T-cells, NK (Natural Killer) cells, and dendritic cells, are time-dependent. Studies suggest vaccines may be more effective when administered in the morning versus the afternoon due to these rhythms.
• Drug Metabolism: Liver enzymes responsible for clearing and degrading drugs peak at specific times. Administering medication (like statins) at the correct circadian time increases effectiveness and reduces toxicity.
• Body Temperature: Reaches its lowest point at approximately $4\text{00}$ ($4\text{AM}$) and its peak at approximately $18\text{00}$ ($6\text{PM}$).
• Mood and Cognition: Neurotransmitters like serotonin and dopamine are regulated by the clock. Disruption can lead to Seasonal Affective Disorder (SAD), particularly in temperate climates where light levels change significantly during winter.

Circadian Rhythm Disruption (CRD)

• Definition: CRD occurs when the internal biological clock is no longer in sync with the external environment.
• Primary Causes:
  • Night Shift Work: Common in nurses, factory workers, and truck drivers. This involves working during the night when melatonin secretion is high.
  • Jet Lag: Occurs when traveling across time zones. The body remains synced to the original location's time, causing imbalances in eating and sleeping (e.g., Nigerian time is approximately $6$ hours ahead of Central US time).
  • Light at Night: Exposure to blue light from phones or screens inhibits melatonin production and signals to the brain that it is still daytime.
  • Eating at Odd Hours: Eating when metabolic hormones are not peak-secreted can cause bloating and microbiota imbalances.
• Global Impact: Between $15\%$ and $20\%$ of the global workforce is involved in night shift work.

Implications for Cancer Pathology

• Classification: The International Agency for Research on Cancer (IARC) has classified shift work as a carcinogen (a cancer-causing agent).
• Risk Factors: Increased years of shift work correlate with a higher risk for breast, colon, and prostate cancers.
• Clock Genes in Cancer:
  • PER Genes: Often act as tumor suppressors; they are frequently silenced or mutated in breast and colorectal cancers.
  • BMAL1: Low levels of BMAL1 expression are associated with poor cancer prognosis because it regulates genes like C-MYC and p21.
  • CLOCK: Overexpression of CLOCK can work with MYC to drive rapid, unscheduled cell growth.
• Recognition: The discovery of these molecular clock mechanisms led to the Nobel Prize in Physiology or Medicine being awarded to Jeffrey Hall, Michael Rosbash, and Michael Young.

Laboratory Case Study: CRD and Triple Negative Breast Cancer (TNBC)

• Research Title: "LILRB4 regulates circadian disruption-induced mammary tumorigenesis via the noncanonical Wnt signaling pathway."
• Research Question: How does clock disruption specifically cause breast cancer to become more aggressive at the molecular and immune levels?
• Methodology:
  • Mouse Model: Researchers used a Genetically Engineered Mouse Model (GEMM) designed to develop aggressive breast cancer spontaneously.
  • CRD Protocol: Chronic life cycle disruption was induced using an "eight-hour phase advance" in light every $2\text{--}3$ days (e.g., giving mice $20$ hours of light and $4$ hours of dark).
• Key Findings on Growth Timing:
  • Mice on a normal schedule developed tumors at approximately $22$ weeks.
  • Mice under CRD developed tumors at approximately $18$ weeks.
  • This $4$-week difference in mice translates to approximately $8\text{--}10$ years in human lifespan.
• Key Findings on Morphology: CRD led to reduced branching in the mammary glands and significantly increased markers for ductal hyperplasia (the initial stage of tumor formation).

The Molecular Link: LILRB4

• Definition: LILRB4 stands for Leukocyte Immunoglobulin-Like Receptor B4.
• Function: It is an immune checkpoint that acts as a "do not attack" signal. Its normal function is to prevent immune cells from attacking healthy tissue.
• Exploitation by Cancer: Tumor cells overexpress LILRB4 to hide from T-cells and NK cells, effectively evading the immune system.
• Tumor Microenvironment (TME):
  • Hot Tumor: A tumor with high immune cell interaction that suppresses growth.
  • Cold Tumor: A tumor that avoids immune detection. CRD causes tumors to develop this "cold" phenotype via LILRB4 expression.
• Metastasis: CRD was found to significantly increase metastasis foci in the lungs. Triple Negative Breast Cancer is particularly prone to spreading to the lungs, brain, and liver.
• Therapeutic Potential: Blocking LILRB4 with an antibody reduced tumor growth and lung metastasis in the study. The FDA has already granted fast-track designation for anti-LILRB4 antibodies in treating Acute Myeloid Leukemia (AML).

Clinical and Future Applications

• Chronotherapy: The practice of timing medical treatments to coincide with the body's internal rhythms to maximize effectiveness and minimize side effects like fatigue or hair loss.
• Precision Immunotherapy: Targeting LILRB4 specifically in patients with circadian disruption could provide a new pathway for treating TNBC, which currently lacks targeted hormonal therapies (estrogen, progesterone, and HER2 receptors).
• Preventative Measures:
  • Maintain proper sleep hygiene.
  • Limit blue light exposure at night.
  • Maintain regular schedules.

Career Opportunities in the Field

• Cancer Biologist: Requires a PhD to study the underlying mechanisms of the disease.
• Oncologist: Can be pursued via an MD or an MD-PhD track; the latter combines medical practice with high-level molecular research.
• Computational Biologist: Utilizes AI, machine learning, and "Big Data" to analyze clock gene expression and develop patient-targeted therapies.

Questions & Discussion

• Student Introductions: Pragya is a rising senior from Austin, Texas, interested in the premed track and biology/computational biology. Raina is a rising senior from Chicago interested in biomedical engineering.
• Question (Raina): What kind of work are you (the speaker) currently doing for your thesis?
• Response (Ola): Research is focused on elucidating the specific interactive partners of LILRB4. This involves techniques such as cell culture, in vivo mouse models, flow cytometry (to define immune populations), and single-cell RNA sequencing to understand which genes are being regulated. Mentorship of undergraduate students and collaboration across departments (like biomedical engineering) is also a major component.
• Advice to Students: Undergraduate students should reach out to professors in their freshman year to get involved in research early. This builds analytical thinking and data-processing skills that are valuable for any clinical or scientific career.