Research paper 2

Question 1

Title?

Answer 1

Identification of FOXN4 as a tumor suppressor of breast carcinogenesis via the activation of TP53 and deactivation of Notch Signaling

Question 2

What is FOX?

Answer 2

• fork head domain-containing superfamily proteins
• transcription factor… regulating expression of genes involved in cell growth, proliferation, differentiation, longevity.
• defining feature is the forkhead box, a sequence of amino acids that binds DNA

Question 3

Why is Fox studied?

Answer 3

• Fox has been found to play a role in biological processes such as carinogenesis…
• dysfunction of Fox = disorder

Question 4

Why FOXN4?

Answer 4

• member of the Fox protein family..
• least studied
• primary function is shown to be involved in determining cell fate in retinal progenitor cells through the use of notch signaling

Question 6

Progenitor cells

Answer 6

• A progenitor cell is a biological cell that can differentiate into a specific cell type

Question 7

Whats the difference between a progenitor cell and a stem cell?

Question 8

Activation of notch signaling

Answer 8

Notch signaling occurs when membrane-bound ligands such as Dll1 and Dll4 on one cell activate Notch receptors on neighboring cells (Figure 1A, trans-activation) (Artavanis-Tsakonas et al., 1999; Nichols et al., 2007b; Bray, 2016). However, other types of interactions are also known to occur.

Question 9

Notch signaling

Answer 9

Notch signaling is an evolutionarily conserved pathway in multicellular organisms that regulates cell-fate determination during development and maintains adult tissue homeostasis (control of proliferation, death, and tissue renewal).

Question 10

Describe figure 1A

Answer 10

• Q: What are the abundance lvls of FOXN4 mRNA in N adjacent vs Tumor tissue?
• Researchers used qPCR (quantify number of nucleic acids) in order to determine the abundance of mRNA.
• They found that NT had a significantly higher amount of FOXN4 mRNA in comparison to tumor tissue
• conclusion:

Question 11

Describe figure 1B

Answer 11

• Q: What are the levels of FOXN4 mRNA in high risk vs low risk breast cancer patients?
• Q: Why low vs high risk?:
• Researchers used breast cancer data from the ATCG database to compare levels of FOXN4 mRNA in low risk vs high risk breast cancer patients.
• Found significantly lower FOXN4 mRNA was associated with higher risk of breast cancer
• conclusion: Low levels of FOXN4 is associated with a higher risk of breast cancer.

Question 12

Describe figure 1C

Question 13

Describe figure 1D

Answer 13

Q: Is there a significant difference in FOXN4 mRNA levels in cell lines from normal tissue vs tumor tissue?
- qPCR to measure mRNA levels in normal cell lines vs breast cancer cell lines
- found lower levels of FOXN4 mRNA in tumor cell lines vs normal cell lines
- found lower levels of FOXN4 mRNA in metastatic cell lines than non-metastatic cell line
- C: FOXN4 levels in breast cancer cell lines are significantly lower than FOXN4 mRNA in normal cell lines.
FOXN4 levels in metastatic breast cancer cell lines are significantly lower than non-metastatic cell lines

Question 14

Describe figure 2A

Answer 14

• Q: What are the levels of FOXN4 mRNA in NM cell line MCF-7 vs M cell line T74D when FOXN4 is overexpressed or silenced?
• In order to further explore the role of FOXN4 on breast cancer cells by gain and loss of function analysis, the researchers first silenced and overexpressed FOXN4 mRNA in cell lines MCF-7 and T74D. why? to use the overexpressed and silenced samples to explore differences in viability and colony formation.
  Silencing: shRNA molecules are processed within the cell to form siRNA which in turn knock down gene expression. The benefit of shRNA is that they can be incorporated into plasmid vectors and integrated into genomic DNA for longer-term or stable expression, and thus longer knockdown of the target mRNA.
  Overexpression: clone it into an expression construct and transfect and retain the plasmid in the cell using a selectable marker.
• found suppression and overexpression of FOXN4 mRNA in both cell lines

Question 15

Describe figure 2B

Answer 15

• Q: How does gene silencing and overexpression in non-metastatic MCF-7 effect cell viabiliy? (# of live, healthy cells)
• Used MTT assay, a colorimetric assay for assessing cell metabolic activity
• found: silencing of FON4 had an increase in cell viability, overexpression of FOXN4 led to a decrease in cell viability
• conclusion: Silencing FOXN4 promotes tumor cell viability, overexpression of FOXN4 inhibits tumor cell viability’

Question 16

Describe figure 2C

Answer 16

• Q; How does gene silencing and overexpression of FOXN4 affect cell viability in in metastatic breast cancer cell lineMDA_MD-486
• used MTT assay, colorimetric assay for assessing cell metabolic activity
• found: silencing of FOXN4 was significantly higher in cell viability than overexpression of FOXN4
• conclusion: silencing FOXN4 promotes tumor cell viability, overexpression of FOXN4 inhibits tumor cell viability

Question 17

Describe figure 2D

Answer 17

• Q: How does FOXN4 affect colony formation in non-metastatic cell line MCF-7 vs metastatic cellline MDA-MB-486
• colony formation assay, propensity of individual cells to survive and propagate over long periods of time
• found: OE led to reduced colony formation, Sh: promote colony formation
• conclusion: FOXN4 may reduces colony formation in tumor cells

Question 18

Describe figure 3A

Answer 18

• Q: What are the levels of ECF biomarkers in non-metastatic cell line MCF-7?
• used RT-PCR analysis
• found: Silenced: reduced E-cadherin, increased N-cadherin and vimentin…. Overexpression: increased E-cadherin, decreased N-cadherin and vimentin…
• conclusion: FOXN4 plays a role in the suppression of ECF biomarkers

Question 19

describe figure 3C

Answer 19

Q: how does FOXN4 affect migration of MCF-7 cells?
- used wound healing assay
- found: Silencing of FOXN4 increased MCF-7 migratory ability, OE reduced migratory ability
conclusion: FOXN4 plays a role in suppression of cancer cell migration

Question 20

Describe figure 3D

Answer 20

Q: How does FOXN4 affect invasion ability of MCF-7?
- transwell assay
- found OE: inhibited invasion ability, Sh: promoted invasion ability
conclusion: FOXN4 reduces the invasion ability of tumor cell line MCF-7.

Question 21

Describe figure 4A?

Answer 21

Q: Which TF does cell line MCF and MDA-MB-486 have the highest affinity for?
- Used a qCHIP assay: chromatin immunoprecipitation sequencing: determine if specific proteins are associated with genomic regions. In this case, the association of different transcription factors with FOXN4 gene in each cell line
- found: highest affinity for TP53
- conclusion: The transcription factor TP53 is a major target for FOXN4 in both MCF and MDA.

Question 22

Describe figure 4B

Answer 22

• Q: How do different FOXN4 mRNA levels affect TP53 promoter luciferase activity?
• used a TP53 promoter assay (dual luciferase reporter assay) to study regulation of tp53 promotor luciferase activity
• found: OE of FOXN4 led to significantly higher levels of TP53 promotor luciferase activity
• conclusion: FOXN4 activates promotor activity in tumor cells.

Question 23

Describe figure 4C

Answer 23

• Q: How is the mRNA abundance of downstream signaling molecules affected by the presence of FOXN4?
• Used qPCR to detect mRNA levels in signaling molecules within OE and Sh cell lines
• found: OE of FOXN4 led to decreased expression of signaling molecules…. Sh led to increased levels of signaling molecule mRNA
  conclusion: FOXN4 suppresses the expression of signaling molecules Survivin, DII4 and Notch1 in tumor cells.

Question 24

Describe figure 5A

Answer 24

• Q: How is the average tumor size over time affected by the inhibition of notch?
• treated the mice subjects with notch inhibitor PF-3084014 measured tumor size over 8 weeks
• notch inhibited mice showed a significantly reduced tumor size
• conclusion: notch may play a role in promoting tumor growth

Question 25

Describe figure 5B

Answer 25

• Q: