EP1066 - MLK4 Knockdown Represses a Network of Oncogenes Responsible for Cell Proliferation and Invasion in Colon Cancer

ePoster Presenter(s)

• 

  HITESH KAPOOR, MBBS

  Research Fellow
  University Of Illinois Chicago
  University of Illinois Chicago
  CHICAGO, IL, United States

  Disclosure information not submitted.

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Author(s)

• SJ

  Saket Jha, PhD

  Post doctoral Fellow
  University of Illinois Chicago
  Chicago, Illinois, United States

  Disclosure information not submitted.
• 

  Nathan Kumar, BS (he/him/his)

  Medical Student
  University of Illinois Chicago College of Medicine
  Chicago, IL, United States

  Disclosure information not submitted.
• RN

  Rakesh Nair, MSc., PhD

  RESEARCH ASST.PROFESSOR
  DEP. OF SURGERY
  CHICAGO, Illinois, United States

  Disclosure information not submitted.
• SS

  Sunil Singh, PhD

  Post Doctoral Research Associate
  University of Illinois Chicago
  Chicago, Illinois, United States

  Disclosure information not submitted.
• AR

  Ajay Rana, n/a

  Endowed Professor
  University of Illinois at Chicago
  Chicago 60612, Illinois, United States

  Disclosure information not submitted.

Senior Author(s)

• GG

  Gerald Gantt, MD

  Associate Professor of Surgery
  University of Illinois Chicago
  Chicago, Illinois, United States

  Disclosure information not submitted.

Purpose/Background: MLK4 is implicated in colon cancer pathology, as evidenced by its increased expression and sensitivity to the inhibitor CEP-1347 in previous studies. To define its molecular role, we created stable MLK4-knockdown (KD) HCT116 cell lines and used the RT² Profiler™ PCR Array to determine how the loss of MLK4 reshapes the transcriptional expression of key oncogenes and tumor suppressor genes in colon cancer.

Methods/Interventions: Lentiviral particles encoding MLK4-specific shRNA constructs were produced in HEK293T packaging cells using a standard third-generation lentiviral system. Following transduction, cells underwent antibiotic selection using puromycin, 1 μg/mL, for 7 days to establish stably transduced populations. Total RNA was isolated, converted to cDNA, and subjected to qPCR using the 96-well gene array. Gene expression was quantified using the ΔΔCt method, with fold changes calculated relative to WT cells.

Results/Outcomes: BCR was the only gene found to be significantly upregulated with a fold change of +2.31. A total of 12 genes showed downregulation, indicating potential suppression of oncogenic signaling pathways or tumor suppressor dysfunction with MYCN (-16.08) and HGF (-12.04), ROS1 (-5.75), KITLG (-5.05), and ESR1 (-4.74) of noteworthy potential. The expression changes were validated using qPCR analysis in the shMLK4-treated cells and WT cells.

Conclusion/Discussion: The stable knockdown of MLK4 in HCT116 cells significantly suppressed the expression of a critical oncogenic network, confirming its role in promoting the malignant phenotype. We observed profound downregulation of key drivers, including the proliferation factor MYCN (∼16-fold) and the invasion ligand HGF (∼12-fold) in CRCs. The overall inhibitory effect on oncogenic transcription, despite the upregulation of BCR, indicates that MLK4 is an upstream regulator of tumor-promoting pathways. Thus, targeting MLK4 represents a high-potential therapeutic strategy for simultaneously disrupting the proliferative and invasive capabilities of colorectal cancer.

MLK4 knockdown suppresses oncogenic transcripts in colon cancer cells