Monga et al. · Journal of Biological Chemistry · 2022

Tribbles 2 pseudokinase confers enzalutamide resistance in prostate cancer by promoting lineage plasticity

Monga Jitender, Adrianto Indra, Rogers Craig, Gadgeel Shirish, Chitale Dhananjay, Alumkal Joshi J., Beltran Himisha, Zoubeidi Amina, Ghosh Jagadananda

Read the paper The product used

The study

What was asked, and what was found

Monga et al., Journal of Biological Chemistry, 2022 asked why prostate cancer stops responding to enzalutamide. They built resistant lines by feeding three human prostate cancer lines rising concentrations of the drug over 3 months, then looked for what had changed. Expression arrays, RT-PCR and immunoblots all pointed at one gene: the pseudokinase TRIB2 was heavily overexpressed in the resistant cells. Immunohistochemistry extended that to 20 patient derived xenografts and to patient tumours, 53 treated with enzalutamide against 75 untreated, where a vast majority of the treated tumours showed an increase in the protein.

The question was then whether TRIB2 is a passenger or the cause. Knockdown in culture killed the resistant cells while leaving benign prostate cells unharmed. Overexpressing TRIB2 alone made sensitive cells resistant, and removing it again made them sensitive. Mechanistically the cells changed identity, losing the luminal markers androgen receptor and cytokeratin 8 and gaining neuroendocrine markers along with the neuronal factor BRN2 and the stemness factor SOX2, and blocking either of those two restored sensitivity to the drug.

The animal test used AUMsilence sdASO against TRIB2, supplied by AUM BioTech, injected directly into the tumour at 2 mg/kg/day every fourth day for 4 weeks once tumours reached about 100 cubic millimetres. It was used in two xenograft experiments, one on enzalutamide resistant LNCaP-ENR tumours and one on TRIB2 overexpressing tumours. For the first the paper states that tumour growth fell. For the second it reports the marker staining and says tumour volumes were measured, but it never states a growth outcome in the text, so none is claimed here. Tumours from the treated animals were stained for the neuroendocrine and stemness markers, tying the in vivo work back to the mechanism. One thing to note when reading this paper: the Results sentence describing the first of those experiments calls the reagent siRNA, while the figure legend for the same panels and the Methods both describe single stranded antisense oligonucleotides from the company.

Key findings

AUMsilence sdASO against TRIB2 was injected into enzalutamide resistant human prostate tumours in nude mice at 2 mg/kg/day every fourth day, and tumour volumes were followed to the end of the study.(Figure 2 caption, panels D and E)
The paper reports that silencing TRIB2 lowered the growth of those enzalutamide resistant tumours, in the one sentence where it states the outcome, and that sentence calls the reagent siRNA while the figure legend it cites and the Methods both describe a chemically modified antisense oligonucleotide from the company.(Results, TRIB2 plays a critical role in enzalutamide-resistant prostate cancer cells, Fig. 2 D and E)
The same oligonucleotide was used against tumours engineered to overexpress TRIB2, and those xenografts were then stained for the neuroendocrine and stemness markers the paper is about.(Figure 4 caption, panel F)
TRIB2 is strongly raised in prostate cancer that has become resistant to enzalutamide, in cell lines, in patient derived xenografts and in patient tumours.(Results, Enzalutamide-resistant prostate cancer cells and tumors overexpress TRIB2)
Knocking TRIB2 down killed the resistant cells while leaving benign prostate cells alone, so the dependency is selective to the resistant state.(Results, TRIB2 plays a critical role in enzalutamide-resistant prostate cancer cells)
Raising TRIB2 alone was enough to make sensitive prostate cancer cells resistant to clinically relevant doses of enzalutamide, and taking it away again restored sensitivity.(Results, TRIB2 enhances prostate cancer cell growth and invasion and confers resistance to enzalutamide)
The mechanism was a change of cell identity: luminal markers fell and neuroendocrine markers rose, along with the neuronal factor BRN2 and the stemness factor SOX2.(Results, TRIB2 confers resistance to enzalutamide by promoting lineage plasticity to develop NE phenotype)

Product: AUMsilence sdASO
Targets: TRIB2 · human · mRNA
Models: athymic nude mouse, subcutaneous xenograft of enzalutamide resistant human LNCaP-ENR prostate cancer cells · in vivoathymic nude mouse, subcutaneous xenograft of TRIB2 overexpressing LNCaP-TRIB2 cells · in vivohuman LNCaP, MDA-PCa-2B and LAPC4 prostate cancer cells and their enzalutamide resistant derivatives, plus BPH-1 benign prostate cells. These arms used shRNA rather than the oligonucleotides. · cell lineprostate tumour tissue microarrays, patient derived xenografts and patient tumours with and without enzalutamide treatment · human samples
Delivery: intratumoral injection, no formulation or delivery reagent described
Readouts: caliper tumour volume, immunohistochemistry, immunoblot, RT-PCR, gene expression array and RNA sequencing, MTS cell viability assay, Annexin V apoptosis assay, soft agar colony formation, Matrigel Boyden chamber invasion
Measured: dose of the TRIB2 antisense oligonucleotide in the mouse xenografts: 2 mg/kg/day (intratumoral injection, every fourth day, for 4 weeks)tumour size at which the mice were randomised to treatment: approximately 100 mm³ (LNCaP-ENR subcutaneous xenograft, 7 week old male athymic nude mice, n = 3)cells implanted per mouse: 3 × 10⁶ cells per mouse in 50% Matrigel in PBS (subcutaneous flank injection)enzalutamide concentration used to make and maintain the resistant lines: 30 μM (increasing concentrations of enzalutamide over 3 months, then maintained)share of enzalutamide resistant prostate cancers with neuroendocrine features: About 10 to 20% (cited from the literature, not measured in this study)patient and xenograft material stained for TRIB2: 20 patient derived xenografts, 53 treated and 75 untreated patient tumours (immunohistochemistry, quantified with QuPath)
Figures: Figure 2 The oligonucleotide arm in enzalutamide resistant xenografts, alongside the shRNA and afatinib work in culture.Figure 4 The lineage switch, including the oligonucleotide treated TRIB2 overexpressing xenografts and their marker staining.
Citation: Monga et al. Tribbles 2 pseudokinase confers enzalutamide resistance in prostate cancer by promoting lineage plasticity. Journal of Biological Chemistry 2022;298:101556. doi:10.1016/j.jbc.2021.101556

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