Jamshed Arslan, Pharm D, PhD
In the United States, 1 in 8 women will be diagnosed with breast cancer in her lifetime.1 Despite the prevalence, cancer genesis is a mystery. The heterogeneity of cancers makes it difficult to study the precise mechanism of breast cancer development in vivo. Yet, research has shown that proteins containing a DNA/RNA-binding cold-shock domain, which have a fundamental role in DNA repair and mRNA transcription and translation, seem to play a key part in malignant transformation and invasiveness.
One such DNA/RNA-binding protein, YB-1, has previously been shown to contribute to the epithelial-mesenchymal transition and metastasis. YB-1 enhances translation of HIF1A and NFE2L2/NRF2 by binding to the 5’ untranslated region (UTR) of their mRNAs. However, studying different stages of breast cancer in vivo is challenging because of lack of human samples. A multicontinental team of researchers overcame this obstacle by transducing normal human mammary cells with a viral vector encoding a modified form of cell growth and maturation proteins, KRASG12D or myristoylated-AKT1 (myrAKT), and transplanting them into immunodeficient mice. In all these experiments, the in vivo transformation, growth, stress response, and metastasis were distinctly correlated with YB-1.
Knockout Validation. Western blot depicting YB-1 expression in lysates from HeLa parental cell line and YB-1 knockout (KO) cell line. After lysates were run, the membrane was probed for YB-1 using the primary antibody Rabbit Anti-Human YB-1 Polyclonal Antibody (Catalog # NBP1-97572) followed by secondary antibody HRP-conjugated Anti-Rabbit IgG (Catalog # HAF008). YB-1 was detected at ~49 kDa in the parental HeLa cell line but is not detectable in the KO HeLa cell line.
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KRAS-YB-1 Interaction in Breast Cancer Development
Before introducing viral vectors into cell lines and mice, the team established the association of KRAS and AKT1 with YB-1 and its target HIF1A using various breast cancer datasets. To determine the role of YB-1 in tumorigenesis and metastasis in vivo, a highly aggressive and invasive triple negative breast cancer (TNBC) cell line called MDA-MB-231, which contains KRAS-mutation and elevated YB-1 expression, was injected into immunodeficient mice. Using a short hairpin (sh)-mediated knockdown of YB-1, the researchers reduced the size and spread of tumors in vivo. Moreover, the immunohistochemical analyses of tumors from mice injected with a less aggressive breast cancer (T47D cells) transduced with KRASG12D, exhibited enhanced levels of YB-1, HIF1A and HIF1A-targets (G3BP1, CAIX). These data suggest that KRASG12D expression promotes YB-1 expression, which activates stress-related genes required for in vivo tumorigenesis.
To further elaborate the contribution of YB-1, the team introduced a doxycycline-inducible KRASG12D vector into normal human mammary cells, which were then injected into mice. Even when KRASG12D expression was stopped at two-weeks by removing doxycycline, the levels of YB-1 and its stress-related targets (HIF1A, G3BP1, CAIX) remained elevated. Injecting mice with mammary cells transduced first with shYB-1 and then with fluorescent-tagged KRASG12D produced smaller tumors with reduced levels of HIF1A, CAIX, VEGF, and a marker of blood vessels, CD34.
Taken together, these data indicate a critical role for YB-1 in KRAS-dependent breast cancer genesis. Next, the researchers investigated the relationship of YB-1 with AKT1 in tumor development.
Critical Role for YB-1 in KRASG12D-induced human mammary tumorigenesis. Representative images of HIF1α (left) and CAIX (right)-stained sections using Rabbit Anti-HIF-1 alpha Polyclonal Antibody (Catalog # NB100-134) and Goat Anti-CAIX Polyclonal Antibody (Catalog # AF2188), respectively, from basal cell- or luminal progenitor-derived tumors developing from KRASG12D+shYB-1 (YB-1 knockout)- or KRASG12D+shScr (control)-transduced cells. Results show reduced tumor size and decreased associated stress-related target expression in the tumors from YB-1 knockout transduced cells. Image adapted from Figure 6 from Lefort, S., El-Naggar, A., Tan, S., et al. De novo and cell line models of human mammary cell transformation reveal an essential role for Yb-1 in multiple stages of human breast cancer [published online ahead of print, 2021 Jul 22]. Cell Death Differ. 2021;10.1038/s41418-021-00836-6. doi:10.1038/s41418-021-00836-6, , provided by CC-BY license.
Role of YB-1 in Pre-Invasive and Metastatic Phases of Breast Cancer
To study the interaction of myrAKT with YB-1 in earlier stages of breast cancer, the team used their own KRASG12D-mediated model of invasive ductal carcinoma, which is the most common form of breast cancer. They injected immunodeficient mice with human mammary cells transduced with bioluminescently tagged myrAKT and YB-1, with or without KRASG12D vector. Although YB-1 knockdown inhibited initial tumor formation, enhanced YB-1 expression alone was not enough for tumorigenicity. Increased YB-1 expression within the myrAKT model could enhance the growth of pre-invasive ductal carcinomas in situ (DCIS) tumors.
In other words, different stages of breast cancer development depend on the levels and interactions of YB-1 with proteins related to cell growth and maturation (KRAS, AKT) and stress response (HIF1A).
Immunohistochemical analysis showing Akt [p Ser473] detected in immersion fixed paraffin-embedded sections of human breast cancer tissue by probing with Rabbit Anti- Phospho-Akt (S473) Antigen Affinity-purified Polyclonal Antibody (Catalog # AF887) (Upper Panel). Following, the tissue was stained with Anti-Rabbit HRP-DAB IHC Detection Kit (Catalog # CTS005) (brown) and counterstained with hematoxylin (blue). The Lower Panel depicts negative control tissue showing a lack of labeling when the primary antibody was omitted and only secondary antibody and detection reagents were used.
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This is the first study showing the role of YB-1 in primary and established cell lines and animal models of breast cancer. Targeted degradation of YB-1 in KRAS-mutated cancers can be a potential future cancer therapy. Additionally, the researcher’s de novo transformation model exhibiting pre-invasive DCIS phase is unique and opens the doors to mechanistic and clinical investigations into breast cancer development.
Lefort et al. used Rabbit Anti-HIF-1 alpha Polyclonal Antibody (NB100-134), Rabbit Anti-G3BP1 Polyclonal Antibody (NBP2-16563), and Goat Anti-Carbonic Anhydrase IX/CA9 Polyclonal Antibody (AF2188).
Jamshed Arslan, Pharm D, PhD
Dr Arslan is an Assistant Professor at Salim Habib University (formerly, Barrett Hodgson University), Pakistan. His interest lies in neuropharmacology and preparing future pharmacists.
Research in Focus
Lefort, S., El-Naggar, A., Tan, S., Colborne, S., Negri, G. L., Pellacani, D., Hirst, M., Gusterson, B., Morin, G. B., Sorensen, P. H., & Eaves, C. J. (2021). De novo and cell line models of human mammary cell transformation reveal an essential role for Yb-1 in multiple stages of human breast cancer. Cell death and differentiation, 10.1038/s41418-021-00836-6. Advance online publication. https://doi.org/10.1038/s41418-021-00836-6