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Cholesterol is a risk factor for breast cancer. However, it is still unclear whether the cholesterol biosynthesis pathway plays any significant role in breast carcinogenesis. 24-Dehydrocholesterol reductase (DHCR24) is a key enzyme in the cholesterol synthesis pathway. Although DHCR24 is reported to have different functions in different cancers, it is not clear whether DHCR24 is involved in breast cancer. In this study, we found that DHCR24 expression was higher in breast cancer especially in luminal and HER2 positive breast cancer tissues compared with normal breast. Changes in DHCR24 expression altered cellular cholesterol content without affecting the adherent growth of breast cancer cells. However, DHCR24 knockdown reduced whereas DHCR24 overexpression enhanced breast cancer stem-like cell populations such as mammosphere and aldehyde dehydrogenase positive cell numbers. In addition, DHCR24 overexpression increased the expression of the Hedgehog pathway-regulated genes. Treating DHCR24 overexpressing breast cancer cell lines with the Hedgehog pathway inhibitor GANT61 blocked DHCR24-induced mammosphere growth and increased mRNA levels of the Hedgehog regulated genes. Furthermore, expression of a constitutively activated mutant of Smoothened, a key hedgehog signal transducer, rescued the decreases in mammosphere growth and Hedgehog regulated gene expression induced by knockdown of DHCR24. These results indicate that DHCR24 promotes the growth of breast cancer stem-like cells in part through enhancing the Hedgehog signaling pathway. Our data suggest that cholesterol contribute to breast carcinogenesis by enhancing Hedgehog signaling and cancer stem-like cell populations. Enzymes including DHCR24 involved in cholesterol biosynthesis should be considered as potential treatment targets for breast cancer., (© 2020 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

As a subtype of acute myeloid leukemia (AML), acute promyelocytic leukemia (APL) is characterized by a chromosomal translocation, most of which result in the production of a PML-RAR alpha fusion protein. Although the overall survival rate of APL patients has improved dramatically due to all-trans retinoic acid (ATRA) treatment, ATRA-resistance remains a clinical challenge in the management of APL. Therefore, alternative agents should be considered for ATRA-resistant APL patients. Here, we report that antimalaria drug primaquine phosphate (PRQ) exhibits an anti-leukemia effect on both ATRA-sensitive cell line NB4 and ATRA-resistant APL cell lines, NB4-LR2, NB4-LR1, and NB4-MR2. Moreover, PRQ significantly inhibited primary colony formation of untreated or relapsed APL patients. Further study showed that PRQ could induce the apoptosis of APL cells by inhibiting NF-κB signaling pathway. The in vivo study showed that PRQ significantly inhibited NB4-LR2 xenograft tumors growth. These results suggest that PRQ is a potential therapeutic agent for ATRA-resistant APL patients., (©2020 Society for Leukocyte Biology.)

SH2-domain containing protein tyrosine phosphatase 1 (Shp1/PTPN6) is mainly expressed in hematopoietic cells and acts a negative signaling regulator. Although Shp1 is also expressed in epithelial cells, the function of shp1 in normal epithelial is still less well understood, especially in regulating the growth of epithelial cells. In this study, different shRNAs and siRNAs against Shp1 were used to knockdown Shp1 expression in MCF10A, an immortalized mammary epithelial cell line. Shp1 knockdown resulted in inhibited cell growth in part due to lower percentage of MCF10A cells entering into S phase and reduced cyclin D1 expression. Accordingly, EGF-induced tyrosyl phosphorylation of EGFR and Stat5 was significantly inhibited in cells with Shp1 knockdown compared with control whereas EGF-induced Akt and Erk phosphorylation was not affected by Shp1 knockdown. Further analysis revealed that Shp1 knockdown lead to decreased EGFR protein expression without affecting EGFR mRNA expression or increasing EGFR protein degradation. Our data indicate that Shp1 functions as a positive regulator and acts in a novel mechanism through promoting EGFR protein expression in mammary epithelial cells., (Copyright © 2017. Published by Elsevier Inc.)

ErbB family of the receptor protein-tyrosine kinase plays an important role in the progression of human cancers including breast cancer. Finding protein-tyrosine phosphatase (PTPs) that can specifically regulate the function of ErbB should help design novel therapies for treatment. By performing a small interfering RNA screen against 43 human PTPs, we find that knockdown of protein-tyrosine phosphatase PTPN9 significantly increases ErbB2 tyrosyl phosphorylation in the SKBR3 breast cancer cell line. In addition, knockdown of PTPN9 expression also enhances tyrosyl phosphorylation of the ErbB1/epidermal growth factor receptor (EGFR) in the MDA-MB-231 breast cancer cell line. Conversely, increasing expression of PTPN9 wild type (WT) inhibits tyrosyl phosphorylation of ErbB2 and EGFR. To test whether ErbB2 and EGFR are substrates of PTPN9, PTPN9 WT, and a substrate trapping mutant (PTPN9 DA) are overexpressed in SKBR3 and MDA-MB-231 cells. Compared with vector control, expression of PTPN9 WT significantly inhibits whereas expression of PTPN9 DA dramatically enhances tyrosyl phosphorylation of ErbB2 and EGFR, respectively. In contrast, expression of PTPN9 WT or DA mutant does not affect tyrosyl phosphorylation of ErbB3 and Shc. Importantly, coimmunoprecipitation and glutathione S-transferase fusion protein pulldown experiments show that tyrosol-phosphorylated ErbB2 or EGFR is preferentially associated with PTPN9 DA compared with PTPN9 WT, indicating that ErbB2 and EGFR are substrates of PTPN9. Furthermore, PTPN9 WT expression specifically impairs EGF-induced STAT3 and STAT5 activation, and inhibits the cell growth in soft agar. Last, PTPN9 WT expression also reduces invasion and MMP2 expression of MDA-MB-231 cells. Our data suggest PTPN9 as a negative regulator of breast cancer cells by targeting ErbB2 and EGFR and inhibiting STAT activation.

The scaffolding adapter Gab2 mediates cell signaling and responses evoked by various extracellular stimuli including several growth factors. Kit, the receptor for stem cell factor (SCF), plays a critical role in the proliferation and differentiation of a variety of cell types, including mast cells. Kit, via Tyr(567) and Tyr(719), activates Src family kinases (SFK) and PI3K respectively, which converge on the activation of a Rac/JNK pathway required for mast cell proliferation. However, how Kit Tyr(567) signals to Rac/JNK is not well understood. By analyzing Gab2(-/-) mast cells, we find that Gab2 is required for SCF-evoked proliferation, activation of Rac/JNK, and Ras. Upon Kit activation in wild-type mast cells, Gab2 becomes tyrosyl-phosphorylated and associates with Kit and Shp-2. Tyr(567), an SFK binding site in Kit, and SFK activity were required for Gab2 tyrosyl phosphorylation and association with Shp-2. By re-expressing Gab2 or a Gab2 mutant that cannot bind Shp-2 in Gab2(-/-) mast cells or acutely by deleting Shp-2 in mast cells, we found that Gab2 requires Shp-2 for SCF-evoked Rac/JNK, Ras activation, and mast cell proliferation. Lastly, by analyzing mast cells from mice with compound Gab2 and Kit Y719F mutations (i.e., Gab2(-/-): KitY719F/Y719F mice), we find that Gab2, acting in a parallel pathway to PI3K from Kit Tyr(719), regulates mast cell proliferation and development in specific tissues. Our data show that Gab2 via Shp-2 is critical for transmitting signals from Kit Tyr(567) to activate the Rac/JNK pathway controlling mast cell proliferation, which likely contributes to mast cell development in specific tissues.

Scaffolding adapter Grb2-associated binder 2 (Gab2) is a key component of FcepsilonRI signaling in mast cells, required for the activation of PI3K. To understand how Gab2 is activated in FcepsilonRI signaling, we asked which protein tyrosine kinase is required for Gab2 phosphorylation. We found that Gab2 tyrosyl phosphorylation requires Lyn and Syk. In agreement with published results, we found that Fyn also contributes to Gab2 tyrosyl phosphorylation. However, Syk activation is defective in Fyn(-/-) mast cells, suggesting that Syk is the proximal kinase responsible for Gab2 tyrosyl phosphorylation. Then, we asked which domains in Gab2 are required for Gab2 tyrosyl phosphorylation. We found that the Grb2-Src homology 3 (SH3) binding sites are required for, whereas the pleckstrin homology (PH) domain contributes to, Gab2 tyrosyl phosphorylation. Using a protein/lipid overlay assay, we determined that the Gab2 PH domain preferentially binds the PI3K lipid products, PI3, 4,5P3 and PI3, 4P2. Furthermore, the Grb2-SH3 binding sites and PH domain binding to PI3K lipid products are required for Gab2 function in FcepsilonRI-evoked degranulation and Akt activation. Our data strongly suggest a model for Gab2 action in FcepsilonRI signaling. The Grb2 SH3 binding sites play a critical role in bringing Gab2 to FcepsilonRI, whereupon Gab2 becomes tyrosyl-phosphorylated in a Syk-dependent fashion. Phosphorylated Gab2 results in recruitment and activation of PI3K, whose lipid products bind the PH domain of Gab2 and acts in positive feedback loop for sustained PI3K recruitment and phosphatidylinositol-3,4,5-trisphosphate production, required for FcepsilonRI-evoked degranulation of mast cells.

Src homology-2 (SH2) domain-containing phosphatases (Shps) are a small, highly conserved subfamily of protein-tyrosine phosphatases, members of which are present in both vertebrates and invertebrates. The mechanism of regulation of Shps by ligand binding is now well understood. Much is also known about the normal signaling pathways regulated by each Shp and the consequences of Shp deficiency. Recent studies have identified mutations in human Shp2 as the cause of the inherited disorder Noonan syndrome. Shp2 mutations might also contribute to the pathogenesis of some leukemias. In addition, Shp2 might be a key virulence determinant for the important human pathogen Helicobacter pylori. Despite these efforts, however, the key targets of each Shp have remained elusive. Identifying these substrates remains a major challenge for future research.

Tyrosine phosphorylation plays an important role in controlling cellular growth, differentiation and function. Abnormal regulation of tyrosine phosphorylation can result in human diseases such as cancer. A major challenge of signal transduction research is to determine how the initial activation of protein-tyrosine kinases (PTKs) by extracellular stimuli triggers multiple downstream signaling cascades, which ultimately elicit diverse cellular responses. Recent studies reveal that members of the Gab/Dos subfamily of scaffolding adaptor proteins (hereafter, "Gab proteins") play a crucial role in transmitting key signals that control cell growth, differentiation and function from multiple receptors. Here, we review the structure, mechanism of action and function of these interesting molecules in normal biology and disease.

The BCR/ABL oncogene causes chronic myelogenous leukemia (CML) in humans and a CML-like disease, as well as lymphoid leukemia, in mice. p210 BCR/ABL is an activated tyrosine kinase that phosphorylates itself and several cellular signaling proteins. The autophosphorylation site tyrosine 177 binds the adaptor Grb2 and helps determine the lineage and severity of BCR/ABL disease: Tyr177 mutation (BCR/ABL-Y177F) dramatically impairs myeloid leukemogenesis, while diminishing lymphoid leukemogenesis. The critical signal(s) from Tyr177 has remained unclear. We report that Tyr177 recruits the scaffolding adaptor Gab2 via a Grb2/Gab2 complex. Compared to BCR/ABL-expressing Ba/F3 cells, BCR/ABL-Y177F cells exhibit markedly reduced Gab2 tyrosine phosphorylation and association of phosphatidylinositol-3 kinase (PI3K) and Shp2 with Gab2 and BCR/ABL, and decreased PI3K/Akt and Ras/Erk activation, cell proliferation, and spontaneous migration. Remarkably, bone marrow myeloid progenitors from Gab2 (-/-) mice are resistant to transformation by BCR/ABL, whereas lymphoid transformation is diminished as a consequence of markedly increased apoptosis. BCR/ABL-evoked PI3K/Akt and Ras/Erk activation also are impaired in Gab2 (-/-) primary myeloid and lymphoid cells. Our results identify Gab2 and its associated proteins as key determinants of the lineage and severity of BCR/ABL transformation.