bims-pideca 2021-04-18 papers

bims-pideca

Biomed News

on Class IA PI3K signalling in development and cancer

Issue of 2021–04–18
eleven papers selected by
Ralitsa Radostinova Madsen, University College London

mTORC2 controls the activity of PKC and Akt by phosphorylating a conserved TOR interaction motif.
CSNK1G2 differently sensitizes tamoxifen-induced decrease in PI3K/AKT/mTOR/S6K and ERK signaling according to the estrogen receptor existence in breast cancer cells.
mTOR activation initiates renal cell carcinoma development by coordinating ERK and p38MAPK.
PIK3CA Mutations as a Molecular Target for Hormone Receptor-Positive, HER2-Negative Metastatic Breast Cancer.
Selective AKT kinase inhibitor capivasertib in combination with fulvestrant in PTEN-mutant ER-positive metastatic breast cancer.
The dynamic mechanism of 4E-BP1 recognition and phosphorylation by mTORC1.
The AMBRA1 E3 ligase adaptor regulates the stability of cyclin D.
Internalized Activation of Membrane Receptors: From Phenomenon to Theory.
Chromothripsis as an on-target consequence of CRISPR-Cas9 genome editing.
Methylglyoxal induces p53 activation and inhibits mTORC1 in human umbilical vein endothelial cells.
PRDM4 inhibits cell proliferation and tumorigenesis by inactivating the PI3K/AKT signaling pathway through targeting of PTEN in cervical carcinoma.

Sci Signal. 2021 Apr 13. pii: eabe4509. [Epub ahead of print]14(678):

mTORC2 controls the activity of PKC and Akt by phosphorylating a conserved TOR interaction motif.

Timothy R Baffi, Gema Lordén, Jacob M Wozniak, Andreas Feichtner, Wayland Yeung, Alexandr P Kornev, Charles C King, Jason C Del Rio, Ameya J Limaye, Julius Bogomolovas, Christine M Gould, Ju Chen, Eileen J Kennedy, Natarajan Kannan, David J Gonzalez, Eduard Stefan, Susan S Taylor, Alexandra C Newton.

The complex mTORC2 is accepted to be the kinase that controls the phosphorylation of the hydrophobic motif, a key regulatory switch for AGC kinases, although whether mTOR directly phosphorylates this motif remains controversial. Here, we identified an mTOR-mediated phosphorylation site that we termed the TOR interaction motif (TIM; F-x3-F-pT), which controls the phosphorylation of the hydrophobic motif of PKC and Akt and the activity of these kinases. The TIM is invariant in mTORC2-dependent AGC kinases, is evolutionarily conserved, and coevolved with mTORC2 components. Mutation of this motif in Akt1 and PKCβII abolished cellular kinase activity by impairing activation loop and hydrophobic motif phosphorylation. mTORC2 directly phosphorylated the PKC TIM in vitro, and this phosphorylation event was detected in mouse brain. Overexpression of PDK1 in mTORC2-deficient cells rescued hydrophobic motif phosphorylation of PKC and Akt by a mechanism dependent on their intrinsic catalytic activity, revealing that mTORC2 facilitates the PDK1 phosphorylation step, which, in turn, enables autophosphorylation. Structural analysis revealed that PKC homodimerization is driven by a TIM-containing helix, and biophysical proximity assays showed that newly synthesized, unphosphorylated PKC dimerizes in cells. Furthermore, disruption of the dimer interface by stapled peptides promoted hydrophobic motif phosphorylation. Our data support a model in which mTORC2 relieves nascent PKC dimerization through TIM phosphorylation, recruiting PDK1 to phosphorylate the activation loop and triggering intramolecular hydrophobic motif autophosphorylation. Identification of TIM phosphorylation and its role in the regulation of PKC provides the basis for AGC kinase regulation by mTORC2.

DOI: https://doi.org/10.1126/scisignal.abe4509
PLoS One. 2021 ;16(4): e0246264

CSNK1G2 differently sensitizes tamoxifen-induced decrease in PI3K/AKT/mTOR/S6K and ERK signaling according to the estrogen receptor existence in breast cancer cells.

Anh Thu Nguyen Hoang, Kwang-Lae Hoe, Sook-Jeong Lee.

Tamoxifen (TAM) is a selective estrogen receptor modulator used for breast cancer patients. Prolonged use of tamoxifen is not recommended for some patients. In this study, we aimed to identify molecular targets sensitive to TAM using a genome-wide gene deletion library screening of fission yeast heterozygous mutants. From the screening, casein kinase 1 gamma 2 (CSNK1G2), a serine-/threonine protein kinase, was the most sensitive target to TAM with a significant cytotoxicity in estrogen receptor-positive (ER+) breast cancer cells but with only a slight toxicity in the case of ER- cells. In addition, tumor sphere formation and expression of breast stem cell marker genes such as CD44/CD2 were greatly inhibited by CSNK1G2 knockdown in ER+ breast cancer cells. Consistently, CSNK1G2 altered ERα activity via phosphorylation, specifically at serine (Ser)167, as well as the regulation of estrogen-responsive element (ERE) of estrogen-responsive genes such as CTSD and GREB1. However, ERα silencing almost completely blocked CSNK1G2-induced TAM sensitivity. In ER+ breast cancer cells, combined treatment with TAM and CSNK1G2 knockdown further enhanced the TAM-mediated decrease in phosphatidylinositol 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR)/ribosomal protein S6 kinase (S6K) signaling but not extracellular signal-regulated kinase (ERK) signaling. Inversely, in ER- cells treated with TAM, only ERK and PI3K signaling was altered by CSNK1G2 knockdown. The CK1 inhibitor, D4476, partly mimicked the CSNK1G2 knockdown effect in ER+ breast cancer cells, but with a broader repression ranging from PI3K/AKT/mTOR/S6K to ERK signaling. Collectively, these results suggest that CSNK1G2 plays a key role in sensitizing TAM toxicity in ER+ and ER- breast cancer cells via differently regulating PI3K/AKT/mTOR/S6K and ERK signaling.

DOI: https://doi.org/10.1371/journal.pone.0246264
Cancer Res. 2021 Apr 16. pii: canres.3979.2020. [Epub ahead of print]

mTOR activation initiates renal cell carcinoma development by coordinating ERK and p38MAPK.

Hongguang Wu, Dan He, Soma Biswas, Md Shafiquzzaman, Xin Zhou, Jean Charron, Yibin Wang, Bijaya K Nayak, Samy L Habib, Huijuan Liu, Baojie Li.

Renal cell carcinoma (RCC) mainly originates from renal proximal tubules. Intriguingly, disruption of genes frequently mutated in human RCC samples thus far has only generated RCC originated from other renal tubule parts in mouse models. This hampers our understanding of the pathogenesis of RCC. Here we show that mTOR signaling, often activated in RCC samples, initiates RCC development from renal proximal tubules. Ablation of Tsc1, encoding an mTOR suppressor, in proximal tubule cells led to multiple precancerous renal cysts. mTOR activation increased MEK1 expression and ERK activation, and Mek1 ablation or inhibition diminished cyst formation in Tsc1-deficient mice. mTOR activation also increased MKK6 expression and p38MAPK activation, and ablation of the p38α-encoding gene further enhanced cyst formation and led to RCC with clear cell RCC features. Mechanistically, Tsc1 deletion induced p53 and p16 expression in a p38MAPK-dependent manner, and deleting Tsc1 and Trp53 or Cdkn2a (encoding p16) enhanced renal cell carcinogenesis. Thus, mTOR activation in combination with inactivation of the p38MAPK-p53/p16 pathway drives RCC development from renal proximal tubules. Moreover, this study uncovers previously unidentified mechanisms by which mTOR controls cell proliferation and suggests the MEK-ERK axis to be a potential target for treatment of RCC.

DOI: https://doi.org/10.1158/0008-5472.CAN-20-3979
Front Oncol. 2021 ;11 644737

PIK3CA Mutations as a Molecular Target for Hormone Receptor-Positive, HER2-Negative Metastatic Breast Cancer.

Nicola Fusco, Umberto Malapelle, Matteo Fassan, Caterina Marchiò, Simonetta Buglioni, Simonetta Zupo, Carmen Criscitiello, Paolo Vigneri, Angelo Paolo Dei Tos, Eugenio Maiorano, Giuseppe Viale.

  Despite the significant achievements in the diagnosis and treatment of metastatic breast cancer (MBC), this condition remains substantially an incurable disease. In recent years, several clinical studies have aimed to identify novel molecular targets, therapeutic strategies, and predictive biomarkers to improve the outcome of women with MBC. Overall, ~40% of hormone receptor (HR)+/HER2- MBC cases harbor alterations affecting the (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathway. This pathway is a major target in oncogenesis, as it regulates growth, proliferation, cell survival, and angiogenesis. Lately, the pharmacologic targeting of PIK3CA in HR+/HER2- MBC has shown significant benefits after the occurrence of endocrine therapy resistance. The orally available α-selective PIK3CA inhibitor, alpelisib, has been approved in this setting. To perform an optimal patients' selection for this drug, it is crucial to adopt a tailored methodology. Clinically relevant PIK3CA alterations may be detected in several biospecimens (e.g. tissue samples and liquid biopsy) using different techniques (e.g. real-time PCR and next-generation sequencing). In this study, we provide an overview of the role of PIK3CA in breast cancer and of the characterization of its mutational status for appropriate clinical management.

Keywords:  HR+/HER2-; PIK3CA; RT-PCR; biomarkers; breast cancer; liquid biopsy; next-generation sequencing; targeted therapy

DOI:  https://doi.org/10.3389/fonc.2021.644737
NPJ Breast Cancer. 2021 Apr 16. 7(1): 44

Selective AKT kinase inhibitor capivasertib in combination with fulvestrant in PTEN-mutant ER-positive metastatic breast cancer.

Lillian M Smyth, Gerald Batist, Funda Meric-Bernstam, Peter Kabos, Iben Spanggaard, Ana Lluch, Komal Jhaveri, Andrea Varga, Andrea Wong, Alison M Schram, Helen Ambrose, T Hedley Carr, Elza C de Bruin, Carolina Salinas-Souza, Andrew Foxley, Joana Hauser, Justin P O Lindemann, Rhiannon Maudsley, Robert McEwen, Michele Moschetta, Myria Nikolaou, Gaia Schiavon, Pedram Razavi, Udai Banerji, José Baselga, David M Hyman, Sarat Chandarlapaty.

Five to ten percent of ER+ metastatic breast cancer (MBC) tumors harbor somatic PTEN mutations. Loss of function of this tumor-suppressor gene defines a highly aggressive, treatment-refractory disease for which new therapies are urgently needed. This Phase I multipart expansion study assessed oral capivasertib with fulvestrant in patients with PTEN-mutant ER+ MBC. Safety and tolerability were assessed by standard methods. Plasma and tumor were collected for NGS and immunohistochemistry analyses of PTEN protein expression. In 31 eligible patients (12 fulvestrant naive; 19 fulvestrant pretreated), the 24-week clinical benefit rate was 17% in fulvestrant-naive and 42% in fulvestrant-pretreated patients, with objective response rate of 8% and 21%, respectively. Non-functional PTEN was centrally confirmed in all cases by NGS or immunohistochemistry. Co-mutations occurred in PIK3CA (32%), with less ESR1 (10% vs 72%) and more TP53 (40% vs 28%) alterations in fulvestrant-naive versus fulvestrant-pretreated patients, respectively. PTEN was clonally dominant in most patients. Treatment-related grade ≥3 adverse events occurred in 32% of patients, most frequently diarrhea and maculopapular rash (both n = 2). In this clinical study, which selectively targeted the aggressive PTEN-mutant ER+ MBC, capivasertib plus fulvestrant was tolerable and clinically active. Phenotypic and genomic differences were apparent between fulvestrant-naive and -pretreated patients.Trial registration number for the study is NCT01226316.

DOI: https://doi.org/10.1038/s41523-021-00251-7
Mol Cell. 2021 Apr 05. pii: S1097-2765(21)00226-4. [Epub ahead of print]

The dynamic mechanism of 4E-BP1 recognition and phosphorylation by mTORC1.

Raphael Böhm, Stefan Imseng, Roman P Jakob, Michael N Hall, Timm Maier, Sebastian Hiller.

  The activation of cap-dependent translation in eukaryotes requires multisite, hierarchical phosphorylation of 4E-BP by the 1 MDa kinase mammalian target of rapamycin complex 1 (mTORC1). To resolve the mechanism of this hierarchical phosphorylation at the atomic level, we monitored by NMR spectroscopy the interaction of intrinsically disordered 4E binding protein isoform 1 (4E-BP1) with the mTORC1 subunit regulatory-associated protein of mTOR (Raptor). The N-terminal RAIP motif and the C-terminal TOR signaling (TOS) motif of 4E-BP1 bind separate sites in Raptor, resulting in avidity-based tethering of 4E-BP1. This tethering orients the flexible central region of 4E-BP1 toward the mTORC1 kinase site for phosphorylation. The structural constraints imposed by the two tethering interactions, combined with phosphorylation-induced conformational switching of 4E-BP1, explain the hierarchy of 4E-BP1 phosphorylation by mTORC1. Furthermore, we demonstrate that mTORC1 recognizes both free and eIF4E-bound 4E-BP1, allowing rapid phosphorylation of the entire 4E-BP1 pool and efficient activation of translation. Finally, our findings provide a mechanistic explanation for the differential rapamycin sensitivity of the 4E-BP1 phosphorylation sites.

Keywords:  NMR spectroscopy; atypical kinase; hierarchical phosphorylation; intrinsically disordered protein; kinetic modelling; mTOR signaling; multi‑site binding; protein dynamics; target of rapamycin; translational control

DOI:  https://doi.org/10.1016/j.molcel.2021.03.031
Nature. 2021 Apr 14.

The AMBRA1 E3 ligase adaptor regulates the stability of cyclin D.

Andrea C Chaikovsky, Chuan Li, Edwin E Jeng, Samuel Loebell, Myung Chang Lee, Christopher W Murray, Ran Cheng, Janos Demeter, Danielle L Swaney, Si-Han Chen, Billy W Newton, Jeffrey R Johnson, Alexandros P Drainas, Yan Ting Shue, Jose A Seoane, Preethi Srinivasan, Andy He, Akihiro Yoshida, Susan Q Hipkins, Edel McCrea, Carson D Poltorack, Nevan J Krogan, J Alan Diehl, Christina Kong, Peter K Jackson, Christina Curtis, Dmitri A Petrov, Michael C Bassik, Monte M Winslow, Julien Sage.

The initiation of cell division integrates a large number of intra- and extracellular inputs. D-type cyclins (hereafter, cyclin D) couple these inputs to the initiation of DNA replication1. Increased levels of cyclin D promote cell division by activating cyclin-dependent kinases 4 and 6 (hereafter, CDK4/6), which in turn phosphorylate and inactivate the retinoblastoma tumour suppressor. Accordingly, increased levels and activity of cyclin D-CDK4/6 complexes are strongly linked to unchecked cell proliferation and cancer2,3. However, the mechanisms that regulate levels of cyclin D are incompletely understood4,5. Here we show that autophagy and beclin 1 regulator 1 (AMBRA1) is the main regulator of the degradation of cyclin D. We identified AMBRA1 in a genome-wide screen to investigate the genetic basis of the response to CDK4/6 inhibition. Loss of AMBRA1 results in high levels of cyclin D in cells and in mice, which promotes proliferation and decreases sensitivity to CDK4/6 inhibition. Mechanistically, AMBRA1 mediates ubiquitylation and proteasomal degradation of cyclin D as a substrate receptor for the cullin 4 E3 ligase complex. Loss of AMBRA1 enhances the growth of lung adenocarcinoma in a mouse model, and low levels of AMBRA1 correlate with worse survival in patients with lung adenocarcinoma. Thus, AMBRA1 regulates cellular levels of cyclin D, and contributes to cancer development and the response of cancer cells to CDK4/6 inhibitors.

DOI: https://doi.org/10.1038/s41586-021-03474-7
Trends Cell Biol. 2021 Apr 12. pii: S0962-8924(21)00054-4. [Epub ahead of print]

Internalized Activation of Membrane Receptors: From Phenomenon to Theory.

Wenjing Wang, Jingwei Bian, Zijian Li.

  Many studies over the past decade have reported that internalized membrane receptors can trigger distinct signal activation, rather than being desensitized inside the cell. Here, we propose the concept of 'internalized activation' as a distinctive component of the receptor theory framework and exhibit its significance and role in diseases.

Keywords:  internalized activation; receptor activation; receptor internalization; signaling

DOI:  https://doi.org/10.1016/j.tcb.2021.03.008
Nat Genet. 2021 Apr 12.

Chromothripsis as an on-target consequence of CRISPR-Cas9 genome editing.

Mitchell L Leibowitz, Stamatis Papathanasiou, Phillip A Doerfler, Logan J Blaine, Lili Sun, Yu Yao, Cheng-Zhong Zhang, Mitchell J Weiss, David Pellman.

Genome editing has therapeutic potential for treating genetic diseases and cancer. However, the currently most practicable approaches rely on the generation of DNA double-strand breaks (DSBs), which can give rise to a poorly characterized spectrum of chromosome structural abnormalities. Here, using model cells and single-cell whole-genome sequencing, as well as by editing at a clinically relevant locus in clinically relevant cells, we show that CRISPR-Cas9 editing generates structural defects of the nucleus, micronuclei and chromosome bridges, which initiate a mutational process called chromothripsis. Chromothripsis is extensive chromosome rearrangement restricted to one or a few chromosomes that can cause human congenital disease and cancer. These results demonstrate that chromothripsis is a previously unappreciated on-target consequence of CRISPR-Cas9-generated DSBs. As genome editing is implemented in the clinic, the potential for extensive chromosomal rearrangements should be considered and monitored.

DOI: https://doi.org/10.1038/s41588-021-00838-7
Sci Rep. 2021 Apr 13. 11(1): 8004

Methylglyoxal induces p53 activation and inhibits mTORC1 in human umbilical vein endothelial cells.

Xinmiao Zhang, Angelica Rodriguez-Niño, Diego O Pastene, Prama Pallavi, Jacob van den Born, Stephan J L Bakker, Bernhard K Krämer, Benito A Yard.

Methylglyoxal (MGO), a precursor of advanced glycation end products (AGEs), is regarded as a pivotal mediator of vascular damage in patients with diabetes. We have previously reported that MGO induces transcriptional changes compatible with p53 activation in cultured human endothelial cells. To further substantiate this finding and to explore the underlying mechanisms and possible consequences of p53 activation, we aimed (1) to provide direct evidence for p53 activation in MGO-treated human umbilical vein endothelial cells (HUVECs), (2) to assess putative mechanisms by which this occurs, (3) to analyze down-stream effects on mTOR and autophagy pathways, and (4) to assess the potential benefit of carnosine herein. Exposure of HUVECs to 800 µM of MGO for 5 h induced p53 phosphorylation. This was paralleled by an increase in TUNEL and γ-H2AX positive cells, indicative for DNA damage. Compatible with p53 activation, MGO treatment resulted in cell cycle arrest, inhibition of mTORC1 and induction of autophagy. Carnosine co-treatment did not counteract MGO-driven effects. In conclusion, our results demonstrate that MGO elicits DNA damage and p53 activation in HUVECs, resulting in modulation of downstream pathways, e.g. mTORC1.

DOI: https://doi.org/10.1038/s41598-021-87561-9
Oncogene. 2021 Apr 12.

PRDM4 inhibits cell proliferation and tumorigenesis by inactivating the PI3K/AKT signaling pathway through targeting of PTEN in cervical carcinoma.

Wen-Ting Yang, Mei Chen, Rui Xu, Peng-Sheng Zheng.

PR domain zinc finger protein 4 (PRDM4) is a transcription factor that plays key roles in stem cell self-renewal and tumorigenesis. However, its biological role and exact mechanism in cervical cancer remain unknown. Here, both immunohistochemistry (IHC) and Western blot assays demonstrated that the expression of PRDM4 in cervical cancer tissues was much lower than that in the normal cervix. A xenograft assay showed that PRDM4 overexpression in the cervical cancer cell lines SiHa and HeLa dramatically inhibited cell proliferation and tumorigenic potential in vivo. Conversely, the silencing of PRDM4 promoted cervical cancer cell proliferation and tumorigenic potential. Mechanistically, PRDM4 induced cell cycle arrest at the transition from G0/G1 phase to S phase by upregulating p27 and p21 expression and downregulating Cyclin D1 and CDK4 expression. Furthermore, the PI3K/AKT signaling pathway was inactivated in PRDM4-overexpressing cells, which decreased the levels of p-AKT and upregulated the expression of PTEN, an inhibitor of the PI3K/AKT signaling pathway, at both the transcriptional and translational levels. Dual-luciferase reporter assays and qChIP assays confirmed that PRDM4 transactivated the expression of PTEN by binding to two specific regions in the PTEN promoter. Furthermore, PTEN silencing or a PTEN inhibitor rescued the cell defects induced by PRDM4 overexpression. Therefore, our data suggest that PRDM4 inhibits cell proliferation and tumorigenesis by downregulating the activity of the PI3K/AKT signaling pathway by directly transactivating PTEN expression in cervical cancer.

DOI: https://doi.org/10.1038/s41388-021-01765-x