The adipocyte-rich microenvironment forms a niche for ovarian cancer metastasis, but the mechanisms driving this process are incompletely understood. Here we show that salt-inducible kinase 2 (SIK2) is overexpressed in adipocyte-rich metastatic deposits compared with ovarian primary lesions. Overexpression of SIK2 in ovarian cancer cells promotes abdominal metastasis while SIK2 depletion prevents metastasis in vivo. Importantly, adipocytes induce calcium-dependent activation and autophosphorylation of SIK2. Activated SIK2 plays a dual role in augmenting AMPK-induced phosphorylation of acetyl-CoA carboxylase and in activating the PI3K/AKT pathway through p85α-S154 phosphorylation. These findings identify SIK2 at the apex of the adipocyte-induced signaling cascades in cancer cells and make a compelling case for targeting SIK2 for therapy in ovarian cancer.
Connexin43 (Cx43), the major protein forming gap junctions in astrocytes, is reduced in high-grade gliomas, where its ectopic expression exerts important effects, including the inhibition of the proto-oncogene tyrosine-protein kinase Src (c-Src). In this work we aimed to investigate the mechanism responsible for this effect. The inhibition of c-Src requires phosphorylation at tyrosine 527 mediated by C-terminal Src kinase (Csk) and dephosphorylation at tyrosine 416 mediated by phosphatases, such as phosphatase and tensin homolog (PTEN). Our results showed that the antiproliferative effect of Cx43 is reduced when Csk and PTEN are silenced in glioma cells, suggesting the involvement of both enzymes. Confocal microscopy and immunoprecipitation assays confirmed that Cx43, in addition to c-Src, binds to PTEN and Csk in glioma cells transfected with Cx43 and in astrocytes. Pull-down assays showed that region 266–283 in Cx43 is sufficient to recruit c-Src, PTEN and Csk and to inhibit the oncogenic activity of c-Src. As a result of c-Src inhibition, PTEN was increased with subsequent inactivation of Akt and reduction of proliferation of human glioblastoma stem cells. We conclude that the recruitment of Csk and PTEN to the region between residues 266 and 283 within the C-terminus of Cx43 leads to c-Src inhibition.
One of the characteristics of gliomas is a decrease in the expression of connexin43, a protein that forms gap junctions. Restoring connexin43 expression in glioma cells reduces their exacerbated rate of cell growth, although it is not yet known how connexin43 modifies the expression of genes involved in cell proliferation. Here, we show that restoring connexin43 to C6 glioma cells impedes their progression from G0/G1 to the S phase of the cell cycle by reducing retinoblastoma phosphorylation and cyclin E expression through the upregulation of p21 and p27. Interestingly, connexin43 diminishes the oncogenic activity of c-Src exhibited by glioma cells. By studying a Tyr247 and Tyr265 mutant connexin43, we show that these residues are required for connexin43 to inhibit c-Src activity and cell proliferation. In conclusion, by acting as a substrate of c-Src, connexin43 reduces its oncogenic activity and decreases the rate of glioma cell proliferation, potentially an early step in the antiproliferative effects of connexin43. Although c-Src is known to phosphorylate connexin43, this study provides the first evidence that connexin43 can also inhibit c-Src activity.
The 40S ribosomal S6 kinase 1 (S6K1) is an important regulator of cell growth. Expression of S6K1 is often elevated in breast cancer cells. However, the transcriptional mechanism of S6K1 overexpression is not understood. In this report, we demonstrate that estrogen activates expression of S6K1 via Estrogen Receptor (ER) α in ER-positive breast cancer cells. We also show that estrogen acts on the proximal promoter of the S6K1 gene in a mechanism involving the transcriptional factor GATA-3. Finally, we provide data that support the importance of estrogenic regulation of S6K1 expression in breast cancer cell proliferation. S6K1 directly phosphorylates and regulates ligand-independent activity of ERα, while ERα upregulates S6K1 expression. This S6K1-ERα relationship creates a positive feed-forward loop in control of breast cancer cell proliferation. Furthermore, the co-dependent association between S6K1 and ERα may be exploited in the development of targeted breast cancer therapies.
Though used widely in cancer therapy, paclitaxel only elicits a response in a fraction of patients. A strong determinant of paclitaxel tumor response is the state of microtubule dynamic instability. However, whether the manipulation of this physiological process can be controlled to enhance paclitaxel response has not been tested. Here, we show a previously unrecognized role of the microtubule-associated protein CRMP2 in inducing microtubule bundling through its carboxy terminus. This activity is significantly decreased when the FER tyrosine kinase phosphorylates CRMP2 at Y479 and Y499. The crystal structures of wild-type CRMP2 and CRMP2-Y479E reveal how mimicking phosphorylation prevents tetramerization of CRMP2. Depletion of FER or reducing its catalytic activity using sub-therapeutic doses of inhibitors increases paclitaxel-induced microtubule stability and cytotoxicity in ovarian cancer cells and in vivo. This work provides a rationale for inhibiting FER-mediated CRMP2 phosphorylation to enhance paclitaxel on-target activity for cancer therapy.
In previous studies, we showed that endothelin-1 increased astrocyte proliferation and glucose uptake. These effects were similar to those observed with other gap junction inhibitors, such as carbenoxolone (CBX). Because 24-h treatment with endothelin-1 or CBX downregulates the expression of connexin43, the main protein forming astrocytic gap junctions, which can also be involved in proliferation, in this study, we addressed the possible role of connexin43 in the effects of endothelin-1. To do so, connexin43 was silenced in astrocytes by siRNA. The knock down of connexin43 increased the rate of glucose uptake, characterized by the upregulation of GLUT-1 and type I hexokinase. Neither endothelin-1 nor CBX were able to further increase the rate of glucose uptake in connexin43-silenced astrocytes. In agreement, no effects of endothelin-1 and CBX on GLUT-1 and type I hexokinase were observed in connexin-43 silenced astrocytes or in astrocytes from connexin43 knock-out (KO) mice. Our previous studies suggested a close relationship between glucose uptake and astrocyte proliferation. Consistent with this, connexin43-silenced astrocytes exhibited an increase in Ki-67, a marker of proliferation. The effects of ET-1 on retinoblastoma phosphorylation on Ser780 and on the upregulation of cyclins D1 and D3 were affected by the levels of connexin43. In conclusion, our results indicate that connexin43 participates in the effects of endothelin-1 on glucose uptake and proliferation in astrocytes. Interestingly, although the rate of growth in connexin43 KO astrocytes has been reported to be reduced, we observed that an acute reduction in connexin43 by siRNA increased proliferation and glucose uptake. V V C
Current screening methods for ovarian cancer can only detect advanced disease. Earlier detection has proved difficult because the molecular precursors involved in the natural history of the disease are unknown. To identify early driver mutations in ovarian cancer cells, we used dense whole genome sequencing of micrometastases and microscopic residual disease collected at three time points over three years from a single patient during treatment for high-grade serous ovarian cancer (HGSOC). The functional and clinical significance of the identified mutations was examined using a combination of population-based whole genome sequencing, targeted deep sequencing, multi-center analysis of protein expression, loss of function experiments in an in-vivo reporter assay and mammalian models, and gain of function experiments in primary cultured fallopian tube epithelial (FTE) cells. We identified frequent mutations involving a 40 kb distal repressor region for the key stem cell differentiation gene SOX2. In the apparently normal FTE, the region was also mutated. This was associated with a profound increase in SOX2 expression (p < 2−16), which was not found in patients without cancer (n = 108). Importantly, we show that SOX2 overexpression in FTE is nearly ubiquitous in patients with HGSOCs (n = 100), and common in BRCA1-BRCA2 mutation carriers (n = 71) who underwent prophylactic salpingo-oophorectomy. We propose that the finding of SOX2 overexpression in FTE could be exploited to develop biomarkers for detecting disease at a premalignant stage, which would reduce mortality from this devastating disease.
Our previous work has shown that tolbutamide increases gap junctional permeability in poorly coupled C6 glioma cells and that this effect is similar and additive to that found with dbcAMP, a well-known activator of gap junctional communication. Furthermore, the increase in gap junctional communication promoted by tolbutamide or dbcAMP is concurrent with the inhibition of proliferation of C6 glioma cells. In the present work, we show that tolbutamide and dbcAMP increase the synthesis of the tumor suppressor protein Cx43 and that they decrease the level of Ki-67, a protein expressed when cells are proliferating. These effects were accompanied by a reduction in the phosphorylation of pRb, mainly on Ser-795, a residue critical for the control of cell proliferation. The decrease in the phosphorylation of pRb is not likely to be mediated by a reduction in the levels of D-type cyclins, since instead of decreasing the expression of cyclins, D1 and D3 increased slightly after treatment with tolbutamide or dbcAMP. However, the Cdk inhibitors p21 and p27 were up-regulated after treatment with tolbutamide and dbcAMP, suggesting that they would be involved in the decrease in pRb phosphorylation. When Cx43 was silenced by siRNA, neither tolbutamide nor dbcAMP were able to up-regulate p21 and consequently to reduce glioma cell proliferation, as judged by Ki-67 expression. In conclusion, tolbutamide and dbcAMP inhibit C6-glioma cell proliferation by increasing Cx43, which correlates with a reduction in pRb phosphorylation due to the up-regulation of the Cdk inhibitors p21 and p27.
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