Attenuation of PITPNM1 signaling cascade can inhibit breast cancer progression

Phosphatidylinositol transfer protein membrane-associated 1 (PITPNM1) contains a highly conserved phosphatidylinositol transfer domain which is involved in phosphoinositide trafficking and signaling transduction under physiological conditions. However, the functional role of PITPNM1 in cancer progression remains unknown. Here, by integrating datasets of The Cancer Genome Atlas (TCGA) and Molecular Taxonomy of Breast Cancer (METABRIC), we found that the expression of PITPNM1 is much higher in breast cancer tissues than in normal breast tissues, and a high expression of PITPNM1 predicts a poor prognosis for breast cancer patients. Through gene set variation analysis (GSEA) and gene ontology (GO) analysis, we found PITPNM1 is mainly associated with carcinogenesis and cell-to-cell signaling ontology. Silencing of PITPNM1, in vitro, significantly abrogates proliferation and colony formation of breast cancer cells. Collectively, PITPNM1 is an important prognostic indicator and a potential therapeutic target for breast cancer

Silencing CTNND1 Mediates Triple-Negative Breast Cancer Bone Metastasis via Upregulating CXCR4/CXCL12 Axis and Neutrophils Infiltration in Bone

Bone metastasis from triple-negative breast cancer (TNBC) frequently results in poorer prognosis than other types of breast cancer due to the delay in diagnosis and intervention, lack of effective treatments and more skeletal-related complications. In the present study, we identified CTNND1 as a most reduced molecule in metastatic bone lesion from TNBC by way of high throughput sequencing of TNBC samples. In vivo experiments revealed that knockdown of CTNND1 enhanced tumor cells metastasis to bones and also increased neutrophils infiltration in bones. In vitro, we demonstrated that knockdown of CTNND1 accelerated epithelial–mesenchymal transformation (EMT) of tumor cells and their recruitment to bones. The involvement by CTNND1 in EMT and bone homing was achieved by upregulating CXCR4 via activating the PI3K/AKT/HIF-1αpathway. Moreover, TNBC cells with reduced expression of CTNND1 elicited cytotoxic T-cells responses through accelerating neutrophils infiltration by secreting more GM-CSF and IL-8. Clinically, patients with triple-negative breast cancer and lower level of CTNND1 had shorter overall survival (OS) and distant metastasis-free survival (DMFS). It was concluded that downregulation of CTNND1 played a critical role in facilitating bone metastasis of TNBC and that CTNND1 might be a potential biomarker for predicting the risk of bone metastases in TNBC

Clinical assessment.

<p>Representative images of legs from group NC, HI, HD and HID on day 28 post-surgery. Necrosis was invisible in group NC (A) and group HI (the right leg, B), but it was present in group HD (the right leg, C) and group HID (the right leg, D). Necrosis was even worse in group HID than group HD. The clinical assessment was evaluated as an appearance score (E). * <i>p</i><0.05, *** <i>p</i><0.001.</p

MiR-92b-3p Inhibits Proliferation of HER2-Positive Breast Cancer Cell by Targeting circCDYL

&lt;jats:sec&gt;&lt;jats:title&gt;Objectives&lt;/jats:title&gt;&lt;jats:p&gt;Circular RNA (circRNA) is a novel class of RNA, which exhibits powerful biological function in regulating cellular fate of various tumors. Previously, we had demonstrated that over-expression of circRNA circCDYL promoted progression of HER2-negative (HER2&lt;jats:sup&gt;–&lt;/jats:sup&gt;) breast cancer &lt;jats:italic&gt;via&lt;/jats:italic&gt; miR-1275-ULK1/ATG7-autophagic axis. However, the role of circCDYL in HER2-positive (HER2&lt;jats:sup&gt;+&lt;/jats:sup&gt;) breast cancer, in particular its role in modulating cell proliferation, one of the most important characteristics of cellular fate, is unclear.&lt;/jats:p&gt;&lt;/jats:sec&gt;&lt;jats:sec&gt;&lt;jats:title&gt;Materials and methods&lt;/jats:title&gt;&lt;jats:p&gt;qRT-PCR and &lt;jats:italic&gt;in situ&lt;/jats:italic&gt; hybridization analyses were performed to examine the expression of circCDYL and miR-92b-3p in breast cancer tissues or cell lines. The biological function of circCDYL and miR-92b-3p were assessed by plate colony formation and cell viability assays and orthotopic animal models. In mechanistic study, circRNAs pull-down, RNA immunoprecipitation, dual luciferase report, western blot, immunohistochemical and immunofluorescence staining assays were performed.&lt;/jats:p&gt;&lt;/jats:sec&gt;&lt;jats:sec&gt;&lt;jats:title&gt;Results&lt;/jats:title&gt;&lt;jats:p&gt;CircCDYL was high-expressed in HER2&lt;jats:sup&gt;+&lt;/jats:sup&gt; breast cancer tissue, similar with that in HER2&lt;jats:sup&gt;–&lt;/jats:sup&gt; breast cancer tissue. Silencing HER2 gene had no effect on expression of circCDYL in HER2&lt;jats:sup&gt;+&lt;/jats:sup&gt; breast cancer cells. Over-expression of circCDYL promoted proliferation of HER2&lt;jats:sup&gt;+&lt;/jats:sup&gt; breast cancer cells but not through miR-1275-ULK1/ATG7-autophagic axis. CircRNA pull down and miRNA deep-sequencing demonstrated the binding of miR-92b-3p and circCDYL. Interestingly, circCDYL did not act as miR-92b-3p sponge, but was degraded in miR-92b-3p-dependent silencing manner. Clinically, expression of circCDYL and miR-92b-3p was associated with clinical outcome of HER2&lt;jats:sup&gt;+&lt;/jats:sup&gt; breast cancer patients.&lt;/jats:p&gt;&lt;/jats:sec&gt;&lt;jats:sec&gt;&lt;jats:title&gt;Conclusion&lt;/jats:title&gt;&lt;jats:p&gt;MiR-92b-3p-dependent cleavage of circCDYL was an essential mechanism in regulating cell proliferation of HER2&lt;jats:sup&gt;+&lt;/jats:sup&gt; breast cancer cells. CircCDYL was proved to be a potential therapeutic target for HER2&lt;jats:sup&gt;+&lt;/jats:sup&gt; breast cancer, and both circCDYL and miR-92b-3p might be potential biomarkers in predicting clinical outcome of HER2&lt;jats:sup&gt;+&lt;/jats:sup&gt; breast cancer patients.&lt;/jats:p&gt;&lt;/jats:sec&gt

Cross-sections of gracilis anterior muscle were stained with H&E.

<p>Representative photographs are shown (A and B). The collaterals were quantified by vessel area (C), lumen area (D), and vessel area/lumen area (E). There were no differences in vessel area, lumen area or vessel area/lumen area of group HD compared with group NC. In group HI and HID, the vessel area increased 7 days post-surgery and further increased 28 days post-surgery. However, in comparison with group NC, the lumen area increased significantly and vessel area/lumen area increased modestly in group HI, but the lumen area decreased and vessel area/lumen area increased dramatically in group HID. * <i>p</i>< 0.05.</p

CD31 positive capillary density.

<p>Capillary density was evaluated using immunostaining for CD31 of tibialis anterior muscle sections in group NC (A), HI (B), HD (C) and HID (D) on day 28 post-surgery and quantified by counting CD31 positive capillaries per square millimeter. The bar graph shows the capillary density (E). Red indicates CD31, and blue indicates nuclei. ** <i>p</i><0.01, *** <i>p</i>< 0.001.</p

Perfusion recovery.

<p>Perfusion recovery is shown by laser Doppler perfusion images (A and B). Perfusion was evaluated in the plantar feet in the same anatomically defined region (shown as A) in all animals (n = 5 per group). The right leg/left leg perfusion ratio of group HI (C), group HD (D) and group HID (E) are shown. In group HI, perfusion ratio fell from 1.02 on baseline to 0.48 immediately post-surgery, but the ratio gradually increased within 28 days and the ratio returned to 1.03 on day 28 post-surgery. In group HD, the perfusion ratio was significantly elevated to 1.47–1.53 immediately post-surgery and each time point within 28 days. In group HID, the perfusion ratio slightly elevated immediately post-surgery and peaked to 1.69 on day 3 but decreased gradually after that and the ratio dropped to 1.25 on day 28 post-surgery.</p

Immunofluorescence staining of the peripheral nerves.

<p>The peripheral nerve was identified using S100 and α-SM-actin immunofluorescence dual staining of gracilis muscle cross-sections from group hindlimb ischemia (HI), group simple denervation (HD) and group ischemia with denervation (HID) on day 7 post-surgery. Green indicates S100, red indicates α-SM-actin, and blue indicates nuclei. S100-positive staining which is indicated with white arrows in group HI (A) was significantly higher than both group HD (B) and group HID (C). The S100-positive staining was evaluated as integrity of density (IOD) value (D). ** <i>p</i><0.01.</p

Collateral number shown by X-ray angiograms.

<p>The collateral arteries in group NC (A) and HI (right leg), HD (right leg), and HID (right leg: ischemic and denervated, left leg: ischemic) 7 and 28 days post-surgery (B) are shown. The number of collateral arteries was obtained by counting the arteries that intersected a line that was perpendicular to the midpoint of the femur (shown as a straight line in A). The remodeling collateral arteries are indicated with black arrows. Few pre-existing collaterals was seen in group NC (A). The number of collaterals in group HI significantly increased compared with group NC on day 7 and further increased on day 28 post-surgery (C). The number of collaterals did not change 7 or 28 days post-surgery in group HD (D) compared with group NC. In group HID, the number of collaterals (the right leg) also increased compared with group NC, but the number was lower than group HI on day 7 and 28 post-surgery (E). n = 4 per group * <i>p</i><0.05, *** <i>p</i><0.001.</p

SMCs and ECs staining of collaterals in cross-sections of gracilis muscle.

<p>Dual immunostaining of ECs with CD31 and SMCs with α-SM-actin in cross-sections of gracilis muscle in group NC, HI, HID. A thin layer of CD31 staining was observed in the collaterals of group NC (A) and HI on day 7 and 28 post-surgery, but the CD31 layer was much thicker on day 7 post-surgery and further thickened dramatically on day 28 post-surgery in group HID compared with group NC and HI (B). Similarly, the layer of α-SM-actin staining was thin in the collaterals of group NC (A). In group HI, the layer of α-SM-actin staining increased compared with group NC on day 7 and further increased slightly on day 28 post-surgery. In group HID, the staining layer increased significantly on day 7 and much further increased on day 28 post-surgery (B). CD31 indicates ECs (green), α-SM-actin indicates SMCs (red), and DAPI indicates nuclei (blue).</p