Immune cells lacking Y chromosome show dysregulation of autosomal gene expression

Funder: Kjell och Märta Beijers Stiftelse (SE)Funder: Hjärnfonden; doi: http://dx.doi.org/10.13039/501100003792Funder: Cancerfonden; doi: http://dx.doi.org/10.13039/501100002794Funder: Vetenskapsrådet; doi: http://dx.doi.org/10.13039/501100004359Funder: Alzheimerfonden; doi: http://dx.doi.org/10.13039/501100008599Funder: Konung Gustaf V:s och Drottning Victorias Frimurarestiftelse (SE)Funder: Science for Life Laboratory (SE)Funder: Fundacja na rzecz Nauki Polskiej (PL)Funder: Uppsala UniversityAbstract: Epidemiological investigations show that mosaic loss of chromosome Y (LOY) in leukocytes is associated with earlier mortality and morbidity from many diseases in men. LOY is the most common acquired mutation and is associated with aberrant clonal expansion of cells, yet it remains unclear whether this mosaicism exerts a direct physiological effect. We studied DNA and RNA from leukocytes in sorted- and single-cells in vivo and in vitro. DNA analyses of sorted cells showed that men diagnosed with Alzheimer’s disease was primarily affected with LOY in NK cells whereas prostate cancer patients more frequently displayed LOY in CD4 + T cells and granulocytes. Moreover, bulk and single-cell RNA sequencing in leukocytes allowed scoring of LOY from mRNA data and confirmed considerable variation in the rate of LOY across individuals and cell types. LOY-associated transcriptional effect (LATE) was observed in ~ 500 autosomal genes showing dysregulation in leukocytes with LOY. The fraction of LATE genes within specific cell types was substantially larger than the fraction of LATE genes shared between different subsets of leukocytes, suggesting that LOY might have pleiotropic effects. LATE genes are involved in immune functions but also encode proteins with roles in other diverse biological processes. Our findings highlight a surprisingly broad role for chromosome Y, challenging the view of it as a “genetic wasteland”, and support the hypothesis that altered immune function in leukocytes could be a mechanism linking LOY to increased risk for disease

The tumour border on contrast-enhanced spectral mammography and its relation to histological characteristics of invasive breast cancer

Contrast-enhanced spectral mammography (CESM) is one of the new diagnostic modalities implemented in clinical practice. In the case of these techniques, there are two major issues to be addressed: (1) their diagnostic usefulness, and (2) the relation between parameters assessed using these techniques and well-known diagnostic/prognostic/predictive markers (histological, clinical, and molecular). Therefore, we studied the relationship between the tumour margin assessed on CESM and (1) tumour borders defined on the basis of macroscopic and microscopic examination, (2) pT, (3) pN, and (4) tumour grade in a group of 82 breast cancer patients. Based on CESM, the tumour border was defined as sharp, indistinct or spiculated, whereas in the case of lesions showing weak or medium enhancement on CESM the borders were classified as unspecified. We found a statistically significant relationship between tumour margin on CESM and (1) macroscopic border (a spiculated margin on CESM was found only in carcinomas with an invasive border on histological examination; p = 0.004), (2) pT (p = 0.016), and (3) pN (nodal involvement was observed most frequently in carcinomas with a spiculated or indistinct margin on CESM; p = 0.045). Moreover, in cases with an undefined margin on CESM (cases showing weak or medium enhancement on CESM), both invasive and pushing borders were found on histological examination. The results of our preliminary study suggest that it is possible to assess macroscopic borders of examined lesions on the basis of CESM imaging. This might be useful in planning the extent of surgical excision. On the other hand, the assessment of the tumour margin on CESM might not be precise in cases showing weak enhancement

Metallothionein-3 Increases Triple-Negative Breast Cancer Cell Invasiveness via Induction of Metalloproteinase Expression.

It has been recently found that metallothionein-3 (MT3) enhances the invasiveness and tumorigenesis of prostate cancer cells. This finding is in contrast to those of earlier studies, which indicated that overexpression of MT3 in breast cancer and prostate cancer cell lines inhibits their growth in vitro. Therefore, to clarify the role of MT3 in breast cancer progression, we analyzed the effect of MT3-overexpression on proliferation, invasiveness, migration, and tumorigenesis of breast cancer MDA-MB-231/BO2 cells. It was found that MDA-MB-231/BO2 cells overexpressing MT3 were characterized by increased invasiveness in vitro, compared to the control cells. Interestingly, this increased invasiveness correlated with a highly increased concentration of MMP3 in the culture supernatants (p<0.0001). Our data suggest that MT3 may regulate breast cancer cell invasiveness by modulating the expression of MMP3. These experimental results, obtained using triple-negative MDA-MB-231/BO2 cells, were further supported by clinical data. It was found that, in triple-negative breast cancer (TNBC), nuclear MT3 immunoreactivity in cancer cells tended to be associated with patients' shorter disease-specific survival, suggesting that nuclear MT3 expression may be a potential marker of poor prognosis of triple-negative TNBC cases

Immune cells lacking Y chromosome show dysregulation of autosomal gene expression.

Epidemiological investigations show that mosaic loss of chromosome Y (LOY) in leukocytes is associated with earlier mortality and morbidity from many diseases in men. LOY is the most common acquired mutation and is associated with aberrant clonal expansion of cells, yet it remains unclear whether this mosaicism exerts a direct physiological effect. We studied DNA and RNA from leukocytes in sorted- and single-cells in vivo and in vitro. DNA analyses of sorted cells showed that men diagnosed with Alzheimer's disease was primarily affected with LOY in NK cells whereas prostate cancer patients more frequently displayed LOY in CD4 + T cells and granulocytes. Moreover, bulk and single-cell RNA sequencing in leukocytes allowed scoring of LOY from mRNA data and confirmed considerable variation in the rate of LOY across individuals and cell types. LOY-associated transcriptional effect (LATE) was observed in ~ 500 autosomal genes showing dysregulation in leukocytes with LOY. The fraction of LATE genes within specific cell types was substantially larger than the fraction of LATE genes shared between different subsets of leukocytes, suggesting that LOY might have pleiotropic effects. LATE genes are involved in immune functions but also encode proteins with roles in other diverse biological processes. Our findings highlight a surprisingly broad role for chromosome Y, challenging the view of it as a "genetic wasteland", and support the hypothesis that altered immune function in leukocytes could be a mechanism linking LOY to increased risk for disease

Univariate analysis of triple-negative breast cancer (TNBC) patients.

<p>Kaplan-Meier disease specific survival curves in regard to MT3 nuclear expression, primary tumor size (<b>B</b>), lymph node status (<b>C</b>), advancement stage (<b>D</b>), malignancy grade (<b>E</b>), patients age (<b>F</b>), menopausal status (<b>G</b>), extent of necrosis (<b>H</b>), fibrosis (<b>I</b>) and lymphocytic infiltration (<b>J</b>). Mantel-Cox test, *p<0.05, **p<0.01.</p

MMP3 is responsible for the increased invasiveness of MT3-overexpressing breast cancer cells.

<p><b>(A)</b> Expression of MMP3 mRNA in control MDA-MB-231/BO2 cells transfected with scrambled siRNA (BO2.MT3.CTRL) and MDA-MB-231/BO2 cells treated with siRNA directed against MMP3 mRNA (BO2.MT3.siRNA8). Real-time PCR was used to analyze MT3 mRNA. Relative expression (RQ) of <i>MMP3</i> gene was normalized against expression of <i>ACTB</i> gene and BO2.MT3.CTRL cells was assigned as a calibrator sample. Results are expressed as mean ± SD. <b>(B)</b> Western blot analysis using anti-MMP3 monoclonal murine antibody on whole cell lysates of control MDA-MB-231/BO2 cells transfected with scrambled siRNA (BO2.MT3.CTRL) and MDA-MB-231 cells treated with siRNA directed against MMP3 mRNA (BO2.MT3.siRNA8). Cell lysates equivalent to 30 μg of protein were separated by SDS-PAGE under reducing conditions on a 12% gel and electrophoretically transferred onto a nitrocellulose membrane. β-Actin served as an internal control<b>. (C)</b> Invasiveness of control MDA-MB-231/BO2 cells transfected with scrambled siRNA (BO2.MT3.CTRL) and MDA-MB-231 cells treated with siRNA directed against MMP3 mRNA (BO2.MT3.siRNA8). Data are presented as mean ± SD. ****p<0.0001, Bonferroni multiple comparison test.</p

MT3 expression affects invasiveness of breast cancer cells <i>in vitro</i>.

<p>Invasiveness <b>(A)</b> and migratory capability <b>(B)</b> of BO2/MT3/LUC/PURO and BO2/LUC/PURO cells. Data are presented as mean ± SD. Bonferroni multiple comparison test, **p<0.01, ****p<0.0001.</p

Effect of MT3 expression on breast cancer cells growth <i>in vitro</i> and tumorigenesis.

<p><b>(A)</b> Proliferation of control BO2/LUC/PURO cells and BO2/MT3/LUC/PURO cells overexpressing MT3. Cell proliferation was determined <i>in vitro</i> using MTT assay, as described in the “Materials and Methods”. The values are shown as the mean ± SD of three independent experiments. <b>(B)</b> Xenograft tumor growth of control BO2/LUC/PURO cells and BO2/MT3/LUC/PURO cells overexpressing MT3. Tumor growth was recorded on a weekly basis using metric calipers. Data are shown as the mean tumor volume for each group of mice (n = 5) at each indicated time point. <b>(C)</b> Haematoxylin and eosin stained tumor sections presenting spindle-like cancer cells with visible mitotic figures and <b>(D)</b> fibrous capsule surrounding the tumor mass. Arrows indicate mitotic figures (<b>C</b> and <b>D</b>); arrowheads, fibrous capsule (<b>D</b>). Magnification ×200.</p

MT3 affects the expression of MMP3 in breast cancer MDA-MB-231 cells.

<p>Expression of MT3 mRNA <b>(A)</b> and MMP3 mRNA <b>(B)</b> in MDA-MB-231 cells treated with scrambled siRNA (MDA-MB-231.CTRL) and MDA-MB-231 cells treated with siRNA directed against MT3 mRNA (MDA-MB-231.siRNA7). Real-time PCR was used to analyze MT3 mRNA and MMP3 mRNA. Relative expression (RQ) of <i>MT3</i> and <i>MMP3</i> genes was normalized against expression of <i>ACTB</i> gene and MDA-MB-231.CTRL cells were assigned as a calibrator sample. Results are expressed as mean ± SD.</p