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. 2010 Jun;8(6):873-84.
doi: 10.1158/1541-7786.MCR-09-0494. Epub 2010 Jun 8.

Autophagy Regulates Keratin 8 Homeostasis in Mammary Epithelial Cells and in Breast Tumors

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Free PMC article

Autophagy Regulates Keratin 8 Homeostasis in Mammary Epithelial Cells and in Breast Tumors

Sameera Kongara et al. Mol Cancer Res. .
Free PMC article

Abstract

Autophagy is activated in response to cellular stressors and mediates lysosomal degradation and recycling of cytoplasmic material and organelles as a temporary cell survival mechanism. Defective autophagy is implicated in human pathology, as disruption of protein and organelle homeostasis enables disease-promoting mechanisms such as toxic protein aggregation, oxidative stress, genomic damage, and inflammation. We previously showed that autophagy-defective immortalized mouse mammary epithelial cells are susceptible to metabolic stress, DNA damage, and genomic instability. We now report that autophagy deficiency is associated with endoplasmic reticulum (ER) and oxidative stress, and with deregulation of p62-mediated keratin homeostasis in mammary cells, allograft tumors, and mammary tissues from genetically engineered mice. In human breast tumors, high phospho(Ser73)-K8 levels are inversely correlated with Beclin 1 expression. Thus, autophagy preserves cellular fitness by limiting ER and oxidative stress, a function potentially important in autophagy-mediated suppression of mammary tumorigenesis. Furthermore, autophagy regulates keratin homeostasis in the mammary gland via a p62-dependent mechanism. High phospho(Ser73)-K8 expression may be a marker of autophagy functional status in breast tumors and, as such, could have therapeutic implications for breast cancer patients.

Figures

Figure 1
Figure 1. Metabolic stress causes preferential upregulation of ER chaperones, mitochondrial enzymes, metabolism-related proteins and keratins in autophagy-deficient mammary cells
A, 2-DIGE gels showing differential regulation of ER chaperones (GRp170, orange arrows), mitochondrial enzymes (PRDX3, yellow arrows), metabolism-related proteins (GPDH, magenta arrows) and keratins (K8, blue arrows) in Bcl-2-expressing beclin 1+/+ (left panel) and beclin 1+/− (right panel) iMMECs in response to metabolic stress (7 days). Total protein from unstressed or metabolically stressed iMMECs were labeled with Cy3 (unstressed) or Cy5 (stressed) and analyzed by 2-DIGE. Images show 2-DIGE gels with proteins that are induced (red), repressed (green) or unchanged (yellow) under stress. 106 differentially expressed protein spots were identified by mass spectroscopy. B, Western blots showing levels of ER chaperones (GRp170, GRp78, calnexin), ATF6, mitochondrial enzymes (PRDX3, SOD2, aconitase), glycolytic enzyme (PGAM) and keratins (K8/18, K17 and K19) in Bcl-2-expressing beclin 1+/+ and beclin 1+/− iMMECs under metabolic stress for 0–5 days. C, PDI (red) and K8 (green) IF in Bcl-2-expressing beclin 1+/+ and beclin 1+/− iMMECs following 5 days metabolic stress and 1 day of recovery. D, SOD2 and PDI IF (red) in mammary acini generated by Bcl-2-expressing beclin 1+/+ and beclin 1+/− iMMECs. β-catenin (green) was used as an epithelial cell marker and DAPI (blue) for counterstaining nuclei. E, ROS (H2O2) measurements in Bcl-2-expressing beclin 1+/+ and beclin 1+/− iMMECs using beclin 1 versus scramble (control) siRNA.
Figure 2
Figure 2. Metabolic stress causes autophagy induction in beclin 1+/+ iMMECs, preferential accumulation of cytoplasmic “garbage” in beclin 1+/− iMMECs and elevated ER stress in autophagy-deficient mammary tumors
A, Representative electron micrographs of Bcl-2-expressing beclin 1+/+ (left column) and beclin 1+/− (right column) iMMECs following 5 days of metabolic stress. Arrows point to autophagosomes (red) in beclin 1+/+ iMMECs and to electron-dense cytoplasmic material (yellow) preferentially accumulating in beclin 1+/− iMMECs. B, Representative electron micrographs of apoptosis-defective beclin 1+/+ (top panel) and beclin 1+/− (bottom panel) iMMEC-generated mammary tumors. An autophagosome is magnified in the red insert (top panel), whereas prominent ER expansion is presented in the green insert (bottom panel). C, GRp170 IHC in mammary tumors generated by beclin 1+/+ (top panel) and beclin 1+/− (bottom panel) iMMECs.
Figure 3
Figure 3. Defective autophagy sensitizes mammary tumor cells to ER stressors and proteasome inhibitors
MTT assays showing sensitivity of Bcl-2-expressing beclin 1+/+ (blue) and beclin 1+/− (red) iMMECs to increasing concentrations of epoxomycin (left column) and tunicamycin (right column) after 2 days of metabolic stress (A), and after a 2-day recovery following metabolic stress for 3 days (B) and 5 days (C). Data is presented as mean ± SD. p-values were calculated by two way ANOVA. ***, <0.001; **, <0.01; *, <0.05.
Figure 4
Figure 4. p62 and keratin accumulation in autophagy-deficient iMMECs under metabolic stress and recovery
A (left top), Western blot showing p62 levels in Bcl-2-expressing beclin 1+/+ and beclin 1+/− iMMECs following 7 days of metabolic stress (D7i) and 2 days of recovery (R2) in the absence and presence of the ROS-scavenger N-acetylcysteine (NAC, N); (left bottom) p62 IF (red) in mammary acini generated by Bcl-2-expressing beclin 1+/+ and beclin 1+/− iMMECs. β-catenin (green) was used as an epithelial cell marker and DAPI (blue) for counterstaining nuclei; (right) p62 IF (red) in Bcl-2-expressing beclin 1+/+ and beclin 1+/− iMMECs following 5 days of metabolic stress (D5i) and 1 day of recovery (R1) in the absence and presence of NAC. B, p62 (red) and K8 (green) IF in Bcl-2-expressing beclin 1+/+ and beclin 1+/− iMMECs following 5 days of metabolic stress (D5i) and 1 day of recovery (R1) in the absence and presence of NAC. C, phospho(Ser73)-K8 (red) IF in Bcl-2-expressing beclin 1+/+ and beclin 1+/− iMMECs stably expressing eGFP-LC3 (green) following 5 days of metabolic stress (D5i) in the absence and presence of bafilomycin. D, Western blots showing levels of p62, phospho(Ser73)-K8, and actin in Bcl-2-expressing beclin 1+/+ and beclin 1+/− iMMECs following metabolic stress for 1–3 days after transfection with control or p62 siRNA.
Figure 5
Figure 5. ER chaperone, p62 and keratin accumulation in beclin 1+/− iMMEC-generated tumors, in autophagy-deficient mammary tissues and in human breast cancers with low Beclin 1 levels
A, p62 and keratin IHC on mammary tumors generated by Bcl-2-expressing beclin 1+/+ (top row) and beclin 1+/− (bottom row) iMMECs. Rectangular insert in top left panel, p62 IHC in normal mammary duct. B, ER chaperone (GRp170, GRp78), p62, and keratin IF, and SOD2 IHC on mammary tissues from 9-month old beclin 1+/+ (top row) and beclin 1+/− (bottom row) female mice. C, phospho(Ser73)-K8 IHC on (left) mammary tissues from 9-month old beclin 1+/+ and beclin 1+/− mice; (middle) mammary tissues from atg7+/+;WAP-cre, atg7F/+;WAP-cre and atg7F/F;WAP-cre mice three weeks after completion of second pregnancy and lactation; (right) liver tissues from 2-year old beclin 1+/+ and beclin 1+/− mice. D, Beclin 1, phospho(Ser73)-K8, GRp78, GRp170, and p62 IHC on human breast tumors. Top row, breast tumor with low (1+) Beclin 1 expression; bottom row, breast tumor with high (3+) Beclin 1 expression.

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