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N-linked glycosylation is a post-translational modification that affects protein function, structure, and interaction with other proteins. The store-operated Ca2+ entry (SOCE) core proteins, Orai1 and STIM1, exhibit N-glycosylation consensus motifs. Abnormal SOCE has been associated to a number of disorders, including cancer, and alterations in Orai1 glycosylation have been related to cancer invasiveness and metastasis. Here we show that treatment of non-tumoral breast epithelial cells with tunicamycin attenuates SOCE. Meanwhile, tunicamycin was without effect on SOCE in luminal MCF7 and triple negative breast cancer (TNBC) MDA-MB-231 cells. Ca2+ imaging experiments revealed that expression of the glycosylation-deficient Orai1 mutant (Orai1N223A) did not alter SOCE in MCF10A, MCF7 and MDA-MB-231 cells. However, expression of the non-glycosylable STIM1 mutant (STIM1N131/171Q) significantly attenuated SOCE in MCF10A cells but was without effect in SOCE in MCF7 and MDA-MB-231 cells. In non-tumoral cells impairment of STIM1 N-linked glycosylation attenuated thapsigargin (TG)-induced caspase-3 activation while in breast cancer cells, which exhibit a smaller caspase-3 activity in response to TG, expression of the non-glycosylable STIM1 mutant (STIM1N131/171Q) was without effect on TG-evoked caspase-3 activation. Summarizing, STIM1 N-linked glycosylation is essential for full SOCE activation in non-tumoral breast epithelial cells; by contrast, SOCE in breast cancer MCF7 and MDA-MB-231 cells is insensitive to Orai1 and STIM1 N-linked glycosylation, and this event might participate in the development of apoptosis resistance.

Secondary lymphoid tissues, such as the spleen and lymph nodes (LNs), contribute to breast cancer development and metastasis in both anti- and pro-tumoral directions. Although secondary lymphoid tissues have been extensively studied, very little is known about the immune conversion in mesenteric LNs (mLNs) during breast cancer development. Here, we demonstrate inflammatory immune conversion of mLNs in a metastatic 4T1 breast cancer model. Splenic T cells were significantly decreased and continuously suppressed IFN-gamma production during tumor development, while myeloid-derived suppressor cells (MDSCs) were dramatically enriched. However, T cell numbers in the mLN did not decrease, and the MDSCs only moderately increased. T cells in the mLN exhibited conversion from a pro-inflammatory state with high IFN-gamma expression to an anti-inflammatory state with high expression of IL-4 and IL-10 in early- to late-stages of breast cancer development. Interestingly, increased migration of CD103(+)CD11b(+) dendritic cells (DCs) into the mLN, along with increased (1 -> 3)-beta-D-glucan levels in serum, was observed even in late-stage breast cancer. This suggests that CD103(+)CD11b(+) DCs could prime cancer-reactive T cells. Together, the data indicate that the mLN is an important lymphoid tissue contributing to breast cancer development.

N-linked glycosylation is a post-translational modification that affects protein function, structure, and interaction with other proteins. The store-operated Ca2+ entry (SOCE) core proteins, Orai1 and STIM1, exhibit N-glycosylation consensus motifs. Abnormal SOCE has been associated to a number of disorders, including cancer, and alterations in Orai1 glycosylation have been related to cancer invasiveness and metastasis. Here we show that treatment of non-tumoral breast epithelial cells with tunicamycin attenuates SOCE. Meanwhile, tunicamycin was without effect on SOCE in luminal MCF7 and triple negative breast cancer (TNBC) MDA-MB-231 cells. Ca2+ imaging experiments revealed that expression of the glycosylation-deficient Orai1 mutant (Orai1N223A) did not alter SOCE in MCF10A, MCF7 and MDA-MB-231 cells. However, expression of the nonglycosylable STIM1 mutant (STIM1N131/171Q) significantly attenuated SOCE in MCF10A cells but was without effect in SOCE in MCF7 and MDA-MB-231 cells. In non-tumoral cells impairment of STIM1 N-linked glycosylation attenuated thapsigargin (TG)-induced caspase-3 activation while in breast cancer cells, which exhibit a smaller caspase-3 activity in response to TG, expression of the non-glycosylable STIM1 mutant (STIM1N131/171Q) was without effect on TG-evoked caspase-3 activation. Summarizing, STIM1 N-linked glycosylation is essential for full SOCE activation in non-tumoral breast epithelial cells; by contrast, SOCE in breast cancer MCF7 and MDA-MB-231 cells is insensitive to Orai1 and STIM1 N-linked glycosylation, and this event might participate in the development of apoptosis resistance.

Secondary lymphoid tissues, such as the spleen and lymph nodes (LNs), contribute to breast cancer development and metastasis in both anti- and pro-tumoral directions. Although secondary lymphoid tissues have been extensively studied, very little is known about the immune conversion in mesenteric LNs (mLNs) during breast cancer development. Here, we demonstrate inflammatory immune conversion of mLNs in a metastatic 4T1 breast cancer model. Splenic T cells were significantly decreased and continuously suppressed IFN-gamma production during tumor development, while myeloid-derived suppressor cells (MDSCs) were dramatically enriched. However, T cell numbers in the mLN did not decrease, and the MDSCs only moderately increased. T cells in the mLN exhibited conversion from a pro-inflammatory state with high IFN-gamma expression to an anti-inflammatory state with high expression of IL-4 and IL-10 in early- to late-stages of breast cancer development. Interestingly, increased migration of CD103(+)CD11b(+) dendritic cells (DCs) into the mLN, along with increased (1 -> 3)-beta-D-glucan levels in serum, was observed even in late-stage breast cancer. This suggests that CD103(+)CD11b(+) DCs could prime cancer-reactive T cells. Together, the data indicate that the mLN is an important lymphoid tissue contributing to breast cancer development.

N-linked glycosylation is a post-translational modification that affects protein function, structure, and interaction with other proteins. The store-operated Ca2+ entry (SOCE) core proteins, Orai1 and STIM1, exhibit N-glycosylation consensus motifs. Abnormal SOCE has been associated to a number of disorders, including cancer, and alterations in Orai1 glycosylation have been related to cancer invasiveness and metastasis. Here we show that treatment of non-tumoral breast epithelial cells with tunicamycin attenuates SOCE. Meanwhile, tunicamycin was without effect on SOCE in luminal MCF7 and triple negative breast cancer (TNBC) MDA-MB-231 cells. Ca2+ imaging experiments revealed that expression of the glycosylation-deficient Orai1 mutant (Orai1N223A) did not alter SOCE in MCF10A, MCF7 and MDA-MB-231 cells. However, expression of the nonglycosylable STIM1 mutant (STIM1N131/171Q) significantly attenuated SOCE in MCF10A cells but was without effect in SOCE in MCF7 and MDA-MB-231 cells. In non-tumoral cells impairment of STIM1 N-linked glycosylation attenuated thapsigargin (TG)-induced caspase-3 activation while in breast cancer cells, which exhibit a smaller caspase-3 activity in response to TG, expression of the non-glycosylable STIM1 mutant (STIM1N131/171Q) was without effect on TG-evoked caspase-3 activation. Summarizing, STIM1 N-linked glycosylation is essential for full SOCE activation in non-tumoral breast epithelial cells; by contrast, SOCE in breast cancer MCF7 and MDA-MB-231 cells is insensitive to Orai1 and STIM1 N-linked glycosylation, and this event might participate in the development of apoptosis resistance.

N-linked glycosylation is a post-translational modification that affects protein function, structure, and interaction with other proteins. The store-operated Ca2+ entry (SOCE) core proteins, Orai1 and STIM1, exhibit N-glycosylation consensus motifs. Abnormal SOCE has been associated to a number of disorders, including cancer, and alterations in Orai1 glycosylation have been related to cancer invasiveness and metastasis. Here we show that treatment of non-tumoral breast epithelial cells with tunicamycin attenuates SOCE. Meanwhile, tunicamycin was without effect on SOCE in luminal MCF7 and triple negative breast cancer (TNBC) MDA-MB-231 cells. Ca2+ imaging experiments revealed that expression of the glycosylation-deficient Orai1 mutant (Orai1N223A) did not alter SOCE in MCF10A, MCF7 and MDA-MB-231 cells. However, expression of the nonglycosylable STIM1 mutant (STIM1N131/171Q) significantly attenuated SOCE in MCF10A cells but was without effect in SOCE in MCF7 and MDA-MB-231 cells. In non-tumoral cells impairment of STIM1 N-linked glycosylation attenuated thapsigargin (TG)-induced caspase-3 activation while in breast cancer cells, which exhibit a smaller caspase-3 activity in response to TG, expression of the non-glycosylable STIM1 mutant (STIM1N131/171Q) was without effect on TG-evoked caspase-3 activation. Summarizing, STIM1 N-linked glycosylation is essential for full SOCE activation in non-tumoral breast epithelial cells; by contrast, SOCE in breast cancer MCF7 and MDA-MB-231 cells is insensitive to Orai1 and STIM1 N-linked glycosylation, and this event might participate in the development of apoptosis resistance.

Co-delivery of anticancer drugs and biologics can provide synergetic effects and outperform single delivery therapies. Here, a nanoparticle (NP) system for co-delivery of methotrexate (MTX) and STAT3 siRNA has been developed and tested in vitro. Mesoporous silica nanoparticles (MSNs) were functionalized with chitosan (ch) by covalent grafting mediated by aminopropyl triethoxysilane (APTES) via glutaraldehyde as the linker. Co-delivery of MTX and STAT3 siRNA to MCF7 cells was demonstrated in cells by flow cytometric analysis and confocal laser scanning fluorescence microscopy for use in breast cancer treatment. MTX either competitively inhibits the dihydrofolate reductase (DHFR) receptor or suppresses the STAT3 metabolic pathway. STAT3 protein plays an essential role in cell division, proliferation and survival. Reduction of the protein by both MTX and STAT3 siRNA, achieved by chMSNs, significantly decreased the viability of breast cancer cells compared to single treatments alone. Cellular uptake of modified NPs was increased over time when additional free MTX was added implicating the DHFR receptor in uptake. In addition, protein corona compositions coated the NPs outer surface, were different between the NPs with and without drug potentially modulating cellular uptake. This study is the first report on co-delivery of MTX and STAT3 siRNA by chitosan modified MSNs.

Reprogramming the tumor stroma is an emerging approach to circumventing the challenges of conventional cancer therapies. This strategy, however, is hampered by the lack of a specific molecular target. We previously reported that stromal fibroblasts (FBs) with high expression of CD36 could be utilized for this purpose. These studies are now expanded to identify the secreted factors responsible for tumor suppression. Methodologies included 3D colonies, fluorescent microscopy coupled with quantitative techniques, proteomics profiling, and bioinformatics analysis. The results indicated that the conditioned medium (CM) of the CD36+ FBs caused growth suppression via apoptosis in the triple-negative cell lines of MDA-MB-231, BT549, and Hs578T, but not in the ERBB2+ SKBR3. Following the proteomics and bioinformatic analysis of the CM of CD36+ versus CD36- FBs, we determined KLF10 as one of the transcription factors responsible for growth suppression. We also identified FBLN1, SLIT3, and PENK as active ligands, where their minimum effective concentrations were determined. Finally, in MDA-MB-231, we showed that a mixture of FBLN1, SLIT3, and PENK could induce an amount of growth suppression similar to the CM of CD36+ FBs. In conclusion, our findings suggest that these ligands, secreted by CD36+ FBs, can be targeted for breast cancer treatment.

Breast cancer is among the most common malignancies in women. From the molecular point of view, breast cancer can be grouped into different categories, including the luminal (estrogen receptor positive (ER+)) and triple negative subtypes, which show distinctive features and, thus, are sensitive to different therapies. Breast cancer cells are strongly dependent on Ca2+ influx. Storeoperated Ca2+ entry (SOCE) has been found to support a variety of cancer hallmarks including cell viability, proliferation, migration, and metastasis. The Ca2+ channels of the Orai family and the endoplasmic reticulum Ca2+ sensor STIM1 are the essential components of SOCE, but the extent of Ca2+ influx is fine-tuned by several regulatory proteins, such as the STIM1 modulators SARAF and EFHB. Here, we show that the expression and/or function of SARAF and EFHB is altered in breast cancer cells and both proteins are required for cell proliferation, migration, and viability. EFHB expression is upregulated in luminal and triple negative breast cancer (TNBC) cells and is essential for full SOCE in these cells. SARAF expression was found to be similar in breast cancer and pre-neoplastic breast epithelial cells, and SARAF knockdown was found to result in enhanced SOCE in pre-neoplastic and TNBC cells. Interestingly, silencing SARAF expression in ER+ MCF7 cells led to attenuation of SOCE, thus suggesting a distinctive role for SARAF in this cell type. Finally, we used a combination of approaches to show that molecular knockdown of SARAF and EFHB significantly attenuates the ability of breast cancer cells to proliferate and migrate, as well as cell viability. In aggregate, SARAF and EFHB are required for the fine modulation of SOCE in breast cancer cells and play an important role in the maintenance of proliferation, migration, and viability in these cells

Reprogramming the tumor stroma is an emerging approach to circumventing the challenges of conventional cancer therapies. This strategy, however, is hampered by the lack of a specific molecular target. We previously reported that stromal fibroblasts (FBs) with high expression of CD36 could be utilized for this purpose. These studies are now expanded to identify the secreted factors responsible for tumor suppression. Methodologies included 3D colonies, fluorescent microscopy coupled with quantitative techniques, proteomics profiling, and bioinformatics analysis. The results indicated that the conditioned medium (CM) of the CD36+ FBs caused growth suppression via apoptosis in the triple-negative cell lines of MDA-MB-231, BT549, and Hs578T, but not in the ERBB2+ SKBR3. Following the proteomics and bioinformatic analysis of the CM of CD36+ versus CD36- FBs, we determined KLF10 as one of the transcription factors responsible for growth suppression. We also identified FBLN1, SLIT3, and PENK as active ligands, where their minimum effective concentrations were determined. Finally, in MDA-MB-231, we showed that a mixture of FBLN1, SLIT3, and PENK could induce an amount of growth suppression similar to the CM of CD36+ FBs. In conclusion, our findings suggest that these ligands, secreted by CD36+ FBs, can be targeted for breast cancer treatment.

Breast cancer is among the most common malignancies in women. From the molecular point of view, breast cancer can be grouped into different categories, including the luminal (estrogen receptor positive (ER+)) and triple negative subtypes, which show distinctive features and, thus, are sensitive to different therapies. Breast cancer cells are strongly dependent on Ca2+ influx. Storeoperated Ca2+ entry (SOCE) has been found to support a variety of cancer hallmarks including cell viability, proliferation, migration, and metastasis. The Ca2+ channels of the Orai family and the endoplasmic reticulum Ca2+ sensor STIM1 are the essential components of SOCE, but the extent of Ca2+ influx is fine-tuned by several regulatory proteins, such as the STIM1 modulators SARAF and EFHB. Here, we show that the expression and/or function of SARAF and EFHB is altered in breast cancer cells and both proteins are required for cell proliferation, migration, and viability. EFHB expression is upregulated in luminal and triple negative breast cancer (TNBC) cells and is essential for full SOCE in these cells. SARAF expression was found to be similar in breast cancer and pre-neoplastic breast epithelial cells, and SARAF knockdown was found to result in enhanced SOCE in pre-neoplastic and TNBC cells. Interestingly, silencing SARAF expression in ER+ MCF7 cells led to attenuation of SOCE, thus suggesting a distinctive role for SARAF in this cell type. Finally, we used a combination of approaches to show that molecular knockdown of SARAF and EFHB significantly attenuates the ability of breast cancer cells to proliferate and migrate, as well as cell viability. In aggregate, SARAF and EFHB are required for the fine modulation of SOCE in breast cancer cells and play an important role in the maintenance of proliferation, migration, and viability in these cells

Reprogramming the tumor stroma is an emerging approach to circumventing the challenges of conventional cancer therapies. This strategy, however, is hampered by the lack of a specific molecular target. We previously reported that stromal fibroblasts (FBs) with high expression of CD36 could be utilized for this purpose. These studies are now expanded to identify the secreted factors responsible for tumor suppression. Methodologies included 3D colonies, fluorescent microscopy coupled with quantitative techniques, proteomics profiling, and bioinformatics analysis. The results indicated that the conditioned medium (CM) of the CD36+ FBs caused growth suppression via apoptosis in the triple-negative cell lines of MDA-MB-231, BT549, and Hs578T, but not in the ERBB2+ SKBR3. Following the proteomics and bioinformatic analysis of the CM of CD36+ versus CD36- FBs, we determined KLF10 as one of the transcription factors responsible for growth suppression. We also identified FBLN1, SLIT3, and PENK as active ligands, where their minimum effective concentrations were determined. Finally, in MDA-MB-231, we showed that a mixture of FBLN1, SLIT3, and PENK could induce an amount of growth suppression similar to the CM of CD36+ FBs. In conclusion, our findings suggest that these ligands, secreted by CD36+ FBs, can be targeted for breast cancer treatment.

Breast cancer is among the most common malignancies in women. From the molecular point of view, breast cancer can be grouped into different categories, including the luminal (estrogen receptor positive (ER+)) and triple negative subtypes, which show distinctive features and, thus, are sensitive to different therapies. Breast cancer cells are strongly dependent on Ca2+ influx. Storeoperated Ca2+ entry (SOCE) has been found to support a variety of cancer hallmarks including cell viability, proliferation, migration, and metastasis. The Ca2+ channels of the Orai family and the endoplasmic reticulum Ca2+ sensor STIM1 are the essential components of SOCE, but the extent of Ca2+ influx is fine-tuned by several regulatory proteins, such as the STIM1 modulators SARAF and EFHB. Here, we show that the expression and/or function of SARAF and EFHB is altered in breast cancer cells and both proteins are required for cell proliferation, migration, and viability. EFHB expression is upregulated in luminal and triple negative breast cancer (TNBC) cells and is essential for full SOCE in these cells. SARAF expression was found to be similar in breast cancer and pre-neoplastic breast epithelial cells, and SARAF knockdown was found to result in enhanced SOCE in pre-neoplastic and TNBC cells. Interestingly, silencing SARAF expression in ER+ MCF7 cells led to attenuation of SOCE, thus suggesting a distinctive role for SARAF in this cell type. Finally, we used a combination of approaches to show that molecular knockdown of SARAF and EFHB significantly attenuates the ability of breast cancer cells to proliferate and migrate, as well as cell viability. In aggregate, SARAF and EFHB are required for the fine modulation of SOCE in breast cancer cells and play an important role in the maintenance of proliferation, migration, and viability in these cells

Breast cancer is among the most common malignancies in women. From the molecular point of view, breast cancer can be grouped into different categories, including the luminal (estrogen receptor positive (ER+)) and triple negative subtypes, which show distinctive features and, thus, are sensitive to different therapies. Breast cancer cells are strongly dependent on Ca2+ influx. Storeoperated Ca2+ entry (SOCE) has been found to support a variety of cancer hallmarks including cell viability, proliferation, migration, and metastasis. The Ca2+ channels of the Orai family and the endoplasmic reticulum Ca2+ sensor STIM1 are the essential components of SOCE, but the extent of Ca2+ influx is fine-tuned by several regulatory proteins, such as the STIM1 modulators SARAF and EFHB. Here, we show that the expression and/or function of SARAF and EFHB is altered in breast cancer cells and both proteins are required for cell proliferation, migration, and viability. EFHB expression is upregulated in luminal and triple negative breast cancer (TNBC) cells and is essential for full SOCE in these cells. SARAF expression was found to be similar in breast cancer and pre-neoplastic breast epithelial cells, and SARAF knockdown was found to result in enhanced SOCE in pre-neoplastic and TNBC cells. Interestingly, silencing SARAF expression in ER+ MCF7 cells led to attenuation of SOCE, thus suggesting a distinctive role for SARAF in this cell type. Finally, we used a combination of approaches to show that molecular knockdown of SARAF and EFHB significantly attenuates the ability of breast cancer cells to proliferate and migrate, as well as cell viability. In aggregate, SARAF and EFHB are required for the fine modulation of SOCE in breast cancer cells and play an important role in the maintenance of proliferation, migration, and viability in these cells

Breast cancer is among the most common malignancies in women. From the molecular point of view, breast cancer can be grouped into different categories, including the luminal (estrogen receptor positive (ER+)) and triple negative subtypes, which show distinctive features and, thus, are sensitive to different therapies. Breast cancer cells are strongly dependent on Ca2+ influx. Storeoperated Ca2+ entry (SOCE) has been found to support a variety of cancer hallmarks including cell viability, proliferation, migration, and metastasis. The Ca2+ channels of the Orai family and the endoplasmic reticulum Ca2+ sensor STIM1 are the essential components of SOCE, but the extent of Ca2+ influx is fine-tuned by several regulatory proteins, such as the STIM1 modulators SARAF and EFHB. Here, we show that the expression and/or function of SARAF and EFHB is altered in breast cancer cells and both proteins are required for cell proliferation, migration, and viability. EFHB expression is upregulated in luminal and triple negative breast cancer (TNBC) cells and is essential for full SOCE in these cells. SARAF expression was found to be similar in breast cancer and pre-neoplastic breast epithelial cells, and SARAF knockdown was found to result in enhanced SOCE in pre-neoplastic and TNBC cells. Interestingly, silencing SARAF expression in ER+ MCF7 cells led to attenuation of SOCE, thus suggesting a distinctive role for SARAF in this cell type. Finally, we used a combination of approaches to show that molecular knockdown of SARAF and EFHB significantly attenuates the ability of breast cancer cells to proliferate and migrate, as well as cell viability. In aggregate, SARAF and EFHB are required for the fine modulation of SOCE in breast cancer cells and play an important role in the maintenance of proliferation, migration, and viability in these cells

[Simple Summary] Breast cancer accounts as the most extended disease among women worldwide. Store-operated calcium entry (SOCE), a major mechanism that allows calcium entry from the extracellular region through the plasma membrane, is required for several physiological processes. In recent years, it has been revealed that several breast cancer types present dysregulated calcium homeostasis, which contribute to their malignancy. Here we show the role of two important regulators of SOCE, SARAF and EFHB, which are necessary for cell viability, proliferation, and migration in breast cancer and pre-neoplastic cells, respectively, thus suggesting that these regulators play a key function in breast cancer development and progression., [Abstract] Breast cancer is among the most common malignancies in women. From the molecular point of view, breast cancer can be grouped into different categories, including the luminal (estrogen receptor positive (ER+)) and triple negative subtypes, which show distinctive features and, thus, are sensitive to different therapies. Breast cancer cells are strongly dependent on Ca2+ influx. Store-operated Ca2+ entry (SOCE) has been found to support a variety of cancer hallmarks including cell viability, proliferation, migration, and metastasis. The Ca2+ channels of the Orai family and the endoplasmic reticulum Ca2+ sensor STIM1 are the essential components of SOCE, but the extent of Ca2+ influx is fine-tuned by several regulatory proteins, such as the STIM1 modulators SARAF and EFHB. Here, we show that the expression and/or function of SARAF and EFHB is altered in breast cancer cells and both proteins are required for cell proliferation, migration, and viability. EFHB expression is upregulated in luminal and triple negative breast cancer (TNBC) cells and is essential for full SOCE in these cells. SARAF expression was found to be similar in breast cancer and pre-neoplastic breast epithelial cells, and SARAF knockdown was found to result in enhanced SOCE in pre-neoplastic and TNBC cells. Interestingly, silencing SARAF expression in ER+ MCF7 cells led to attenuation of SOCE, thus suggesting a distinctive role for SARAF in this cell type. Finally, we used a combination of approaches to show that molecular knockdown of SARAF and EFHB significantly attenuates the ability of breast cancer cells to proliferate and migrate, as well as cell viability. In aggregate, SARAF and EFHB are required for the fine modulation of SOCE in breast cancer cells and play an important role in the maintenance of proliferation, migration, and viability in these cells.

Breast cancer is among the most common malignancies in women. From the molecular point of view, breast cancer can be grouped into different categories, including the luminal (estrogen receptor positive (ER+)) and triple negative subtypes, which show distinctive features and, thus, are sensitive to different therapies. Breast cancer cells are strongly dependent on Ca2+ influx. Storeoperated Ca2+ entry (SOCE) has been found to support a variety of cancer hallmarks including cell viability, proliferation, migration, and metastasis. The Ca2+ channels of the Orai family and the endoplasmic reticulum Ca2+ sensor STIM1 are the essential components of SOCE, but the extent of Ca2+ influx is fine-tuned by several regulatory proteins, such as the STIM1 modulators SARAF and EFHB. Here, we show that the expression and/or function of SARAF and EFHB is altered in breast cancer cells and both proteins are required for cell proliferation, migration, and viability. EFHB expression is upregulated in luminal and triple negative breast cancer (TNBC) cells and is essential for full SOCE in these cells. SARAF expression was found to be similar in breast cancer and pre-neoplastic breast epithelial cells, and SARAF knockdown was found to result in enhanced SOCE in pre-neoplastic and TNBC cells. Interestingly, silencing SARAF expression in ER+ MCF7 cells led to attenuation of SOCE, thus suggesting a distinctive role for SARAF in this cell type. Finally, we used a combination of approaches to show that molecular knockdown of SARAF and EFHB significantly attenuates the ability of breast cancer cells to proliferate and migrate, as well as cell viability. In aggregate, SARAF and EFHB are required for the fine modulation of SOCE in breast cancer cells and play an important role in the maintenance of proliferation, migration, and viability in these cells

Breast cancer is among the most common malignancies in women. From the molecular point of view, breast cancer can be grouped into different categories, including the luminal (estrogen receptor positive (ER+)) and triple negative subtypes, which show distinctive features and, thus, are sensitive to different therapies. Breast cancer cells are strongly dependent on Ca2+ influx. Storeoperated Ca2+ entry (SOCE) has been found to support a variety of cancer hallmarks including cell viability, proliferation, migration, and metastasis. The Ca2+ channels of the Orai family and the endoplasmic reticulum Ca2+ sensor STIM1 are the essential components of SOCE, but the extent of Ca2+ influx is fine-tuned by several regulatory proteins, such as the STIM1 modulators SARAF and EFHB. Here, we show that the expression and/or function of SARAF and EFHB is altered in breast cancer cells and both proteins are required for cell proliferation, migration, and viability. EFHB expression is upregulated in luminal and triple negative breast cancer (TNBC) cells and is essential for full SOCE in these cells. SARAF expression was found to be similar in breast cancer and pre-neoplastic breast epithelial cells, and SARAF knockdown was found to result in enhanced SOCE in pre-neoplastic and TNBC cells. Interestingly, silencing SARAF expression in ER+ MCF7 cells led to attenuation of SOCE, thus suggesting a distinctive role for SARAF in this cell type. Finally, we used a combination of approaches to show that molecular knockdown of SARAF and EFHB significantly attenuates the ability of breast cancer cells to proliferate and migrate, as well as cell viability. In aggregate, SARAF and EFHB are required for the fine modulation of SOCE in breast cancer cells and play an important role in the maintenance of proliferation, migration, and viability in these cells

Orai1 plays a major role in store-operated Ca 2+ entry (SOCE) in triple-negative breast cancer (TNBC) cells. This channel is inactivated via different mechanisms, including protein kinase C (PKC) and protein kinase A (PKA)-dependent phosphorylation at Ser-27 and Ser-30 or Ser-34, respectively, which shapes the Ca 2+ responses to agonists. The Ca 2+ calmodulin-activated adenylyl cyclase type 8 (AC8) was reported to interact directly with Orai1, thus mediating a dynamic interplay between the Ca 2+ - and cyclic adenosine monophosphate (cAMP)-dependent signaling pathways. Here, we show that the breast cancer cell lines MCF7 and MDA-MB-231 exhibit enhanced expression of Orai1 and AC8 as compared to the non-tumoral breast epithelial MCF10A cell line. In these cells, AC8 interacts with the Orai1α variant in a manner that is not regulated by Orai1 phosphorylation. AC8 knockdown in MDA-MB-231 cells, using two different small interfering RNAs (siRNAs), attenuates thapsigargin (TG)-induced Ca 2+ entry and also Ca 2+ influx mediated by co-expression of Orai1 and the Orai1-activating small fragment (OASF) of STIM1 (stromal interaction molecule-1). Conversely, AC8 overexpression enhances SOCE, as well as Ca 2+ entry, in cells co-expressing Orai1 and OASF. In MDA-MB-231 cells, we found that AC8 overexpression reduces the Orai1 phosphoserine content, thus suggesting that AC8 interferes with Orai1 serine phosphorylation, which takes place at residues located in the AC8-binding site. Consistent with this, the subset of Orai1 associated with AC8 in naïve MDA-MB-231 cells is not phosphorylated in serine residues in contrast to the AC8-independent Orai1 subset. AC8 expression knockdown attenuates migration of MCF7 and MDA-MB-231 cells, while this maneuver has no effect in the MCF10A cell line, which is likely attributed to the low expression of AC8 in these cells. We found that AC8 is required for FAK (focal adhesion kinase) phosphorylation in MDA-MB-231 cells, which might explain its r

Purpose: Investigation of effects on DNA of γ-irradiated human cancer cells pretreated with free radical scavengers is aimed to create reference data which would enable assessment of the relative efficiency of high linear energy transfer (LET) radiations used in hadron therapy, i.e. protons and carbon ions. Materials and methods: MCF-7 breast and HTB177 lung cancer cells are irradiated with γ-rays. To minimize indirect effects of irradiation, dimethyl sulfoxide (DMSO) or glycerol are applied as free radical scavengers. Biological response to irradiation is evaluated through clonogenic cell survival, immunocytochemical and cell cycle analysis, as well as expression of proteins involved in DNA damage response. Results: Examined cell lines reveal similar level of radioresistance. Application of scavengers leads to the rise of cell survival and decreases the number of DNA double strand breaks in irradiated cells. Differences in cell cycle and protein expression between the two cell lines are probably caused by different DNA damage repair mechanisms that are activated. Conclusion: The obtained results show that DMSO and glycerol have good scavenging capacity, and may be used to minimize DNA damage induced by free radicals. Therefore, they will be used as the reference for comparison with high LET irradiations, as well as good experimental data suitable for validation of numerical simulations. © 2019, © 2019 Taylor & Francis Group, LLC.

Background: Pro-oxidant drugs have been proposed for treating certain cancers but the resistance developed by cancer cells to oxidative stress limits its potential use in clinics. To understand the mechanisms underlying resistance to oxidative stress, we found that the chronic exposure to an H2O2-generating system (ascorbate/menadione, Asc/Men) or catalase overexpression (CAT3 cells) increased the resistance of cancer cells to oxidative stress, likely by increasing the antioxidant status of cancer cells. Methods: Modulation of catalase expression was performed by either protein overexpression or protein down-regulation using siRNA against catalase and aminotriazole as pharmacological inhibitor. The former approach was done by transfecting cells with a plasmid construct containing human catalase cDNA (CAT3 cells, derived from MCF-7 breast cancer cell line) or by generating resistant cells through chronic exposure to an oxidant injury (Resox cells). Cell survival was monitored by using the MTT reduction assay and further calculation of IC50 values. Protein expression was done by Western blots procedures. The formation of reactive oxygen species was performed by flow cytometry. The transcriptional activity of human catalase promoter was assessed by using transfected cells with a plasmid containing the - 1518/+ 16 promoter domain. Results: Using Resox and CAT3 cells (derived from MCF-7 breast cancer cell line) as models for cancer resistance to pro-oxidative treatment, we found that arsenic trioxide (ATO) remarkably sensitized Resox and CAT3 cells to Asc/Men treatment. Since catalase is a key antioxidant enzyme involved in detoxifying Asc/Men (as shown by siRNA-mediated catalase knockdown) that is overexpressed in resistant cells, we hypothesized that ATO might regulate the expression levels of catalase. Consistently, catalase protein level is decreased in Resox cells when incubated with ATO likely by a decreased transcriptional activity of the catalase promoter. Conclu

The influence of cell membrane fluidity on cancer progression has been established in different solid tumors. We previously reported that "cancer-associated fibroblasts" (CAFs) induced epithelial-mesenchymal transition and increased cell membrane fluidity and migration in poorly (MCF-7) and highly invasive (MDA-MB-231) breast cancer cells. We also found that the membrane fluidity regulating enzyme stearoyl-CoA desaturase 1 (SCD1) was upregulated in tumor cells co-cultured with CAFs and established its essential role for both intrinsic and CAF-driven tumor cell motility. Here, we further explored the mechanisms involved in the SCD1-based modulation of breast cancer cell migration and investigated the role of the other human SCD isoform, SCD5. We showed that the addition of oleic acid, the main SCD1 product, nullified the inhibitory effects produced on MCF-7 and MDA-MB-231 cell migration by SCD1 depletion (pharmacological or siRNA-based). Conversely, SCD5 seemed not involved in the regulation of cancer cell motility. Interestingly, a clear induction of necrosis was observed as a result of the depletion of SCD5 in MCF-7 cells, where the expression of SCD5 was found to be upregulated by CAFs. The necrotic effect was rescued by a 48-h treatment of cells with oleic acid. These results provide further insights in understanding the role of SCD1 in both intrinsic and CAF-stimulated mammary tumor cell migration, unveiling the metabolic basis of this desaturase-triggered effect. Moreover, our data suggest the ability of CAFs to promote the maintenance of tumor cell survival by the induction of SCD5 levels.

Cells are commonly used in research to evaluate toxicity and efficiency of drugs. However, to further increase the usefulness of cells as well as the understandings of effects of different interventions, new methods must constantly be developed and refined. Today, many assays use end-point analysis of large populations of cells, to evaluate the research question. However, there are many cases when this kind of analysis hides important effects or behaviour of individual cells. Therefore, quantitative analysis of individual cells over long time periods is important for the complete understanding of the heterogeneity of cell populations. Dogital holographic imaging is a non-toxic quantitative method that can be used for analysis of individual cells over long periods of time. It is the major analysis method of this thesis. In cancer, a small population of cells has gained the interest of cancer researchers since the cells resist treatment and have increased capability to migrate and form metastases. Those cells are called cancer stem cells, due to their many similarities to normal stem cells. The interest in drugs that specifically target cancer stem cells has dramatically increased during the last decade. One of the drugs found to target cancer stem cells in multiple cancers is salinomycin, an ionophore which has been used as an antibiotic for more than 30 years. Almost immediately after addition to the medium of cells, salinomycin is found in the endoplasmatic reticulum resulting in increases the cytosolic Ca2+. This leads to further down-stream effects, which among others includes mesenchymal to epithelial transition. We have used longitudinal tracking of cells in time-lapses acquired using digital holographic imaging to evaluate cell cycle times and movement of different cancer cell lines as well as normal cell lines. We found that small sub-populations of cells behaved differently than the rest of the individually tracked cells. The existence of these cells could n

BACKGROUND/AIM: The survival rate of women diagnosed with triple-negative breast-cancer (TNBC) remains low. Hence, this study aimed at the chemical and biological optimization of furanosteroid derivatives for the treatment of this type of malignancy using TNBC cells. MATERIALS AND METHODS: Semi-synthetic analogs of wortmannolone (1-6) that negatively affected the aberrant pathways in tumor cells were evaluated in hormone-independent breast cancer cells using western blot and cell-cycle analysis. RESULTS: Wortmannolone derivatization generated NF-ĸB inhibitors as new lead structures for further development. Compound (3) was found to be the most significantly active lead. CONCLUSION: Structure-activity analysis in the present study showed that acetylation of the hydroxyl groups and substitution on C3 and C17 of wortmannolone enhanced biological activity. Alpha-substitution of the acetyl group in C3 on ring A (compound 3) resulted in ROS inducing effect; however, presence of an acetyl group in β-position of C3 displayed the highest NF-ĸB p65 inhibitory activity (0.60 μM).

The role of direct cell-cell interactions mediating selective bone metastasis by breast cancer cells (BCCs) niche is still mostly unknown., SCOPUS: ar.j, info:eu-repo/semantics/published

Computational techniques have been used to design a novel class of RNA architecture with expected improved resistance to nuclease degradation, while showing interference RNA activity. The in silico designed structure consists of a 24–29 bp duplex RNA region linked on both ends by N-alkyl-N dimeric nucleotides (BCn dimers; n = number of carbon atoms of the alkyl chain). A series of N-alkyl-N capped dumbbell-shaped structures were efficiently synthesized by double ligation of BCn-loop hairpins. The resulting BCn-loop dumbbells displayed experimentally higher biostability than their 3′-N-alkyl-N linear version, and were active against a range of mRNA targets. We studied first the effect of the alkyl chain and stem lengths on RNAi activity in a screen involving two series of dumbbell analogues targeting Renilla and Firefly luciferase genes. The best dumbbell design (containing BC6 loops and 29 bp) was successfully used to silence GRB7 expression in HER2+ breast cancer cells for longer periods of time than natural siRNAs and known biostable dumbbells. This BC6-loop dumbbell-shaped structure displayed greater anti-proliferative activity than natural siRNAs., Instituto de Salud Carlos III [Miguel Servet Program, CP13/00211, 205024141 to M.T.]; Spanish MINECO [BIO2012–32869 and BIO2015-64802-R toM.O.]; AGAUR (toM.O.); ERCCouncil (SimDNA, grant 291433, to M.O.). M.O. is an ICREA Academia fellow. Funding for open access charge: ERC Council [grant 291433 (simDNA)]., Peer Reviewed, Postprint (published version)

Sulforafan är en isothiocyanat-förening som finns i stora mängder i olika kålväxter. Sulforafan har setts ha en kemopreventiv inverkan bland annat genom att inducera fas II-enzymer styrda av transkriptionsfaktorn nuclear factor E2-related factor-2 (Nrf2). Nrf2 är mycket viktig i cellers skydd mor oxidativ- och elektrofil stress och inducerar uttrycket av många olika gener, bland annat olika tranportproteiner. Breast cancer resistance protein (BCRP) är en av transportörerna som tros induceras av Nrf2. BCRP finns i stora mängder i lakterande juvervävnad men också i många andra vävnader där den transporterar läkemedel ut ur cellerna. Om sulforafan kan inducera genuttrycket av BCRP skulle det kunna innebära att halten av läkemedelsrester ökar i mjölken hos kor och människor som äter en kost rik på sulforafan. Det är därför viktigt att veta om så är fallet eftersom det kan påverka livsmedelssäkerheten för konsumenter och spädbarn. Med anledning av detta har jag tittat på om sulforafan kan uppreglera genuttrycket av BCRP och om det kan aktivera Nrf2 i bröstcancercellinjen MCF-7. Ingen uppreglering av genuttrycket av BCRP kunde ses med realtids-PCR och vid 20 μM sulforafan kunde till och med en sänkning av uttrycket ses. Med en Nrf2-luciferasreporter-assay sågs däremot en ökning av Nrf2 aktiviteten med ökande sulforafan koncentration. Dock har studien haft problem med toxicitet av sulforafan som inte detekterades i MTS-assay och inga säkra slutsatser kan därför dras om påverkan av sulforafan på Nrf2 och BCRP. Sammanfattningsvis har jag inte kunnat bevisa att sulforafan inducerar BCRP eller aktiverar Nrf2 men har inte heller bevisat motsatsen., Sulforaphane is an isothiocyanate compound that is abundant in cruciferous vegetables. Sulforaphane has been shown to have a chemo preventive effect by, among other things, inducing phase II enzymes governed by the transcription factor nuclear factor E2-related factor-2 (Nrf2). Nrf2 plays an important part in the protection of cells from oxidative and electrophilic stress and induce many different genes including various transport proteins. Breast cancer resistance protein (BCRP) is one of the transporters believed to be induced by Nrf2. BCRP is present in great quantity in lactating mammary glands but also exists in various other tissues were it transports drugs out of the cells. If sulforaphane is able to induce gene expression of BCRP that might lead to an increase of dug residues in milk from cows and humans consuming a diet rich in sulforaphane and thus be a safety hazard for consumers of milk and for infants. With that in mind I have made a study where I investigated if sulforaphane can induce gene expression of BCRP and if it can activate Nrf2 in the breast cancer cell line MCF-7. I was not able to detect any induction of BCRP expression with real-time PCR and at a concentration of 20 μM of sulforaphane a reduction in expression was seen. With a Nrf2-luciferase reporter assay I was, on the other hand, able to detect an increase in Nrf2 activity with rising sulforaphane concentrations. Alas the study had problems with toxicity of sulforaphane that was not detected by MST-assay. Because of this no safe conclusions about the effect of sulforaphane on the Nrf2 mediated expression of BCRP can be drawn. To summarise I have not been able to show that sulforaphane induces BCRP or that it activates Nrf2 but have not proven the opposite either.

Sulforafan är en isothiocyanat-förening som finns i stora mängder i olika kålväxter. Sulforafan har setts ha en kemopreventiv inverkan bland annat genom att inducera fas II-enzymer styrda av transkriptionsfaktorn nuclear factor E2-related factor-2 (Nrf2). Nrf2 är mycket viktig i cellers skydd mor oxidativ- och elektrofil stress och inducerar uttrycket av många olika gener, bland annat olika tranportproteiner. Breast cancer resistance protein (BCRP) är en av transportörerna som tros induceras av Nrf2. BCRP finns i stora mängder i lakterande juvervävnad men också i många andra vävnader där den transporterar läkemedel ut ur cellerna. Om sulforafan kan inducera genuttrycket av BCRP skulle det kunna innebära att halten av läkemedelsrester ökar i mjölken hos kor och människor som äter en kost rik på sulforafan. Det är därför viktigt att veta om så är fallet eftersom det kan påverka livsmedelssäkerheten för konsumenter och spädbarn. Med anledning av detta har jag tittat på om sulforafan kan uppreglera genuttrycket av BCRP och om det kan aktivera Nrf2 i bröstcancercellinjen MCF-7. Ingen uppreglering av genuttrycket av BCRP kunde ses med realtids-PCR och vid 20 μM sulforafan kunde till och med en sänkning av uttrycket ses. Med en Nrf2-luciferasreporter-assay sågs däremot en ökning av Nrf2 aktiviteten med ökande sulforafan koncentration. Dock har studien haft problem med toxicitet av sulforafan som inte detekterades i MTS-assay och inga säkra slutsatser kan därför dras om påverkan av sulforafan på Nrf2 och BCRP. Sammanfattningsvis har jag inte kunnat bevisa att sulforafan inducerar BCRP eller aktiverar Nrf2 men har inte heller bevisat motsatsen., Sulforaphane is an isothiocyanate compound that is abundant in cruciferous vegetables. Sulforaphane has been shown to have a chemo preventive effect by, among other things, inducing phase II enzymes governed by the transcription factor nuclear factor E2-related factor-2 (Nrf2). Nrf2 plays an important part in the protection of cells from oxidative and electrophilic stress and induce many different genes including various transport proteins. Breast cancer resistance protein (BCRP) is one of the transporters believed to be induced by Nrf2. BCRP is present in great quantity in lactating mammary glands but also exists in various other tissues were it transports drugs out of the cells. If sulforaphane is able to induce gene expression of BCRP that might lead to an increase of dug residues in milk from cows and humans consuming a diet rich in sulforaphane and thus be a safety hazard for consumers of milk and for infants. With that in mind I have made a study where I investigated if sulforaphane can induce gene expression of BCRP and if it can activate Nrf2 in the breast cancer cell line MCF-7. I was not able to detect any induction of BCRP expression with real-time PCR and at a concentration of 20 μM of sulforaphane a reduction in expression was seen. With a Nrf2-luciferase reporter assay I was, on the other hand, able to detect an increase in Nrf2 activity with rising sulforaphane concentrations. Alas the study had problems with toxicity of sulforaphane that was not detected by MST-assay. Because of this no safe conclusions about the effect of sulforaphane on the Nrf2 mediated expression of BCRP can be drawn. To summarise I have not been able to show that sulforaphane induces BCRP or that it activates Nrf2 but have not proven the opposite either.

Human breast cancers, like other cancers, frequently harbor amplifications of chromosomal regions containing multiple genes. Many such multi-gene amplifications are recurrent across many individual instances of breast cancer, suggesting they contain oncogenes critical for breast cancer development or progression. In some cases, traditional laboratory methods have identified more than one candidate driver oncogene within usuch amplified regions. This research proposal is designed to test the hypothesis that some recurrent amplicons actually function through the concerted action of multiple simultaneously overexpressed oncogenes. We are testing this hypothesis by attempting to create cell line models of such multigene amplifications, prospectively. We have designed an experimental strategy to target a dominantly selectable IMPDH gene to specific chromosomal regions using gene targeting technology. Targeted cells will then be selected in increasing concentrations of the IMPDH inhibitors mycophenolic acid and mizoribine to provide selective pressure for the targeted cells to amplify the targeted region, co-amplifying multiple genes simultaneously. Here we report the design of a functional, efficient gene targeting vector and the successful delivery of the IMPDH selection marker to two different chromosomal loci, ZNF703 and ERBB2, in two different human breast cancer cell lines, MCF-7, and T47D. In MCF-7, we have succeeded in creating subclones with focal, overlapping amplification of regions of chromosome 8p11-12, where ZNF703 is located. We demonstrate amplification by array CGH, digital PCR, and fluorescent in situ hybridization. Amplification is shown to cause coordinate overexpression of co-amplified genes at the mRNA and protein level., The original document contains color images.

The Ron receptor tyrosine kinase is over-expressed and over-activated in a cohort of human cancers, with the most compelling data yet found in breast cancer. Specifically, Ron is overexpressed in approximately 50% of human breast cancers, and has been shown to be an independent predictor of both metastases and poor prognosis in women with this disease. While Ron overexpression appears to be an important factor in human breast cancer growth and metastasis, a significant gap exists in our knowledge about the signaling pathways that Ron activates in breast tumors, and about the importance of these pathways with respect to overall tumor growth and metastatic dissemination. Our laboratories have shown that mammary tumors from mice overexpressing Ron selectively in the mammary epithelium exhibit increased levels of the DEK proto-oncogene. In addition, we also show that ligand-induced Ron activation in human and murine breast cancer cell lines induces the accumulation of DEK protein. This accumulation of DEK is significant as DEK overexpression in breast cancer cell lines leads to increases in cell growth and migration while DEK depletion in breast cancer cells leads to dramatic reductions in cell growth and migration. Moreover, we also show that DEK deficient cells are more susceptible to DNA damage. Based on these data, our goal is to test the hypothesis that Ron-mediated DEK upregulation contributes functionally to breast cancer development, dissemination and resistance to clastogenic therapies and that targeting the Ron-DEK signaling axis may represent an important new therapeutic option for the treatment of breast cancer. To test this hypothesis, two Specific Aims were proposed. In Aim 1, we will determine the requirement of DEK in Ron overexpressing breast cancers utilizing a combination of DEK loss of function and Ron transgenic overexpression. Aim 2 will examine the therapeutic utility of targeting Ron and DEK on beta-catenin activation and breast cancer growth., The original document contains color images.

Abnormal nuclear shape and structure has long been recognized as a characteristic feature of cancer cells, but the underlying molecular basis and functional consequences have remained elusive. It is now emerging that many breast cancer cells have reduced expression of lamins A/C, which negatively correlates with disease-free survival. To investigate the consequences of altered lamin expression in more detail, we modulated the expression of lamins in a panel of breast cancer cells. By developing and using novel microfluidic devices that mimic the conditions encountered during perfusion through capillaries or migration through interstitial spaces, we demonstrated that nuclear deformability, governed by levels of lamins A/C, constitutes a rate-limiting factor in the ability of cells to pass through openings smaller than the nucleus. Furthermore, we found that metastatic cells can dynamically adjust their nuclear envelope composition during migration through confining 3-D environments, facilitating transit through narrow constrictions. Interestingly, cells with reduced levels of lamins A/C were more prone to repetitive nuclear rupture, which could result in increased DNA damage and chromosomal rearrangements. Loss of lamins A/C also disturbed MKL1/SRF signaling. Our findings could provide novel diagnostic and prognostic markers for the treatment of cancer patients; ultimately, a better understanding of the molecular mechanisms underlying the ability of breast cancer cells to dynamically alter their nuclear envelope composition may lead to the identification of new therapeutic targets., The original document contains color images.

Our long-term goal is to understand the impact of high levels of SNAI repressor proteins in the etiology, progression and pathogenicity of breast cancer. Our central hypothesis is that combinatorial treatment of SNAI-high breast tumor cells with the SNAI inhibitors will not only diminish their aggressiveness but also make these cells sensitive to the inhibition of some of the conventional anticancer agents such as vitamin D and anti-estrogens. Specific Aims: (1) To evaluate the levels of SNAI proteins in relation to the levels of the proteins that impart resistance against vitamin D and anti-estrogens in human breast cancer tissues; (2) To determine the effects of alterations of the levels of SNAI proteins in breast cancer cells on their sensitivity towards vitamin D and/or 4HT; and (3) To evaluate the efficacy of RNA and peptide-based inhibitors of SNAI-protein functions on the aggressiveness, metastatic ability and drug sensitivity of SNAI-high human breast cancer cells in a mouse xenograft model., The original document contains color images.

Background: MicroRNA-34 is a family of three miRNAs that have been reported to function as tumor suppressor miRNAs and show decreased expression in various cancers. Here, we examine functions of miR-34c in basal-like breast cancer cells. Methods: Data from The Cancer Genome Atlas (TCGA) were used for evaluation of expression in primary breast cancers. Cellular processes affected by miR-34c were investigated by thymidine incorporation, Annexin V-assays and cell cycle analysis using breast cancer cell lines. Effects on potential targets were analyzed with qPCR and Western blot. Results: TCGA data revealed that miR-34c was expressed at lower levels in basal-like breast cancer tumors and low expression was associated with poor prognosis. Ectopic expression of miR-34c in basal-like breast cancer cell lines resulted in suppressed proliferation and increased cell death. Additionally, miR-34c influenced the cell cycle mainly by inducing an arrest in the G2/M phase. We found that expression levels of the known cell cycle-regulating miR-34 targets CCND1, CDK4 and CDK6, were downregulated upon miR-34c expression in breast cancer cell lines. In addition, the levels of CDC23, an important mediator in mitotic progression, were suppressed following miR-34c expression, and siRNAs targeting CDC23 mimicked the effect of miR-34c on G2/M arrest. However, protein levels of PRKCA, a predicted miR-34c target and a known regulator of breast cancer cell proliferation were not influenced by miR-34c. Conclusions: Together, our results support the role of miR-34c as a tumor suppressor miRNA also in breast cancer.

PURPOSE: Radiation therapy (RT) is thought to produce clinical responses in cancer patients, not only through direct toxicity to cancer cells and supporting tumor stroma cells, but also through activation of immunologic effectors. More recently, RT has potentiated the local and systemic effects of cancer immunotherapy (IT). However, combination regimens that maximize immunologic and clinical efficacy remain undefined. METHODS AND MATERIALS: We evaluated the impact of local RT on adenoviral-mediated vaccination against the colorectal cancer antigen GUCY2C (Ad5-GUCY2C) in a murine subcutaneous tumor model using mouse CT26 colon cancer cells (CT26-GUCY2C). Immune responses were assessed by ELISpot, and clinical responses were assessed by tumor size and incidence. RESULTS: The specific sequence of tumor-directed RT preceding Ad5-GUCY2C IT transformed inactive therapeutic Ad5-GUCY2C vaccination into a curative vaccine. GUCY2C-specific T cell responses were amplified (P CONCLUSIONS: Optimal sequencing of RT and IT amplifies antigen-specific local and systemic immune responses, revealing novel acute and long-term therapeutic antitumor protection. These observations underscore the importance of modality sequence optimization before the initiation of clinical trials of RT and IT to maximize immune and antitumor responses.

The Ron receptor tyrosine kinase is over-expressed and over-activated in a cohort of human cancers, with the most compelling data yet found in breast cancer. Specifically, Ron is overexpressed in approximately 50% of human breast cancers, and has been shown to be an independent predictor of both metastases and poor prognosis in women with this disease. While Ron overexpression appears to be an important factor in human breast cancer growth and metastasis, a significant gap exists in our knowledge about the signaling pathways that Ron activates in breast tumors, and about the importance of these pathways with respect to overall tumor growth and metastatic dissemination. Our laboratories have shown that mammary tumors from mice overexpressing Ron selectively in the mammary epithelium exhibit increased levels of the DEK proto-oncogene. In addition, we also show that ligand-induced Ron activation in human and murine breast cancer cell lines induces the accumulation of DEK protein. This accumulation of DEK is significant as DEK overexpression in breast cancer cell lines leads to increases in cell growth and migration while DEK depletion in breast cancer cells leads to dramatic reductions in cell growth and migration. Moreover, we also show that DEK deficient cells are more susceptible to DNA damage. Based on these data, our goal is to test the hypothesis that Ron-mediated DEK upregulation contributes functionally to breast cancer development, dissemination and resistance to clastogenic therapies and that targeting the Ron-DEK signaling axis may represent an important new therapeutic option for the treatment of breast cancer. To test this hypothesis, two Specific Aims were proposed. In Aim 1, we will determine the requirement of DEK in Ron overexpressing breast cancers utilizing a combination of DEK loss of function and Ron transgenic overexpression. Aim 2 will examine the therapeutic utility of targeting Ron and DEK on beta-catenin activation and breast cancer growth. Th, The original document contains color images.

BRCA1 is a multifunctional tumor suppressive protein. Knockout of WT BRCA1 in breast cancer cells resulted in an increase in cell proliferation, anchorage-independent growth, cell migration, invasion and a loss of p21/Waf1 and P27Kip1 expression. Further, in BRCA1 knocked-down cells, the expression of survivin was significantly up-regulated with a decrease in cellular sensitivity to paclitaxel. Cells that harbor endogenous mutant or defective BRCA1 (such as MDA-MB-436 and HCC1937) were highly proliferative and expressed a relatively low levels of p21/Waf1 and p27Kip1 and high level of survivin and were resistant to paclitaxel. Thus, mutated BRCA1 or loss of WT BRCA1 up-regulates the malignant cell behavior. However, it is still not clear how tumor cells expressing mutant BRCA1 have enhance tumorogenicity in vivo., The original document contains color images.

Interactions occurring between malignant cells and stromal microenvi-ronment greatly influence progression of breast cancer. In a previous study, we co-cultured mammary cancer cells exhibiting different degrees of metastatic potential (MDA-MB-231>MCF-7) with fibroblasts isolated from breast healthy skin (normal fibroblasts, NFs) or breast tumor stroma (cancer-associated fibroblasts, CAFs). In this system, we demonstrated the influence exerted in particular by CAFs on malignant cells, leading to the acquisition of a more aggressive/invasive phenotype (i.e. reduced adhesion, epithelial-mesenchymal transition, enhanced plasma membrane fluidity and migration velocity/directness). In the present study, we evaluated the reciprocal effect of breast tumor cells and fibroblasts in co-culture on the expression of the main enzyme regulating the fatty acids membrane composition, Stearoyl-CoA desaturase 1 (SCD1). Abnormally high levels of SCD1 have been reported in different cancers and transformed cells and the enzyme has been recently raised to the role of key regulator of cell growth, programmed cell death and car-cinogenesis. In our experience, Western blot analysis demonstrated a strong increase in SCD1 expression in both MCF-7 and MDA-MB-231 cells, resulting from their interaction with CAFs and, at a lesser extent, with NFs. High levels of SCD1 were also observed in both NFs and CAFs when co-cultured with MCF-7 cells. MDA-MB-231 cells more slightly up-regulated the enzyme expression in NFs or even induced a certain inhi-bition in CAFs. The fibroblast-triggered up-regulation of SCD1 in cancer cells could rea-sonably be considered the molecular event underlying the increase of membrane fluidity, previously observed in tumor cells co-cultured with NFs and, notably, with CAFs. This change might downstream promote the pre-viously described increase in tumor cell motility.

Interactions occurring between malignant cells and stromal microenvi-ronment greatly influence progression of breast cancer. In a previous study, we co-cultured mammary cancer cells exhibiting different degrees of metastatic potential (MDA-MB-231>MCF-7) with fibroblasts isolated from breast healthy skin (normal fibroblasts, NFs) or breast tumor stroma (cancer-associated fibroblasts, CAFs). In this system, we demonstrated the influence exerted in particular by CAFs on malignant cells, leading to the acquisition of a more aggressive/invasive phenotype (i.e. reduced adhesion, epithelial-mesenchymal transition, enhanced plasma membrane fluidity and migration velocity/directness). In the present study, we evaluated the reciprocal effect of breast tumor cells and fibroblasts in co-culture on the expression of the main enzyme regulating the fatty acids membrane composition, Stearoyl-CoA desaturase 1 (SCD1). Abnormally high levels of SCD1 have been reported in different cancers and transformed cells and the enzyme has been recently raised to the role of key regulator of cell growth, programmed cell death and car-cinogenesis. In our experience, Western blot analysis demonstrated a strong increase in SCD1 expression in both MCF-7 and MDA-MB-231 cells, resulting from their interaction with CAFs and, at a lesser extent, with NFs. High levels of SCD1 were also observed in both NFs and CAFs when co-cultured with MCF-7 cells. MDA-MB-231 cells more slightly up-regulated the enzyme expression in NFs or even induced a certain inhi-bition in CAFs. The fibroblast-triggered up-regulation of SCD1 in cancer cells could rea-sonably be considered the molecular event underlying the increase of membrane fluidity, previously observed in tumor cells co-cultured with NFs and, notably, with CAFs. This change might downstream promote the pre-viously described increase in tumor cell motility.

Interactions occurring between malignant cells and the stromal microenvironment heavily influence tumor progression. We investigated whether this cross-talk affects some molecular and functional aspects specifically correlated with the invasive phenotype of breast tumor cells (i.e. adhesion molecule expression, membrane fluidity, migration) by co-culturing mammary cancer cells exhibiting different degrees of metastatic potential (MDA-MB-231>MCF-7) with fibroblasts isolated from breast healthy skin (normal fibroblasts, NFs) or from breast tumor stroma (cancer-associated fibroblasts, CAFs) in 2D or 3D (nodules) cultures. Confocal immunofluorescence analysis of the epithelial adhesion molecule E-cadherin on frozen nodule sections demonstrated that NFs and CAFs, respectively, induced or inhibited its expression in MCF-7 cells. An increase in the mesenchymal adhesion protein N-cadherin was observed in CAFs, but not in NFs, as a result of the interaction with both kinds of cancer cells. CAFs, in turn, promoted N-cadherin up-regulation in MDA-MB-231 cells and its de novo expression in MCF-7 cells. Beyond promotion of "cadherin switching", another sign of the CAF-triggered epithelial-mesenchymal transition (EMT) was the induction of vimentin expression in MCF-7 cells. Plasma membrane labeling of monolayer cultures with the fluorescent probe Laurdan showed an enhancement of the membrane fluidity in cancer cells co-cultured with NFs or CAFs. An increase in lipid packing density of fibroblast membranes was promoted by MCF-7 cells. Time-lapsed cell tracking analysis of mammary cancer cells co-cultured with NFs or CAFs revealed an enhancement of tumor cell migration velocity, even with a marked increase in the directness induced by CAFs.Our results demonstrate a reciprocal influence of mammary cancer and fibroblasts on various adhesiveness/invasiveness features. Notably, CAFs' ability to promote EMT, reduction of cell adhesion, increase in membrane fluidity, and migration veloci

A comparison among different advanced extraction techniques such as microwave-assisted extraction (MAE), supercritical fluid extraction (SFE) and pressurized liquid extraction (PLE), together with traditional solid-liquid extraction, was performed to test their efficiency towards the extraction of phenolic compounds from leaves of six Tunisian olive varieties. Extractions were carried out at the best selected conditions for each technique; the obtained extracts were chemically characterized using high-performance liquid chromatography (HPLC) coupled to electrospray time-of-flight mass spectrometry (ESI-TOF-MS) and electrospray ion trap tandem mass spectrometry (ESI-IT-MS 2). As expected, higher extraction yields were obtained for PLE while phenolic profiles were mainly influenced by the solvent used as optimum in the different extraction methods. A larger number of phenolic compounds, mostly of a polar character, were found in the extracts obtained by using MAE. Best extraction yields do not correlate with highest cytotoxic activity against breast cancer cells, indicating that cytotoxicity is highly dependent on the presence of certain compounds in the extracts, although not exclusively on a single compound. Therefore, a multifactorial behavior is proposed for the anticancer activity of olive leaf compounds. © 2012 Elsevier Ltd.

The histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA, vorinostat) is undergoing clinical trials as an antitumor drug and has received regulatory approval for cancer treatment. Here, we show that pre-treatment of human breast cancer cells with SAHA makes them susceptible to apoptosis induced by TRAIL (tumour necrosis factor-related apoptosis-inducing ligand). The apoptosis of breast tumour cells induced by TRAIL is blocked at the level of apical activation of caspase-8 and SAHA enhances the TRAIL-induced processing of procaspase-8. Consequently, a TRAIL associated pathway of apoptosis operated via mitochondria is activated in cells treated with SAHA. Interestingly, degradation of cellular FLICE-inhibitory proteins (cFLIPL and cFLIPS) by an ubiquitin/proteasome-dependent Itch/AIP4-independent mechanism is observed upon exposure to SAHA. Targeting cFLIPL directly with siRNA oligonucleotides also sensitizes human breast tumour cells to TRAIL-induced apoptosis. Furthermore, cFLIPL over-expression significantly inhibits the apoptosis elicited through the combined effects of SAHA and TRAIL. Together, these results indicate that SAHA sensitizes breast cancer cells to TRAIL-induced apoptosis by facilitating the activation of early events in the apoptotic TRAIL pathway. Therefore, the combination of TRAIL and SAHA may represent a therapeutic tool to combat breast tumours.

Binding of a targeting agent in tumor tissue is influenced by many factors such as molecular weight, charge and affinity of the targeting agent and vascularization of the tumor. In this study, we analyzed tumor cell binding of three HER2-specific and radiolabeled Affibody molecules with different affinities. The Affibody molecules had affinities in the range of 0.12-3.8 nM. Cellular binding was analyzed, after 2 h of incubation, in tumor spheroids composed of BT474 breast cancer cells, which highly express HER2. Binding was, due to the binding-site barrier,limited to the outer 15 +/- 5 mu m rim of the spheroids, independent of affinity when the concentration of the substances was low. When the concentration was high, the binding site barrier was overcome and the binding occurred approximately 35 +/- 5 mu m into the spheroids for the two high affinity substances and 50 +/- 5 mu m for the low affinity substance. The lower affinity might allow for penetration into deeper regions due to less firm binding. We conclude that there is a binding site barrier within tumor spheroids which can be overcome by increased concentration of substance and modified by affinity.

The growing role of the reciprocal interaction between epithelial and stromal cells in the development and progression of breast cancer has been recognized. In particular, the migratory/invasive behaviour of tumor cells seems to be strongly influenced by their dialogue with neighbouring stromal cells. To verify if this cross-talk may affect some molecular and functional aspects of the cell biology correlated with the metastasizing vocation of the tumor cells (i.e. adhesion molecule expression, membrane fluidity, migration), we co-cultured estrogen receptor (ER)-positive, poorly invasive and low metastasizing (MCF-7) or ER-negative, highly invasive and metastatic (MDA-MB-231) breast cancer cells with fibroblasts isolated from breast healthy skin (normal fibroblasts, NFs) or from breast tumor stroma (cancer associated fibroblasts, CAFs) in monolayer or in a three-dimensional system (nodules). We previously reported the ability of NFs and CAFs to respectively induce or inhibit the epithelial adhesion molecule, E-cadherin, expression in MCF-7 cells. In the present study, the expression of the mesenchymal adhesion protein N-cadherin (N-cad) was investigated by confocal immunofluorescence microscopy on frozen nodule sections. An increase in N-cad levels was observed in CAFs, but not in NFs, as a result of the interaction with both kinds of epithelial cancer cells. CAFs, in turn, promoted an increase in N-cad level of MDA-MB-231 cells and induced its expression in MCF-7 cells, originally negative for N-cad. Two-photon microscopy imaging of cells labeled with Laurdan, a membrane fluorescent probe, was used to investigate fluidity changes in plasma membranes of all the cell types in monolayer cultures. Tumor cell/fibroblast interaction enhanced fluidity of cancer cell membrane while tumor cells generally promoted an increase in fibroblast membrane packing density. Cell tracking by confocal microscopy demonstrated that the interaction of mammary cancer cells with NFs or CAFs

The autocrine-paracrine effects of IGF2 are important in the growth and differentiation of normal breast. In breast cancer (BC), IGF2 is initially stimulated by estrogen, progesterone and prolactin to regulate proliferation and cancer progression. These actions are mediated by the IGF-1R and insulin receptor A (IR-A) both members of the tyrosine- kinase receptors family. The activation of Estrogen Receptor (ER) is also very important in BC growth and progression. As BC progresses to estrogen-independent growth, the IGF-1R and the estrogen receptor (ER) interact in crosstalk mechanisms that are synergistic and results in enhanced activation of both receptors signaling cascades. This mechanism plays a central role in the transition of estrogen-dependent to estrogen-independent breast cancer (BC) progression. Basal-like BC (BLBC) is a sub-group of estrogen-independent tumors that have a very aggressive clinical behavior and are resistant to hormone-based therapy resulting in reduced disease-free survival period and increasing the mortality of breast cancer (BC) patients. Our BC research team has elucidated how IGF2 crosstalk signaling results in the activation of ER pathways independent of estrogen. Central to our investigation is how this mechanism is associated to the survival disparity observed among African American (AA) BC patients. BLBC accounts for nearly 15-20% of all breast cancers, however it represents 45% of all BC observed in AA patients. Analyses of subcellular compartments, Western-Blot and siRNA demonstrated that IGF2 activates ER-α and ER-β in ER negative BLBC cells Hs578t and CRL-2335. Our studies show that both IGF-1R and IR crosstalk with ER-α and ER-β promoting BLBC progression. This novel mechanism offers new therapeutic targets that will significantly impact treatment and diagnosis of BLBC patients.

The autocrine-paracrine effects of IGF2 are important in the growth and differentiation of normal breast. In breast cancer (BC), IGF2 is initially stimulated by estrogen, progesterone and prolactin to regulate proliferation and cancer progression. These actions are mediated by the IGF-1R and insulin receptor A (IR-A) both members of the tyrosine- kinase receptors family. The activation of Estrogen Receptor (ER) is also very important in BC growth and progression. As BC progresses to estrogen-independent growth, the IGF-1R and the estrogen receptor (ER) interact in crosstalk mechanisms that are synergistic and results in enhanced activation of both receptors signaling cascades. This mechanism plays a central role in the transition of estrogen-dependent to estrogen-independent breast cancer (BC) progression. Basal-like BC (BLBC) is a sub-group of estrogen-independent tumors that have a very aggressive clinical behavior and are resistant to hormone-based therapy resulting in reduced disease-free survival period and increasing the mortality of breast cancer (BC) patients. Our BC research team has elucidated how IGF2 crosstalk signaling results in the activation of ER pathways independent of estrogen. Central to our investigation is how this mechanism is associated to the survival disparity observed among African American (AA) BC patients. BLBC accounts for nearly 15-20% of all breast cancers, however it represents 45% of all BC observed in AA patients. Analyses of subcellular compartments, Western-Blot and siRNA demonstrated that IGF2 activates ER-α and ER-β in ER negative BLBC cells Hs578t and CRL-2335. Our studies show that both IGF-1R and IR crosstalk with ER-α and ER-β promoting BLBC progression. This novel mechanism offers new therapeutic targets that will significantly impact treatment and diagnosis of BLBC patients.

The growing role of the reciprocal interaction between epithelial and stromal cells in the development and progression of breast cancer has been recognized. In particular, the migratory/invasive behaviour of tumor cells seems to be strongly influenced by their dialogue with neighbouring stromal cells. To verify if this cross-talk may affect some molecular and functional aspects of the cell biology correlated with the metastasizing vocation of the tumor cells (i.e. adhesion molecule expression, membrane fluidity, migration), we co-cultured estrogen receptor (ER)-positive, poorly invasive and low metastasizing (MCF-7) or ER-negative, highly invasive and metastatic (MDA-MB-231) breast cancer cells with fibroblasts isolated from breast healthy skin (normal fibroblasts, NFs) or from breast tumor stroma (cancer associated fibroblasts, CAFs) in monolayer or in a three-dimensional system (nodules). We previously reported the ability of NFs and CAFs to respectively induce or inhibit the epithelial adhesion molecule, E-cadherin, expression in MCF-7 cells. In the present study, the expression of the mesenchymal adhesion protein N-cadherin (N-cad) was investigated by confocal immunofluorescence microscopy on frozen nodule sections. An increase in N-cad levels was observed in CAFs, but not in NFs, as a result of the interaction with both kinds of epithelial cancer cells. CAFs, in turn, promoted an increase in N-cad level of MDA-MB-231 cells and induced its expression in MCF-7 cells, originally negative for N-cad. Two-photon microscopy imaging of cells labeled with Laurdan, a membrane fluorescent probe, was used to investigate fluidity changes in plasma membranes of all the cell types in monolayer cultures. Tumor cell/fibroblast interaction enhanced fluidity of cancer cell membrane while tumor cells generally promoted an increase in fibroblast membrane packing density. Cell tracking by confocal microscopy demonstrated that the interaction of mammary cancer cells with NFs or CAFs

The combination of two or more chemopreventive agents is currently being used to achieve greater inhibitory effects on breast cancer cells. Anacardic acid and lunasin are two plant-derived compounds that have been associated with anti-carcinogenic properties. These compounds show inhibitory effects on cell proliferation and inducing properties of apoptosis in human breast cancer MDA-MB-231 cell line. Both lunasin and anacardic acid exert their effects through the modulation of expression of several genes involved in cell cycle, apoptosis and signal transduction. Their combination arrests the cell cycle in S-phase and induces apoptosis at higher levels than that observed when each compound is used individually. This combination also promotes the inhibition of ERBB2, AKT1, JUN and RAF1 signalling gene expression, whose up-regulation has been reported as responsible for breast cancer cells growth and resistance to apoptosis. Our results introduce these two compounds as a promising strategy to prevent/treat breast cancer.