Proteomic Characterization of Ovarian and Breast Cancer Microenvironments for Improved Diagnostics and Therapeutic Targeting

Cancers exist within complex microenvironments formed by heterogeneous cell types. This diversity creates significant challenges for detection, diagnosis and treatment. Mass spectrometry-based proteomics is a powerful approach capable of characterizing complex biological systems which are characteristic of cancer biology. In this thesis, proteomics was utilized to answer several questions related to ovarian cancer diagnosis and detection, and the effects of NODAL, an embryonic morphogen, on the breast cancer secretome and stromal cell recruitment. First, I compared multiple sample preparation techniques and found high-pH/low-pH fractionation to yield the greatest proteome coverage over commonly used approaches. Second, I compared the proteomes from two ovarian cancer subtypes (high-grade serous and endometrioid) for which histological discrimination remains difficult in a proportion of cases. I documented several unknown proteins, including KIAA1324, which were validated and confirmed to improve the differential diagnosis of endometrial ovarian cancer. Third, I extensively characterized extracellular vesicle proteomes from biological fluids (conditioned media, plasma and ascites) to catalogue potential biomarkers associated with malignant ovarian cancer. I detected many factors associated with advanced stage, high-grade serous ovarian cancer including CFHR4, MUC1, APCS and PZP that may be useful for early detection. Last, I characterized the global effects of the Transforming Growth Factor-β superfamily member NODAL on the breast cancer secretome and stromal cell recruitment in vitro. I found a previously unknown role for NODAL in modulating pro-inflammatory factors, including CXCL1 and IL6 that were correlated with multipotent stromal cell recruitment. In summary, this work represents a significant contribution to the histological assessment and detection of ovarian cancer and our understanding of the malignant properties of NODAL within the breast cancer microenvironment

Embryonic Morphogen Nodal Promotes Breast Cancer Growth and Progression

Breast cancers expressing human embryonic stem cell (hESC)-associated genes are more likely to progress than well-differentiated cancers and are thus associated with poor patient prognosis. Elevated proliferation and evasion of growth control are similarly associated with disease progression, and are classical hallmarks of cancer. In the current study we demonstrate that the hESC-associated factor Nodal promotes breast cancer growth. Specifically, we show that Nodal is elevated in aggressive MDA-MB-231, MDA-MB-468 and Hs578t human breast cancer cell lines, compared to poorly aggressive MCF-7 and T47D breast cancer cell lines. Nodal knockdown in aggressive breast cancer cells via shRNA reduces tumour incidence and significantly blunts tumour growth at primary sites. In vitro, using Trypan Blue exclusion assays, Western blot analysis of phosphorylated histone H3 and cleaved caspase-9, and real time RT-PCR analysis of BAX and BCL2 gene expression, we demonstrate that Nodal promotes expansion of breast cancer cells, likely via a combinatorial mechanism involving increased proliferation and decreased apopotosis. In an experimental model of metastasis using beta-glucuronidase (GUSB)-deficient NOD/SCID/mucopolysaccharidosis type VII (MPSVII) mice, we show that although Nodal is not required for the formation of small (\u3c100 cells) micrometastases at secondary sites, it supports an elevated proliferation:apoptosis ratio (Ki67:TUNEL) in micrometastatic lesions. Indeed, at longer time points (8 weeks), we determined that Nodal is necessary for the subsequent development of macrometastatic lesions. Our findings demonstrate that Nodal supports tumour growth at primary and secondary sites by increasing the ratio of proliferation:apoptosis in breast cancer cells. As Nodal expression is relatively limited to embryonic systems and cancer, this study establishes Nodal as a potential tumour-specific target for the treatment of breast cancer. © 2012 Quail et al

Embryonic protein NODAL regulates the breast tumor microenvironment by reprogramming cancer-derived secretomes

The tumor microenvironment (TME) is an important mediator of breast cancer progression. Cancer-associated fibroblasts constitute a major component of the TME and may originate from tissue-associated fibroblasts or infiltrating mesenchymal stromal cells (MSCs). The mechanisms by which cancer cells activate fibroblasts and recruit MSCs to the TME are largely unknown, but likely include deposition of a pro-tumorigenic secretome. The secreted embryonic protein NODAL is clinically associated with breast cancer stage and promotes tumor growth, metastasis, and vascularization. Herein, we show that NODAL expression correlates with the presence of activated fibroblasts in human triple-negative breast cancers and that it directly induces Cancer-associated fibroblasts phenotypes. We further show that NODAL reprograms cancer cell secretomes by simultaneously altering levels of chemokines (e.g., CXCL1), cytokines (e.g., IL-6) and growth factors (e.g., PDGFRA), leading to alterations in MSC chemotaxis. We therefore demonstrate a hitherto unappreciated mechanism underlying the dynamic regulation of the TME

Embryonic Morphogen Nodal Promotes Breast Cancer Growth and Progression

<div><p>Breast cancers expressing human embryonic stem cell (hESC)-associated genes are more likely to progress than well-differentiated cancers and are thus associated with poor patient prognosis. Elevated proliferation and evasion of growth control are similarly associated with disease progression, and are classical hallmarks of cancer. In the current study we demonstrate that the hESC-associated factor Nodal promotes breast cancer growth. Specifically, we show that Nodal is elevated in aggressive MDA-MB-231, MDA-MB-468 and Hs578t human breast cancer cell lines, compared to poorly aggressive MCF-7 and T47D breast cancer cell lines. Nodal knockdown in aggressive breast cancer cells via shRNA reduces tumour incidence and significantly blunts tumour growth at primary sites. <em>In vitro</em>, using Trypan Blue exclusion assays, Western blot analysis of phosphorylated histone H3 and cleaved caspase-9, and real time RT-PCR analysis of <em>BAX</em> and <em>BCL2</em> gene expression, we demonstrate that Nodal promotes expansion of breast cancer cells, likely via a combinatorial mechanism involving increased proliferation and decreased apopotosis. In an experimental model of metastasis using beta-glucuronidase (GUSB)-deficient NOD/SCID/mucopolysaccharidosis type VII (MPSVII) mice, we show that although Nodal is not required for the formation of small (<100 cells) micrometastases at secondary sites, it supports an elevated proliferation:apoptosis ratio (Ki67:TUNEL) in micrometastatic lesions. Indeed, at longer time points (8 weeks), we determined that Nodal is necessary for the subsequent development of macrometastatic lesions. Our findings demonstrate that Nodal supports tumour growth at primary and secondary sites by increasing the ratio of proliferation:apoptosis in breast cancer cells. As Nodal expression is relatively limited to embryonic systems and cancer, this study establishes Nodal as a potential tumour-specific target for the treatment of breast cancer.</p> </div

Nodal inhibition alters proliferation:apoptosis ratios in micrometastases.

<p>(A) GUSB staining of pulmonary micrometastases from MDA-MB-231 cells transfected with a Control shRNA (231+shControl) or a shRNA to Nodal (231+shNodal) in NOD/SCID/MPSVII mice 4 weeks post-intravenous injection (red and outlined with white dotted line). (B) Scatter plot representing the average number of micrometastases (<100 cells) per section of lung from NOD/SCID/MPSVII mice 4 weeks after injection with 231+shControl or 231+shNodal cells. The number of 231+shNodal micrometastases that formed after 4 weeks in NOD/SCID/MPSVII mice was not significantly reduced compared to the number of 231+shControl micrometastases (n≥5, p>0.05). Each point represents the average mean number of micrometastases per section per mouse. Black bars represent the median number of micrometastases per section per mouse. (C) Immunohistochemical analysis of Ki67 expression (brown) and TUNEL (brown) staining in pulmonary micrometastases from 231+shControl cells or 231+shNodal cells in NOD/SCID/MPSVII mice 4 weeks post-intravenous injection. Proliferation is indicated by Ki67 staining and apoptotic nuclei were detected with TUNEL. (D) Proliferation:apoptosis ratios in 4 week micrometastases were determined with immunohistochemical localization of Ki67 and TUNEL. At 4 weeks, lesions from 231+shControl cells had a positive proliferation ratio (1.57) whereas lesions from 231+shNodal cells had a negative proliferation:apoptosis ratio (0.74) (n≥3, p<0.05). Values represent mean average proliferation:apoptosis ratio in tumour lesions per mouse ± S.E.M.</p

Nodal knockdown decreases proliferation and increases apoptosis in aggressive MDA-MB-231 breast cancer cells.

<p>(A) Trypan Blue exclusion was used to count live cells daily to generate growth curves over 3 days, in response to altered Nodal expression. MDA-MB-231 cells transfected with a Nodal-targeted shRNA (231+shNodal) exhibited a significant decrease in proliferation over 3 days compared to cells transfected with a scrambled Control shRNA (231+shControl) (n = 3; p = 0.047). (B) Representative histogram of mean fluorescence intensity (mfi) in 231+shNodal and 231+shControl cells labelled with Cell Trace Violet (CTV) for 4 days after synchronization via serum starvation. There was a greater loss of CTV in 231+shControl compared to 231+shNodal cells (4801 and 6006, respectively) indicating that control cells proliferated more than their shNodal-treated counterparts. (C) Western blots demonstrating decreased phosphorylated histone H3 at 4 different sites, including Thr11, Ser10, Ser28 and Thr3 in 231+shNodal cells compared to 231+shContol cells. Total histone H3 and β-Actin are used as controls. (D) Representative images of TUNEL staining and corresponding quantification of percent TUNEL-positive cells in 231+shNodal and 231+shControl cells grown <i>in vitro</i>. TUNEL positive cells are delineated by arrows and nuclei are counterstained blue. Micron bars equal 25 µm. The percentage of TUNEL positive cells is significantly higher in 231+shNodal cells as compared to 231+shControl cells (n = 23; p = 0.001). (E) Western blot demonstrating that cleavage of caspase-9 is elevated in 231+shNodal compared to 231+shControl cells. Uncleaved caspase-9 and β-Actin are used as controls. (F) Real time RT-PCR analysis demonstrating that <i>BAX</i> mRNA expression is significantly higher in 231+shNodal cells compared to 231+shControl cells (n = 4, p = 0.029). (G) Real time RT-PCR analysis demonstrating that <i>BCL2</i> mRNA expression is significantly lower in 231+shNodal cells compared to 231+shControl cells (n = 4, p = 0.029). All bar graphs are presented as mean ± S.E.M. for replicate values. Asterisks indicate a significant difference as specified compared to controls.</p

Nodal over-expression increases proliferation and decreases apoptosis in poorly aggressive T47D breast cancer cells.

<p>(A) Western blot demonstrating that Nodal protein is elevated in T47D cells transfected with a Nodal expression construct (T47D+Nodal) compared to empty vector controls (T47D+EV). The ∼39 kDa Pro-Nodal band is presented and Actin is used as a loading control. Trypan Blue exclusion was used to count live cells daily to generate growth curves over 3 days, in response to altered Nodal expression. T47D+Nodal cells exhibited a significant increase in proliferation compared to T47D+EV cells over 3 days (n = 3, p = 0.046). (B) Representative histogram of mfi in T47D+EV and T47D+Nodal cells labelled with CTV for 6 days after synchronization via serum starvation. There was a greater loss of CTV in T47D+Nodal compared to T47D+EV cells (2738 and 3319, respectively) indicating that proliferation increased with Nodal over-expression. (C) Western blots demonstrating increased phosphorylated histone H3 at 4 different sites, including Thr11, Ser10, Ser28 and Thr3 in T47D+Nodal cells compared to T47D+EV cells. Total histone H3 and β-Actin are used as controls. (D) Representative images of TUNEL staining and corresponding quantification of percent TUNEL-positive cells in T47D+EV and T47D+Nodal cells grown <i>in vitro</i>. TUNEL positive cells are delineated by arrows and nuclei are counterstained blue. Micron bars equal 25 µm. The percentage of TUNEL positive cells is significantly higher in T47D+EV cells as compared to T47D+Nodal cells (n = 8; p = 0.006). (E) Western blot demonstrating that cleavage of caspase-9 is reduced in T47D+Nodal cells compared to T47D+EV cells. Uncleaved caspase-9 and β-Actin are used as controls. (F) Real time RT-PCR analysis demonstrating that <i>BAX</i> mRNA expression is significantly lower in T47D+Nodal cells compared to T47D+EV cells (n = 5, p = 0.016). (G) Real time RT-PCR analysis demonstrating that <i>BCL2</i> mRNA expression is significantly higher in T47D+Nodal cells compared to T47D+EV cells (n = 5, p = 0.016). All data are presented as mean ± S.E.M. for replicate values. Asterisks indicate a significant difference compared to controls.</p

The effects of Nodal on proliferation and apoptosis are dependent on activation of the type 1 receptor (ALK4/7).

<p>(A) Western blot analysis demonstrating that phosphorylation of SMAD2 decreases with SB431542 treatment (1–10 µM) in MDA-MB-231 cells. SMAD2/3 and Actin are used as loading controls. (B) Immunofluorescence localization of SMAD2/3 (red) demonstrating that in MDA-MB-231 cells and in MDA-MB-468 cells, SMAD2/3 localization to the nucleus is reduced upon treatment with SB431542 (10 µM). DAPI (blue) is used to stain nuclei and bar equals 10 µm. (C) Growth curves for MDA-MB-231 cells or (D) MDA-MB-468 cells over 4 days demonstrating that inhibition of the Nodal type 1 receptor (ALK4/7) with SB431542 (10 µM) causes reduced growth over time, and this is not rescued with addition of rhNodal (100 ng/mL). Time points indicated by an asterisk (*) demonstrated a significant difference between controls and all other treatments. (E) PCR analysis demonstrating that treatment of MDA-MB-231 cells with 10 µM SB431542 causes a significant increase in <i>BAX</i> expression (n = 3, p = 0.002), and (F) a significant decrease in <i>BCL2</i> expression (n = 3, p = 0.022), indicative of elevated apoptosis compared to vehicle controls. Recombinant human Nodal (100 ng/mL) was used in combination with SB431542, and no rescue in either <i>BAX</i> or <i>BCL2</i> expression was observed. (G) PCR analysis demonstrating that treatment of MDA-MB-468 cells with 10 µM SB431542 causes a significant increase in <i>BAX</i> expression (n = 3, p = 0.020), and (H) a significant decrease in <i>BCL2</i> expression (n = 3, p = 0.046) compared to vehicle controls. Recombinant human Nodal (100 ng/mL) was used in combination with SB431542, and no rescue in either <i>BAX</i> or <i>BCL2</i> expression was observed. Data are presented as mean ± S.E.M. for replicate values. All experiments were repeated ≥3 times. Values indicated by an asterisk (*) are significantly different from controls.</p

Nodal knockdown reduces breast cancer tumour growth <i>in vivo</i>.

<p>(A) Western blot confirming Nodal knockdown by shRNA in MDA-MB-468 cells. The ∼39 kDa Pro-Nodal band is presented and Actin is used as a loading control. (B) 2.5 million MDA-MB-468 cells transfected with Control shRNA (468+shControl) or a Nodal-targeted shRNA (468+shNodal) were injected with Matrigel into the flanks of nude mice, and tumour diameter was measured over the course of 6 weeks. 468+shControl cells formed significantly larger tumours compared to 468+shNodal cells (n = 8, p<0.05). Values represent mean tumour diameter (mm) ± standard error of the mean (S.E.M.). (C) Tumour volume of MDA-MB-468-derived tumours excised after 6 weeks. Bars represent mean tumour volume (mm³) ± S.E.M. (D) Western blot confirming Nodal knockdown by shRNA in MDA-MB-231 cells. The ∼39 kDa Pro-Nodal band is presented and Actin is used as a loading control. (E) 0.5 million MDA-MB-231 cells transfected with Control shRNA (231+shControl) or Nodal-targeted shRNA (231+shNodal) were orthotopically injected through the nipple into the mammary fat pads of nude mice, and tumour diameter was measured over the course of 9 weeks. 231+shControl cells formed significantly larger tumours compared to 231+shNodal cells (n = 10, p<0.05). Values represent mean tumour diameter (mm) ± S.E.M. (F) Tumour volume of MDA-MB-231-derived tumours excised after 9 weeks. Bars represent mean tumour volume (mm³) ± S.E.M.</p