Marina A. Guvakova

Type I insulin-like growth factor receptor function in breast cancer.

Experimental evidence suggests an important role of the type I IGF receptor (IGF-IR) in breast cancer development. Breast tumors and breast cancer cell lines express the IGF-IR. IGF-IR levels are higher in cancer cells than in normal breast tissue or in benign mammary tumors. The ligands of the IGF-IR are potent mitogens promoting monolayer and anchorage-independent growth of breast cancer cells. Interference with IGF-IR activation, expression, or signaling inhibits growth and induces apoptosis in breast cancer cells. In addition, recent studies established the involvement of the IGF-IR in the regulation of breast cancer cell motility and adhesion. We have demonstrated that in MCF-7 cells, overexpression of the IGF-IR promotes E-cadherin-dependent cell aggregation, which is associated with enhanced cell proliferation and prolonged survival in three-dimensional culture.The expression or function of the IGF-IR in breast cancer cells is modulated by different humoral factors, such as estrogen, progesterone, IGF-II, and interleukin-1. The IGF-IR and the estrogen receptor (ER) are usually co-expressed and the two signaling systems are engaged in a complex functional cross-talk controlling cell proliferation.Despite the convincing experimental evidence, the role of the IGF-IR in breast cancer etiology, especially in metastatic progression, is still not clear. The view emerging from cellular and animal studies is that abnormally high levels of IGF-IRs may contribute to the increase of tumor mass and/or aid tumor recurrence, by promoting proliferation, cell survival, and cell-cell interactions. However, in breast cancer, except for the well established correlation with ER status, the associations of the IGF-IR with other prognostic parameters are still insufficiently documented.

Differential roles of IRS-1 and SHC signaling pathways in breast cancer cells

Several polypeptide growth factors stimulate breast cancer growth and may be involved in tumor progression. However, the relative importance of diverse growth factor signaling pathways in the development and maintenance of the neoplastic phenotype is largely unknown. The activation of such growth factor receptors as the insulin‐like growth factor I receptor (IGF‐I R), erbB‐type receptors (erbB Rs) and FGF receptors (FGF Rs) controls the phenotype of a model breast cancer cell line MCF‐7. To evaluate the function of 2 post‐receptor signaling molecules, insulin receptor substrate‐1 (IRS‐1) (a major substrate of the IGF‐IR) and SHC (a common substrate of tyrosine kinase receptors), we developed several MCF‐7‐derived cell clones in which the synthesis of either IRS‐1 or SHC was blocked by antisense RNA. In MCF‐7 cells, down‐regulation of IRS‐1 by 80–85% strongly suppressed anchorage‐dependent and ‐independent growth and induced apoptotic cell death under growth factor‐ and estrogen‐reduced conditions. The reduction of SHC levels by approximately 50% resulted in the inhibition of monolayer and anchorage‐independent growth but did not decrease cell survival. Importantly, cell aggregation and the ability of cells to survive on the extracellular matrix were inhibited in MCF‐7/anti‐SHC clones, but not in MCF‐7/anti‐IRS‐1 clones. Cell motility toward IGF was not attenuated in any of the tested cell lines, but motility toward EGF was decreased in MCF‐7/anti‐SHC clones. Our results suggest that in MCF‐7 cells: 1) both IRS‐1 and SHC are implicated in the control of monolayer and anchorage‐independent growth; 2) IRS‐1 is critical to support cell survival; 3) SHC is involved in EGF‐dependent motility; and 4) normal levels of SHC, but not IRS‐1, are necessary for the formation and maintenance of cell‐cell interactions. Int. J. Cancer 72:828–834, 1997.

Insulin receptor substrate 1 is a target for the pure antiestrogen ICI 182,780 in breast cancer cells.

The pure antiestrogen ICI 182,780 inhibits insulin‐like growth factor (IGF)‐dependent proliferation in hormone‐responsive breast cancer cells. However, the interactions of ICI 182,780 with IGF‐I receptor (IGF‐IR) intracellular signaling have not been characterized. Here, we studied the effects of ICI 182,780 on IGF‐IR signal transduction in MCF‐7 breast cancer cells and in MCF‐7‐derived clones overexpressing either the IGF‐IR or its 2 major substrates, insulin receptor substrate 1 (IRS‐1) or src/collagen homology proteins (SHC). ICI 182,780 blocked the basal and IGF‐I‐induced growth in all studied cells in a dose‐dependent manner; however, the clones with the greatest IRS‐1 overexpression were clearly least sensitive to the drug. Pursuing ICI 182,780 interaction with IRS‐1, we found that the antiestrogen reduced IRS‐1 expression and tyrosine phosphorylation in several cell lines in the presence or absence of IGF‐I. Moreover, in IRS‐1‐overexpressing cells, ICI 182,780 decreased IRS‐1/p85 and IRS‐1/GRB2 binding. The effects of ICI 182,780 on IGF‐IR protein expression were not significant; however, the drug suppressed IGF‐I‐induced (but not basal) IGF‐IR tyrosine phosphorylation. The expression and tyrosine phosphorylation of SHC as well as SHC/GRB binding were not influenced by ICI 182,780. In summary, downregulation of IRS‐1 may represent one of the mechanisms by which ICI 182,780 inhibits the growth of breast cancer cells. Thus, overexpression of IRS‐1 in breast tumors could contribute to the development of antiestrogen resistance. Int. J. Cancer 81:299–304, 1999.

Functional role of alpha-actinin, PI 3-kinase and MEK1/2 in insulin-like growth factor I receptor kinase regulated motility of human breast carcinoma cells

Within epithelial tissue, cells are held together by specialized lateral junctions. At particular stages of development and in pathological processes such as metastasis, cells break down the intercellular junctions, separate from the epithelial sheet and migrate individually. Despite the importance of these processes, little is understood about the regulatory mechanisms of active cell separation. In view of the effects of insulin-like growth factor I (IGF-I) on mammary gland development and cancer, we developed a model using MCF-7 human breast cancer cells in which the process of cell separation can be induced by IGF-I. The separation was enhanced in MCF-7 cells overexpressing the IGF-IR and blocked in the cells expressing a dead-kinase mutant of this receptor. Activation of the IGF-IR resulted in a rapid formation of motile actin microspikes at the regions of cell-cell contacts, disorganization of mature adherens junctions and the onset of cell migration. In cell separation, the signaling between the IGF-IR kinase and actin required phosphatidylinositol 3 (PI 3)-kinase-generated phospholipids but not MAP kinases and was mediated by α-actinin. The activity of MEK1/2 kinases was needed for consecutive cell migration. This work also defined a new function for α-actinin. Upon IGF-IR activation, green fluorescence protein (GFP)-labeled α-actinin concentrated at the base of actin microspikes. Deletion of the N-terminal actin-binding domain ofα -actinin prevented this redistribution, indicating that this domain is necessary. Delection of the C-terminal tail of α-actinin reduced the number of microspikes, showing that α-actinin has a role in the development of microspikes and is not passively reorganized with filamentous actin. We suggest that the signaling pathway from the IGF-IR kinase through the PI-3 kinase to α-actinin participates in the rapid organization of actin into microspikes at the cell-cell junctions and leads to active cell separation, whereas signaling through ERK1/2 MAP kinases controls cell migration following cell separation.

Induction of fascin spikes in breast cancer cells by activation of the insulin-like growth factor-I receptor.

Insulin-like growth factor-I receptor (IGF-IR) signaling contributes to the formation of mammary carcinomas and has chiefly been studied with regard to the proliferative and anti-apoptotic effects of IGF-IR signaling. However, IGF-IR activation also affects the actin cytoskeleton and alterations in cell migratory behavior are of known importance for the malignant conversion and metastasis of epithelial cells. The actin-binding protein fascin is found in cell projections and spikes that are involved in the locomotion of mesenchymal cells. Fascin expression is typically low in normal epithelial cells, but is markedly upregulated in several types of carcinomas. Here, we also demonstrate increased fascin expression in breast carcinoma cell lines and adopt MCF-7 human mammary carcinoma cells that over-express wild-type or kinase-inactivated forms of the IGF-IR as a model system to test the hypothesis that IGF-IR activation induces fascin projections. We show that the time-dependent dissociation of cell colonies that occurs upon receptor activation by IGF-I involves the formation of dynamic, fascin-containing lateral cell projections that co-localize with ruffling membranes in association with protrusive activity and cell migratory phenotype. The molecular mechanism of these effects is completely dependent on IGF-IR tyrosine kinase activity and is mediated by a phosphatidylinositol (PI) 3-kinase-dependent process. In demonstrating transduction of fascin spike assembly by activation of a peptide growth factor receptor, these novel data reveal a wide role for fascin spikes in cell motility and provide new insight into the complex effects of IGF-IR signaling on actin cytoskeletal organization.

Rap2A Is Upregulated in Invasive Cells Dissected from Follicular Thyroid Cancer.

The development of molecular biomarkers (BMs) of follicular thyroid carcinoma is aimed at advancing diagnosis of follicular neoplasm, as histological examination of those tumors does not lend itself to definitive diagnosis of carcinoma. We assessed the relative levels of expression of 6 genes: CCND2, PCSK2, PLAB, RAP2A, TSHR, and IGF-1R in archived thyroid tissue. The quantitative real-time PCR analysis revealed a significant change in 3 genes: PSCK2 (a 22.4-fold decrease, P = 2.81E − 2), PLAB (an 8.3-fold increase, P = 9.81E − 12), and RAP2A (a 6.3-fold increase, P = 9.13E − 10) in carcinoma compared with adenoma. Expression of PCSK2 was equally low, PLAB was equally high, whereas RAP2A expression was significantly higher (25.9-fold, P = 0.039) in microdissected carcinoma cells that have invaded through the thyroid capsule and entered blood vessels than in thyroid tumor cells growing under the capsule. Thus, RAP2A appeared as a unique and worthy of further evaluation candidate BM associated with invasion of thyroid follicular cells.

Vav2 protein overexpression marks and may predict the aggressive subtype of ductal carcinoma in situ.

BackgroundA subset of patients with ductal carcinoma in situ (DCIS) will develop invasive breast cancer (IBC). To date, there are no effective predictive biomarkers for identifying this subset with worse prognosis whose lesions are essentially indistinguishable histologically from those with favorable outcomes. We hypothesized that measurable parameters that discriminate DCIS from DCIS with concurrent invasion may serve as diagnostic biomarkers (BM) of progressive cancer in situ (CIS).ResultsUsing a novel imaging-based method of tissue testing, we measured the relative expression levels of three candidate BM proteins specifically implicated in IBC progression - the insulin-like growth factor I receptor (IGF-IR), Ras-related protein 1 (Rap1), and Vav2 oncoprotein. Protein profiles were compared in 42 histologically normal mammary epithelial samples, 71 CIS (35 without/36 with invasion either on diagnostic biopsy or final surgical excision), and 98 IBC of known estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2) status. The levels of the IGF-IR and Rap1 protein expression were significantly elevated in ER-positive (ER+/PR+/-/HER2 –) DCIS relative to normal epithelium (P <0.0001). The IGF-IR protein expression was also significantly up regulated in HER2-positive (ER+/-/PR+/-/HER2+) DCIS relative to normal epithelium (P = 0.0002). IGF-IR and Rap1 protein expression levels were similar among DCIS patients without or with concurrent invasion. Vav2 upregulation in DCIS relative to normal group was not associated with steroid hormone receptor and HER2 status, but was associated with the presence of concurrent invasion, including microinvasion (invasive foci of less than 1 mm). DCIS with high Vav2 were more than twice as likely to progress to invasive cancers as DCIS with low Vav2 (odds ratio, 2.42; 95% CI, 1.26-4-65; P =0.008). Furthermore, a receiver operating characteristic curve analysis revealed moderate ability of Vav2 protein expression measurements in DCIS to predict the existence of invasion concurrent with DCIS (area under the curve, 0.71; 95% CI, 0.59- 0.84).ConclusionsOur novel findings hold promise for utilizing Vav2 protein as a predictive BM for differentiating progressive from non-progressive DCIS.

Abstract B24: The diagnostic power of direct mRNA profiling in breast cancer core biopsy

Background: Worldwide, biopsy is the gold standard for pathological assessment of cancer. Routinely, immunohistochemistry (IHC) can be performed to evaluate various tumor markers on serial sections of formalin-fixed paraffin-embedded (FFPE) tissue. Comprehensive molecular analysis of biopsy is limited by the microscopic size of samples and the scarcity of quantitative tools for FFPE tissue examination. Development of the new tools for molecular analysis of biopsy is needed to stratify, as early as possible, a patient’s individual risk for tumor aggressiveness and sensitivity to therapy to avoid potential over- and under treatment. Objective: To assess the feasibility of direct mRNA expression profiling of all three classic clinical markers of cancer progression - estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2) in routinely collected FFPE core needle biopsy (CNB) of the breast. Methods: We obtained tissue from the Department of Pathology and Laboratory Medicine at the University of Pennsylvania, the University of Pennsylvania Tumor Tissue and Biospecimen Bank and the Cooperative Human Tissue Network. Steroid hormone receptor and HER2 status, determined by FDA-approved methods, was obtained from the surgical pathology reports. For direct (i.e. without amplification) mRNA profiling, we ran the Panomics/Affymetrix QuantiGene Plex 2.0 assay on Luminex according to manufacturer’s protocol. After the removal of low-abundance housekeeping genes expressing samples, the relative activity of target genes, ERS1, PGR, ERBB2 encoding ER alpha, PR and HER2 was detected. mRNA profiling was completed in 107 breast samples: 6 clinically normal (N) cases from reduction mammoplasty, 17 ductal carcinoma in situ ( DCIS) on CNB , 42 invasive ductal carcinoma (IDC) and 42 non-malignant matched tissue from surgical excisions. Groups were compared using one-way analysis of variance (ANOVA). Results: A pair-wise comparison among tissue groups revealed 3-fold higher expression levels of ERS1 in ER+ DCIS on CNB (P=. 055), and statistically significantly higher expression levels of ERS1 in the ER+/PR+ (P= .021) and ER+/PR- (P= .004) subgroups of IDC than in the N group. Remarkably, compared with N tissue, ERS1 levels were already decreased in non-malignant tissue adjacent to ER-/PR-/HER2- subgroup of IDC and were further significantly decreased in ER-/PR-/HER2- IDC itself (P= .023). Compared to the N group, significant decreases in PGR levels were found only in ER-/PR-/HER2- subgroup of IDC (P= .003), whereas significant increases in ERBB2 levels were found in both HER2+ DCIS on CNB (P= . 037) and HER2+IDC in excisions (P= .005). Conclusions: Our study, for the first time, proved the feasibility of using FFPE CNB as an RNA source for direct mRNA expression profiling. These findings support the notion that IHC evaluation of ER, PR, and HER2 status in the CNB DCIS may be complemented by concurrent detection of ESR1/PGR/ERBB2 mRNA levels. Citation Format: Indira Prabakaran, Paul J. Zhang, Marina Guvakova. The diagnostic power of direct mRNA profiling in breast cancer core biopsy. [abstract]. In: Proceedings of the Fourth AACR International Conference on Frontiers in Basic Cancer Research; 2015 Oct 23-26; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2016;76(3 Suppl):Abstract nr B24.

Abstract 5283: Vav2 oncoprotein up regulation may predict the aggressive subtype of ductal carcinoma in situ

Background: A subset of patients with ductal carcinoma in situ (DCIS) will develop invasive breast cancer (IBC). To date, there are no effective predictive biomarkers for identifying this subset with worse prognosis whose lesions are essentially indistinguishable histologically from those with favorable outcomes. We hypothesized that measurable parameters that discriminate DCIS from DCIS with concurrent invasion may serve as diagnostic biomarkers (BM) of progressive cancer in situ (CIS). Methods: Using a novel imaging-based method of tissue testing, we measured the relative expression levels of three candidate BM proteins specifically implicated in IBC progression - the insulin-like growth factor I receptor (IGF-IR), Ras-related protein 1 (Rap1), and Vav2 oncoprotein. Protein profiles were compared in 42 histologically normal mammary epithelial samples, 71 CIS (35 without/36 with invasion either on diagnostic biopsy or final surgical excision), and 98 IBC of known estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2) status. Groups were compared using one-way analysis of variance (ANOVA) followed by pair-wise t-tests with Tukey9s correction for multiple testing. Receiver operating characteristic (ROC) curves were constructed to evaluate how well each BM can predict tumor type. Results: The levels of the IGF-IR and Rap1 protein expression were significantly elevated in ER-positive (ER+/PR+/-/HER2 -) DCIS relative to normal epithelium (P Conclusions: This study describes the use of a novel imaging method for archival tissue testing, which may inform the status of protein BM in archived tissue and may help to stratify a women9s individual risk for tumor invasiveness to avoid potential over- or under-treatment. Despite the apparent limitation of our study DCIS cohort size, our novel findings hold promise for utilizing Vav2 as a predictive BM of progressive DCIS and a target for cancer therapy. Citation Format: Marina A. Guvakova, Yun Qing Jiang, Indira Prabakaran, Fei Wan, Nandita Mitra, Paul J. Zhang, Douglas L. Fraker. Vav2 oncoprotein up regulation may predict the aggressive subtype of ductal carcinoma in situ. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5283. doi:10.1158/1538-7445.AM2015-5283

Abstract B40: The quest for biomarkers of progressive DCIS using archived tissue.

Background: Presently, an indolent ductal carcinoma in situ (DCIS) cannot be distinguished from a progressive tumor, making the appropriate treatment of DCIS patients a major clinical dilemma. Here, we tested if measuring protein levels in archived tissue could facilitate the development of biomarkers (BMs) accurately discriminating progressive from non-invasive DCIS. We theorized that BMs of progressive lesion are measurable parameters that distinguish pure DCIS from DCIS with concurrent invasive breast cancer (IBC). To test the hypothesis, we studied the insulin-like growth factor I receptor (IGF-IR), Ras oncogene-like protein 1 (Rap1), and oncoprotein Vav2 implicated in the progression of IBC. Methods: We developed and applied a new quantitative imaging-based uniplex (IBU) method to measure in-tissue protein expression. The quantification was performed on a continuous scale, and multiple repeated measurements of relative (rather than absolute) intensity of immunohistochemical staining took into account fluctuations of background. Protein profiling was completed in 211 breast samples: 42 histologically normal, 71 cancer in situ (with/without associated invasion), and 98 IBC. Groups were compared using one-way analysis of variance (ANOVA) followed by pair-wise t-tests with Tukey9s correction for multiple testing. Receiver operating characteristic (ROC) curves were constructed to evaluate how well each BM can predict tumor type. Results: A pair-wise comparison among tissue groups revealed statistically significantly higher levels of IGF-IR and Rap1 in the in situ and IBC groups than in the normal group (P Conclusions: The application of our IBU method identified previously undetected changes in IGF-IR, Rap1, and Vav2 that co-evolve with IBC progression. Vav2 may be a valuable BM for the diagnosis of progressive DCIS. Improvement in earlier detection of DCIS with aggressive phenotype would go a long way in advancing efforts towards prevention of IBC. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B40. Citation Format: Marina A. Guvakova, YunQing Jiang, Indira Prabakaran, Fei Wan, Nandita Mitra, Douglas L. Fraker, Paul J. Zhang. The quest for biomarkers of progressive DCIS using archived tissue. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B40.