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Fernandez, M.K.;  Sinha, M.;  Renz, M. Differential Intracellular Protein Distribution in Cancer and Cells. Encyclopedia. Available online: https://encyclopedia.pub/entry/29975 (accessed on 15 December 2025).
Fernandez MK,  Sinha M,  Renz M. Differential Intracellular Protein Distribution in Cancer and Cells. Encyclopedia. Available at: https://encyclopedia.pub/entry/29975. Accessed December 15, 2025.
Fernandez, Maria Kristha, Molika Sinha, Malte Renz. "Differential Intracellular Protein Distribution in Cancer and Cells" Encyclopedia, https://encyclopedia.pub/entry/29975 (accessed December 15, 2025).
Fernandez, M.K.,  Sinha, M., & Renz, M. (2022, October 18). Differential Intracellular Protein Distribution in Cancer and Cells. In Encyclopedia. https://encyclopedia.pub/entry/29975
Fernandez, Maria Kristha, et al. "Differential Intracellular Protein Distribution in Cancer and Cells." Encyclopedia. Web. 18 October, 2022.
Differential Intracellular Protein Distribution in Cancer and Cells
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This entry is adapted from the peer-reviewed paper 10.3390/cancers14194788

It is well-established that cancer and normal cells can be differentiated based on the altered sequence and expression of specific proteins. There are only a few examples, showing that cancer and normal cells can be differentiated based on the altered distribution of proteins within intracellular compartments. there are available data on shifts in the intracellular distribution of two proteins, the membrane associated beta-catenin and the actin-binding protein CapG. Both proteins show altered distributions in cancer cells compared to normal cells. These changes are noted (i) in steady state and thus can be visualized by immunohistochemistry—beta-catenin shifts from the plasma membrane to the cell nucleus in cancer cells; and (ii) in the dynamic distribution that can only be revealed using the tools of quantitative live cell microscopy—CapG shuttles faster into the cell nucleus of cancer cells. Both proteins may play a role as prognosticators in gynecologic malignancies: beta-catenin in endometrial cancer and CapG in breast and ovarian cancer. Thus, both proteins may serve as examples of altered intracellular protein distribution in cancer and normal cells.

steady-state and dynamic distribution gynecological cancers quantitative fluorescence microscopy

1. Introduction

Cells are individual compartments that differ in composition from the exterior. These compartments are generally maintained by lipid membrane systems that prevent homogenous mixing of interior and exterior. Furthermore, various intracellular compartments exist that may or may not be enclosed by lipid membranes. These compartments limit the exchange of molecules and permit the buildup of concentration gradients, but also employ mechanisms that allow transport across membranes and thus the exchange between compartments. Thereby, certain molecules distribute differentially between cellular compartments as well as exterior and cell interior. Examples of differential molecular distribution are symmetry-breaking and polarization events during embryonic development. Similarly, shifts in compartmentalization occur during carcinogenesis. Although less studied, these shifts in compartmentalization could be used to differentiate normal and cancer cells.

2. Beta-Catenin

2.1. Beta-Catenin and Cancer

Alterations on any level of the Wnt/beta-catenin signaling pathway can promote cancer development: overexpression of the Wnt ligands, alterations in the Fzd receptor or Lrp co-receptor, mutations in the destruction complex, and mutations in beta-catenin itself. A prime example for alterations in the destructions complex are APC mutations resulting in decreased beta-catenin phosphorylation. Mutations in APC can cause familial adenomatous polyposis (FAP), the main hereditary factor in colorectal cancer. It was noted that the retention and not the complete loss of the first 20 amino acid repeat of APC that can bind beta-catenin and regulate its activity to some extent is favoring colon cancer formation. The unrestricted activity of beta-catenin by the complete loss of inactivating APC, however, results in further increased signaling and risk of cell death. This was postulated as the ‘just-right’ signaling model [1]. In a ‘three-hit’ hypothesis it has been stated that in some colorectal cancers Wnt signaling is modulated by copy number changes or other ‘third-hits’ of APC [2]. The common end pathway of alterations in the canonical Wnt/beta-catenin signaling pathway is that beta-catenin accumulates in the cytoplasm and then shuttles into the cell nucleus.
Aside from its role in colon cancer, beta catenin was described to play a role in many cancers including breast cancer and the breast cancer microenvironment [3][4][5][6], hepatocellular carcinoma [7], and pancreatic cancer [8].

2.2. Change of the Steady-State Distribution of Beta-Catenin in Endometrial Cancer

In 2013, The Cancer Genome Atlas (TCGA) suggested a subclassification of endometrial cancers into four groups based on their molecular profiles [9]. One of the four groups, the microsatellite-stable, low-copy number group showed CTNNB1 mutations in the high frequency of 52%. Most mutations were found in exon 3 of the beta-catenin gene, which is thus a mutational hotspot. A mutation in this gene location results in a missense of the GSK3β consensus site, thus prevents the destruction complex from binding and stabilizes beta-catenin. This stabilization results in the re-distribution of beta-catenin from the plasma membrane to the cell nucleus. Wright et al. found this mutation in 16% of all endometrioid endometrial adenocarcinomas [10]. The redistribution of beta-catenin can be readily detected using immunohistochemistry. Costigan et al. reported a high correlation (p < 0.0001) of exon 3 mutations identified by sequencing with the beta-catenin redistribution into the cell nucleus detected by immunohistochemistry in endometrial cancer. Nuclear beta-catenin immunohistochemistry stain showed a 91% sensitivity and an 89% specificity. The positive and negative predictive value were 86% and 93%, respectively [11]. Thus, IHC can be used as a sufficiently reliable tool to detect exon 3 beta-catenin mutations in endometrial cancer cells. 
Based on clinical data, the prognostic relevance of a beta catenin mutation and redistribution in endometrial cancer is still discussed. However, limited data suggest that a beta-catenin mutation in endometrial cancer increases the risk of recurrence in low-risk and low-stage endometrial cancers including the risk of distant recurrences [11][12]. To establish beta-catenin as negative prognosticator of low-grade, low-stage endometrioid endometrial cancer, further and larger clinical studies are needed.

3. CapG

CapG is a relatively small 39 kDa actin-binding protein of the Gelsolin family [13][14]. In contrast to other Gelsolin-related actin-binding proteins CapG lacks a nuclear export sequence. Thus, it is the only member of its family that distributes diffusively all throughout the cell, i.e., in the cytoplasm and the cell nucleus. The function of CapG in the cytoplasm has been well-established. It binds Ca2-dependent the rapidly growing plus ends of actin filaments, by placing a ‘cap’ on actin filaments (capping like Gelsolin). CapG does not sever [13] or nucleate new actin filaments. The capping stops the linear elongation of actin filament but promotes new filament nucleation by Arp 2/3 and results in the dense dendritic actin meshwork at the leading edge of a cell [15]. Cytoplasmic CapG has been shown in many studies to be involved in the actin-based cell motility and membrane ruffling [16][17]. The function of CapG in the cell nucleus, however, remains unknown. It has been hypothesized that the nuclear fraction of CapG and not only the cytoplasmic fraction promotes cell motility and invasiveness [18][19]. The underlying mechanisms of such hypothesized function of the nuclear CapG fraction are unclear.

3.1. CapG and Cancer

In comparison with beta-catenin, few is known about the role of CapG in cancer. CapG was described within the German Human Genome project to be overexpressed in breast and ovarian cancer [20][21]. It may be particularly upregulated in breast cancer cells that form bone metastases [22][23][24]. There are reports of overexpression of CapG in pancreatic cancer with a reportedly immunohistochemistry nuclear stain that correlated with increasing tumor size [25]. In bladder cancer and its surrounding tumor microenvironment, CapG was found to be overexpressed compared to normal bladder tissue. CapG expression correlated with stage, grade, tumor size and shorter time to recurrence in bladder cancer [26]. Other studies report overexpression of CapG in ocular melanoma [27], clear cell renal cell carcinoma [28], mesothelioma [29], glioblastoma [30][31][32][33], and hepatocellular carcinoma with vascular invasion [34].

3.2. Change in the Dynamic Distribution of CapG in Breast Cancer

As in other cells, CapG distributes diffusively in both the cell nucleus and cytoplasm of the highly invasive breast cancer cell line MDA-MB-231 and the nearly normal breast epithelial cell line MCF-12A. Transfected with CapG-GFP, there is no apparent difference in fluorescence intensity in cytoplasm and nucleus in both cell lines. However, the nucleocytoplasmic shuttling in these two cell lines was found to be different [35]. CapG-GFP shuttles faster into the cancer cell nucleus of the MDA-MB-213 cell than into the cell nucleus of MCF-12A. This difference in dynamic distribution was revealed by Fluorescence after Photobleaching (FRAP) experiments and cannot be detected by merely recording the steady-state distribution of fluorescent molecules. FRAP or fluorescence photobleaching recovery (FPR) examines the ensemble dynamics of molecules and was originally introduced in the mid-1970s to determine the dynamics of plasma membrane proteins [36][37]. The advent of the green fluorescent protein (GFP) and advances in laser scanning microscopes have allowed the technique to be more broadly applicable since the mid-1990s [38]. Using FRAP, fluorescent molecules in a cellular compartment or region of interest are irreversibly bleached with high laser intensity. Thereby, the equilibrium of fluorescent molecules is disturbed, and two distinct populations of (i) irreversibly bleached and (ii) still fluorescent molecules are created. Then, it is monitored over time with low laser intensity how the equilibrium of fluorescent molecules recovers. This fluorescence recovery reveals the underlying ensemble dynamics of a protein of interest. For both cancer and normal cells, the Cap-GFP molecules in the cell nucleus were irreversibly bleached.
The experimentally determined fluorescence intensity over time F(t) can be fit to a single-exponential function as below [38] or, likely more precisely, to a Bessel function [39]. 
In a single-exponential function, a is the fraction of fluorescence intensity initially bleached, b the fraction that recovers over time, and IF the immobile fraction that does not recover on the timescale of the experiment, i.e., the fraction of immobile irreversibly bleached molecules that is not replaced by still fluorescent molecules. The reciprocal of λ is the characteristic recovery time τ, i.e., the time by which two-thirds of the initial fluorescence intensity have been regained. The slope of the curve gives the characteristic recovery time τ. Both parameters, τ and IF, distinguish the CapG-GFP transport kinetics in MDA-MB-231 as compared to MCF-12A [35]. 
In fact, τ and IF, are independently distinguishing parameters of cancer and normal cells. IF remains different, even if the recovery times vary as shown in the analysis of different subgroups [35]. The prognostic and clinical relevance of these proof-of-principle findings is at the time uncertain and will require further studies. The described difference of nucleocytoplasmic CapG shuttling has not been tested in clinical samples.

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Subjects: Biochemistry & Molecular Biology
Contributors MDPI registered users' name will be linked to their SciProfiles pages. To register with us, please refer to https://encyclopedia.pub/register : Maria Kristha Fernandez , Molika Sinha , Malte Renz
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