CHAPTER 6
Contact Normalization: Mechanisms and Pathways to Biomarkers and Chemotherapeutic Targets
JHON ALBERTO OCHOA-ALVAREZ, CANDACY GEORGE, HARINI KRISHNAN, XIAOXUAN WU AND GARY S. GOLDBERG*
Molecular Biology Department, University of Medicine and Dentistry
of New Jersey, Science Center, and Graduate School of Biomedical Sciences, 2 Medical Center Dr., Stratford, NJ 08084, USA
6.1 Introduction
About 12% of American women are likely to be diagnosed with breast cancer within their lifetimes.1 However, studies of healthy women, without any apparent risk for breast cancer, find that nearly half of them harbor genotypically abnormal and possibly premalignant mammary epithelial cells.2 Other studies have identified microadenomas in intestines3 and skin4,5 from significant numbers of healthy humans and animals. Interestingly, most microadenomas do not progress into macroscopic tumors. Histological studies have found that microadenomas surrounded by normal cells retain their transformed genotype, but assume a normal morphology.6–8 This phenomenon, first reported in polyoma transformed cells by Stoker et al. in 1966, is known as contact
RSC Drug Discovery Series No. 10 Extracellular and Intracellular Signaling
Edited by James D. Adams, Jr. and Keith K. Parker r Royal Society of Chemistry 2011
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normalization.9 It is the process by which tumor cell growth is normalized by contact with neighboring non-transformed cells.
6.2 Contact Normalization
Contact normalization is a powerful process. To become malignant and metastatic tumor cells must overcome inhibition by contact normalization. Cells transformed by a variety of chemicals,10 viral agents9 and oncogenes including Src11–15 can be normalized by contact with non-transformed cells. This process is clearly exemplified by malignant tumor cells that form normal adult organs when injected into mouse blastocysts.16 Demonstrations that a
population of tumor cells can be normalized by as little as three times as many non-transformed cells illustrate the power of this process.17,18
Contact normalization is an important process in vivo. Genetically trans-
formed cells can assume a normal morphology and reside in many organs including skin,4,5,19 breast2,20 and intestine.3 Moreover, since these ‘‘occult
tumor’’ cells are phenotypically normal, they tend to resist chemotherapy.21–23 As stated above, contact normalization is a powerful process; transformed keratinocytes that comprise up to 4% of epidermal volume can be controlled in human skin for decades.24
Taken together, a number of studies indicate that contact normalization is
mediated through direct contact between transformed and non-transformed cells.9,15,25–29 Direct contact between cells is mediated by intercellular junctions
such as adherens and gap junctions. Though much remains to be elucidated, progress has been made in identifying mediators involved in this process.
While not essential, gap junctions appear to facilitate the process of contact normalization.7,8,15,29 In addition, a number of genes associated with contact
normalization have been identified.29–33
6.3 Cadherins
Cadherins form intercellular junctions that are required to maintain normal cell architecture. Cadherins are tethered by catenins to the actin cytoskeleton. Cadherin junctions are disrupted by tyrosine phosphorylation of b-catenin. In addition to disturbing cell morphology, disruption of cadherin junctions allows b-catenin to enter the nucleus and participate in mitogenic transcriptional signaling events.34,35
Cadherins are tumor suppressors. Aberrant cadherin expression is often found in tumor cells.36,37 Interestingly, loss of cadherin expression was found
necessary, but not su cient, for tumor cell invasion in genetic screens for metastatic behavior of tumor suppressor genes in a Drosophila model.38 As expected, expression of wild-type cadherins can suppress transformed cell growth,39 while expression of dominant negative cadherins can enhance cell invasion.40
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Epithelial-mesenchymal transition (EMT) is a characteristic |
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in many epithelial malignant tumors. The loss or decrease of E-cadherin expression results in the dedi erentiation, loss of epithelial morphology and increase of cellular motility. In the process of EMT, di erentiated epithelial cells acquire attributes that are similar to embryonic mesenchymal cells.41
Like E-cadherin, N-cadherin is also involved in cell adhesion, di erentiation and invasion.42 As with other ‘‘classic’’ cadherins, protein kinase, exemplified by Src, can phosphorylate b-catenin to break N-cadherin junctions with adjacent cells.43–45 In addition, tumor promoting proteases can disrupt N-cadherin junctions to induce b-catenin nuclear signaling.46 It should be noted that N-cadherin can promote the growth of some epithelial tumor cells.42 However, N-cadherin can suppress the growth of many other tumor cells including osteosarcoma,47 ovarian carcinoma,48 glioblastoma and astrocytoma.49–51
6.4 Gap Junctions
Gap junctions form aqueous channels that connect the cytoplasm of adjacent cells. These channels are formed by integral membrane proteins called connexins.
Connexins have evolved into a family of at least 20 mammalian members, which are commonly named by their predicted molecular weights.52–54
Gap junctions allow adjacent cells to share intracellular signals and function in a coordinated fashion.55–57
Evidence indicates that connexins play an important role in cell growth control.58 Like cadherins, experiments have identified connexins as tumor suppressor genes.59–61 In general, gap junctional communication is blocked between transformed cells.62,63 For example, Cx43 expression is robust in normal glial and mammary epithelial cells, but repressed in some human glioma
and mammary carcinoma cells. Moreover, restoration of Cx43 expression can normalize the growth of human glioma and mammary carcinoma cells.61,64,65
Results from experiments with connexin knockout cells and chemical blockers indicate that gap junctional communication is not required for contact normalization.29 This is consistent with previous reports of contact normalization in the absence of dye transfer between transformed and non-transformed cells.14,66 However, while gap junctions are not absolutely required for contact normalization, many reports suggest that gap junctional communication
augments the ability of normal cells to control the growth of neighboring tumor cells.15,26,64,67,68 This has been demonstrated for cells transformed by a variety of agents.12–15
Cx43 can augment the contact normalization of some transformed cells. We have previously shown that inhibiting gap junctional communication with a Cx43 anti-sense construct curtails the ability of non-transformed cells to normalize Src transformed cells in coculture.15 We have also shown that non-transformed cells normalize gap junctional communication with adjacent Src transformed cells.15,69 Thus, signals passed between transformed and non-transformed cells may help normalize the growth of coupled cells.
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For example, we have shown that Cx43 can e ectively equilibrate levels of the high-energy metabolite ATP throughout a population of cells. In this way, gap junctional communication could prevent an individual tumor cell from acquiring a level of metabolic energy required to undergo mitosis.55,56
Src phosphorylates Connexin43, but may require downstream events to block gap junctional communication. Like Cas and b-catenin, Cx43 is a functionally relevant Src substrate. Src phosphorylates Cx43 on critical tyrosine residues, and this event can reduce intercellular communication.70–73 However, modification of tyrosine residues 247 and 265 to glutamate does not a ect channel function.74 It has become apparent that Src requires other factors to block gap junctional communication mediated by Cx43. For example, MAPK acts downstream of Src to phosphorylate Cx43 and actually close the gap junction channel. In addition, other components may be involved since potential SH3 binding domains on Cx43 are required for channel closure in Src transformed cells.75 Src and the focal adhesion adaptor protein Cas both possess SH3 domains.76 In addition to Src, Cas associates with Cx43, and Src
utilizes Cas to block gap junctional communication between transformed cells.77,78
As described above, junctions formed by Cx43 and cadherins are disrupted by oncogenic protein kinases in transformed cells. In addition, tumor promoters such as TPA also disrupt Cx43 and cadherin junctions.79 Interestingly, cadherins are required for Cx43 assembly and function in some cells. Disruption of junctions formed by N-cadherin can block gap junctional communication.80 Moreover, induction of N-cadherin has been shown to increase Cx43 expression while inhibiting tumor cell growth.81
6.5 Contact Normalization and Tumor Suppressors
Comprehensive analysis of gene expression has found that less than about 0.01% of the transcriptome is a ected by contact normalization. The expression of most of these gene products is inhibited in transformed cells and induced during contact normalization. Some of these genes can act as tumor suppressors.29,30
Fhl1 and Sdpr provide examples of tumor suppressors that are induced by contact normalization. Fhl1 consists of four-and-a-half LIM domains.
Fhl1 can move between intercellular junctions,82 focal adhesions and the nucleus,83 to a ect gene expression.84,85 For example, Fhl1 associates with the
RBP-J DNA binding protein to modulate gene transcription.84,85 Sdpr is a phosphatidylserine-binding protein86 that is induced during growth arrest by serum deprivation of non-transformed cells, but not transformed cells.87 Fhl1 and Sdpr expression is suppressed in some human tumors including those of the breast, kidney and prostate.32,33 Fhl1 is a functionally relevant protein that inhibits anchorage-independent growth and migration of transformed cells.33 Interestingly, in addition to blocking gap junctional communication,77,78 Src utilizes the Cas adaptor protein to suppress Fhl1 expression.33 This relationship