Tetraspanins regulate tumor progression and metastasis. But the mechanisms remain largely unknown. At the cellular level, tetraspanins modulate cell adhesion, migration, proliferation, and fusion. The adhesiveness and motility of tumor cells partially determine tumor metastatic potential. Hence, at the cellular level, tetraspanins probably regulate tumor metastasis by modulating the abilities of tumor cells to adhere and move. At the molecular level, tetraspanins associate with integrins, IgSF proteins, growth factors and their receptors, proteases, and intracellular signaling proteins to form TEM 
. Hence, at the molecular level, tetraspanins regulate tumor progression and metastasis probably by altering the functions of the associated proteins 
The expression of tetraspanin CO-029 is typically linked to poor prognosis of digestive system cancers 
, CO-029 is upregulated upon the progression of colorectal, liver, pancreatic, and esophageal cancers 
, and the increased expression of CO-029 promotes the liver or lung metastasis of these cancers 
. Tumor cell migration and invasion are indispensable for metastasis. The movement of tumor cells is involved in at least two phases in the metastasis cascade 
. The first phase is that tumor cells migrate away from the primary tumor and invade the circulatory system; the second phase is that tumor cells migrate out of blood vessels and into target tissues. The reduced cell movement upon CO-029 silencing indicates that CO-029 is required for the efficient migration and invasion of colorectal cancer cells and suggests that CO-029 likely promotes both stages of tumor metastasis.
CO-029 appears to promote cell movement by altering cell-matrix and -cell adhesions. It is well established that cell-cell and -matrix adhesion directly determines cell motility. For example, the loss or reduction of E-cadherin expression and/or activity in tumor cells leads to the dispatch of tumor cells from primary tumor mass 
. The increased total and Ca++
-dependent cell-cell adhesion upon CO-029 silencing is consistent with the reduced motility of HT29-KD cells and also strongly suggests that CO-029 promotes cell movement by reducing cell-cell adhesion. The enhanced cell-cell adhesion, caused by CO-029 silencing, likely results from the increased activity of cadherins and/or other calcium-dependent cell-cell adhesion molecules. E-cadherin, however, appears to be unaltered because the cell surface level of E-cadherin and the recruitment of E-cadherin to adherens junction remain unchanged. In collective cell migration such as wound healing, cell-cell adhesion directly regulates cell migration; and increased calcium-dependent cell-cell adhesiveness upon CO-029 silencing is likely to be responsible for or at least contribute to the decreased collective migration or healing process of HT29-KD cells. While in solitary cell migration such as transwell cell migration, the increased surface presence of integrins α3β1 and α5β1 and upregulated activity of integrins α6β1 and/or α6β4 probably cause decreased migration onto extracellular matrices of HT29-KD cells.
For cell-matrix adhesion, the selectively increased adhesion on laminins but not on fibronectin upon CO-029 silencing suggests that CO-029 likely inhibits cell adhesion on laminins. Because laminins are the constituents of basement membranes that typically keep epithelial and endothelial cells in stationary, the role of CO-029 in inhibiting cell adhesion on laminins is consistent with its role in facilitating cell movement. The increased adhesion on laminin 111, which is ubiquitously expressed in epithelia, implies the activation of its receptors of integrins α6β1 and/or α6β4 because the surface levels of these integrins were not altered in HT29-KD cells, while increased adhesion on laminin 332, which is found mainly in skin, intestine, respiratory, and urinary epithelia, likely reflects the enhanced surface level of its receptor integrin α3β1, which is the major component of TEMs, and probably also the enhanced activity of its receptor integrin α6β4. Although the level of fibronectin-binding integrin α5β1 at the cell surface is upregulated upon CO-029 silencing, cell adhesion on fibronectin was not altered in HT29-KD cells, which is presumably due to a reduced functional activity of integrin α5 β 1 or lower levels and/or activities of other fibronectin-binding integrins like αV integrins. The altered levels of cell adhesion proteins at the cell surface upon CO-029 silencing could result from the altered endocytosis and/or recycling of these transmembrane proteins. In other words, CO-029 probably regulates the trafficking of these proteins through TEMs.
Since the levels of α6 integrins remain unaltered upon CO-029 silencing, the increased cell adhesion onto laminins observed in our study could result from the upregulated activity of integrin α6β4. Because HT-29 colon cancer cells form hemidesmosomes 
and integrin α6β4 is the only integrin in hemidesmosomes, the upregulated activity of integrin α6β4 may lead to the formation of stronger or more hemidesmosomes. In addition to the cell-matrix adhesive structures that can be visualized microscopically such as focal adhesions and hemidesmosomes, cell-matrix adhesion is also mediated by the direct engagement to matrices of the cell adhesion molecules that do not form microscopic adhesive structure. This type of cell-matrix adhesion mechanism is diffusely and evenly distributed at the interface of the basal plasma membrane of a cell and the underlying matrices, while focal adhesions and hemidesmosomes are spotted at this interface. Hence, the increased adhesiveness onto laminins could also reflect the upregulation in level or activity of the laminin-binding integrins α3β1, α6β1, and α6β4 that do not form microscopic adhesive structures.
Reduced cell adhesion at the early stage of metastasis helps release tumor cells from the primary tumor. Cell-cell adhesion needs to be disrupted so that invasive tumor cells can dissociate from the primary tumor and infiltrate interstitial tissue. Thus, cell-cell adhesion is an important factor in tumor cell invasiveness and metastasis 
. In addition, tumor cells need to reduce the adhesiveness on laminins to effectively breach or pass through various epithelium and endothelium basement membranes that tumor cells encounter during metastasis. Therefore, we extrapolate that CO-029 likely facilitates 1) the dispatch of tumor cells by reducing cell-cell adhesion at the early stage of metastasis, and 2) the infiltration, intravasation, and extravasation of tumor cells by confining the adhesion on laminins in the basement membranes of epithelium and endothelium. Moreover, CO-029 perturbs other molecules, such as CD44 and MelCAM, important for metastasis. Besides directly engaging cell-matrix and -cell adhesions, CD44 facilitates cancer progression and serves as a marker for cancer-initiating cells 
. CD44 could be one of the key molecules through which CO-029 promotes cell motility and cancer metastasis, especially given that CO-029 and CD44 form a complex in colorectal cancer cells and correlate with the progression of this cancer 
In summary, the cell migration capability is markedly diminished upon the silencing of CO-029. Because CO-029 regulates cell-cell and cell-matrix adhesions, the reduced motility of HT29-KD cells likely results from the deregulated cell adhesions. The altered cell-matrix adhesion is caused by the altered surface expression or activity of integrins and CD44, while the altered cell-cell adhesion is caused mainly by the malfunction of calcium-dependent cell-cell adhesion molecules (Figure S2
). Hence, CO-029 likely promotes the progression and metastasis of colorectal cancer by enhancing tumor cell movement and deregulating cell adhesions.