**2. Results**

*Advances in the Molecular Understanding of Colorectal Cancer*

illness [3, 4].

Colorectal cancer progression is driven by increasing or recurring growth of the primary carcinoma as well as hematogenic and lymphatic spread. For hematogenic spread, the liver is most important as it constitutes the first vascular bed in which disseminating CRC cells can be trapped after their dissemination. Hence, this organ is affected in up to 10–20% of CRC patients at the time of presentation. Another 20–25% will develop overt liver metastasis during the course of their

The main purpose of our experimental studies was first to develop a suitable model for investigating the efficacy of novel drugs [5–7]. One of the few wellcharacterized animal models for hepatic CRC benefits from the rat CC531 cell line. After injecting the cells, liver metastases develop and their growth has been frequently used for studying effects of various anti-cancer treatments [8–10].

The second aim was to identify genes, which are instrumental in the survival and metastasis formation of disseminated CRC cells. In addition, we reasoned that there are genes, which are necessary for the primary tumor as well as those, which are essential for metastasis initiation and formation. We furthermore hypothesized that the latter genes would be modulated in expression during the cells' colonization of the liver. Consequently, temporal changes in gene expression of CRC cells homing into the liver were investigated using an *in vivo* rat model, which is characterized by a definite metastatic proliferation-onset in rat liver after intra-portal inoculation of CC531 rat colorectal cancer cells. This model relies on the successful reisolation of CC531 cells at various time intervals after their injection into the mesenteric vein of syngeneic rats and allows exploring the chronological modulation of gene expression, from the very beginning of cancer cell homing into the liver to their final colonization of the whole organ. Based on this procedure, a cDNA microarray was performed to analyze gene expression profiles of several thousand genes in the reisolated CC531 cells. Upon analysis of microarray's data, candidates from gene families being significantly up- or down-regulated were chosen for further study by using different *in vitro* models. These candidate genes included claudins and insulin like-growth factor binding proteins. It was hoped that the emerging genes or their

products would be useful as target of a specific therapy or as a biomarker.

diagnostic and therapeutic approaches.

dins were proven to be useful as biomarkers [13, 14].

The National Institutes of Health defined a biomarker as "a characteristic that is objectively measured and evaluated as an indicator of normal biological processes, pathogenic processes, or pharmacologic responses to a therapeutic intervention [11]. From a therapeutic point of view, genome variations are recognized as the main cause of variable response to and side effects of drugs and a "one size fits all" approach is not the best solution any more. The individual's genetic and molecular makeup will be devoted to improve and develop more specific and "personal"

Claudins (CLDNs) are tight junction (TJ) proteins that serve an intercellular adhesion function. The aberrant expression of individual claudins is well documented in different stages of various human cancers [12]. In addition, some clau-

The insulin-like growth factor (IGF) axis attracts increasing attention since it is involved in several stages of cancer [15–17], and for its vital role in regulating cell survival and growth [18, 19] as well as the possible use of constituents of this axis as tumor and/or metastasis markers, which is becoming the focus of increasing

The insulin-like growth factors IGF-I and –II orchestrate their roles through the interaction with other members in this system, namely their receptors IGF-IR and -IIR, their binding proteins (IGFBPs) and the IGFBP proteases including matrix metalloproteinases (MMPs), cathepsins, and kallikreins [20]. Type I receptor mediates the growth promoting effects of IGFs [21], which are further modulated by 6 binding

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research activities.

### **2.1 Modulation of selected genes in reisolated CC531 tumor cells**

For identifying genes, which enable tumor cells to metastasize and colonize the liver, the CC531 cells were reisolated from rats, which had been implanted intraportally with these tumor cells. After various periods following tumor cell implantation, the CC531 cells were reisolated with a specific technique [33]. In subsequent experiments, these cells were used for mRNA and protein isolation and the mRNA screened by cDNA microarray and RT-PCR, and the proteins by Western blot.

As shown in **Table 1**, the microarray analysis revealed a significantly increased expression of insulin like growth factor binding proteins (*Igfbp3* and *Igfbp7)* and significantly decreased expression levels of claudins (*Cldn1* and *Cldn4*) in the beginning of liver colonization (days 3 and 6 after tumor implantation). These results were further confirmed by RT-PCR (for all four genes) and Western blot (for the two claudins and igfbp7) (**Figure 1**).

#### **2.2 Effects of genes' knockdown in colorectal cancer cells**

To investigate the knockdown effect(s) of each gene on various functions of colorectal cancer cell lines, siRNA experiments for transient knockdown were performed.


*The day of tumor cell implantation was counted day 0.*

*b The number denotes the fold change in expression versus an in vitro control.*

#### **Table 1.**

*Gene expression profiles from members of two gene families, chosen from the microarray analysis of reisolated CC531 cells.*

#### **Figure 1.**

*Expression of Igfbp3, Igfbp7, cldn1, and cldn4 in reisolated CC531 cells. (A and C): Expression of Igfbp3 and Igfbp7 (A) and of Cldn1 and Cldn4 (C) in reisolated CC531 cells as shown by RT-PCR compared to the expression of the housekeeping gene γ-tubulin. (B and D): Expression of the proteins IGFBP7 (B), CLDN1 and CLDN4 (D) in the reisolated CC531 cells as shown by western blot compared to the expression of ERK2 loading control. 1st lane: CC531 cells (control); 2nd, 3rd, 4th, 5th and 6th lanes: CC531 cells (reisolated from rat livers after 3, 6, 9, 14 and 21 days, respectively); 7th and 8th lanes: CC531 cells (reisolated after 21 days and cultured in vitro for further 14 and 22 days, respectively).*

Colorectal cancer cells (CC531, Caco2 or SW480) cultured in 6-well-plates were transfected with specific siRNA (200 nM) or negative control using Lipofectamine 2000 (Invitrogen) following the manufacturer's instructions. The cells were harvested at 24, 48 and 72 h after treatment.

As shown for overview in **Table 2** (for details see **Figures 2**–**4**), knockdown of *igfbp3* or *igfbp7* was induced in cell lines with relevant expression only and caused significantly reduced proliferation rates (**Figure 2A–C**). Similarly, colony formation (**Figure 3A**–**C**) of CRC cells was diminished. Finally, cell migration was reduced in SW480 cells (**Figure 4B**), but not in CC531 (**Figure 4A**) and Caco2 (**Figure 4C**) cells.

Interestingly, different effects were noticed after *cldn1* or *cldn4* knockdown in CC531 cells. No significant effect on cell proliferation was observed, while a significant inhibition of colony formation and significant stimulation of cell migration resulted from the siRNA knockdown of each claudin (**Table 2**).


*↓\* and ↑\* denote significant (p < 0.05) inhibition or stimulation of the investigated cell function (proliferation, migration or colony formation), respectively; ns denotes a nonsignificant effect.*

#### **Table 2.**

*Overview of the siRNA knockdown effects of insulin like growth factor binding proteins 3 and 7 (Igfbp3 and 7) and claudins 1 and 4 (cldn1 and 4) on cellular functions (cell proliferation, migration and colony formation) of colorectal cancer cell lines (SW480, Caco2 and CC531).*

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**Figure 2.**

*formation.*

*Experimental Results Help Shape the Development of Personalized Medicine...*

*Effects of Igfbp7 or Igfbp3 knockdown on proliferation of colorectal cancer cells. (A) Reduced proliferation of rat CC531 colorectal cancer cells after si.Igfbp7 treatment. (B) Reduced proliferation of human SW480 colorectal cancer cells after si.Igfbp3 treatment. (C) Reduced proliferation of human Caco2 colorectal cancer cells after si.Igfbp7 treatment. Data (n = 3) are shown as means ± S.D. in percentage of nonsensetreated cells. Asterisk (\*) denotes a significant difference to controls (p < 0.05). Abbreviations: CF; colony* 

*DOI: http://dx.doi.org/10.5772/intechopen.80752*

*Experimental Results Help Shape the Development of Personalized Medicine... DOI: http://dx.doi.org/10.5772/intechopen.80752*

#### **Figure 2.**

*Advances in the Molecular Understanding of Colorectal Cancer*

harvested at 24, 48 and 72 h after treatment.

*in vitro for further 14 and 22 days, respectively).*

from the siRNA knockdown of each claudin (**Table 2**).

*migration or colony formation), respectively; ns denotes a nonsignificant effect.*

*of colorectal cancer cell lines (SW480, Caco2 and CC531).*

(**Figure 4C**) cells.

**Figure 1.**

Colorectal cancer cells (CC531, Caco2 or SW480) cultured in 6-well-plates were transfected with specific siRNA (200 nM) or negative control using Lipofectamine 2000 (Invitrogen) following the manufacturer's instructions. The cells were

*Expression of Igfbp3, Igfbp7, cldn1, and cldn4 in reisolated CC531 cells. (A and C): Expression of Igfbp3 and Igfbp7 (A) and of Cldn1 and Cldn4 (C) in reisolated CC531 cells as shown by RT-PCR compared to the expression of the housekeeping gene γ-tubulin. (B and D): Expression of the proteins IGFBP7 (B), CLDN1 and CLDN4 (D) in the reisolated CC531 cells as shown by western blot compared to the expression of ERK2 loading control. 1st lane: CC531 cells (control); 2nd, 3rd, 4th, 5th and 6th lanes: CC531 cells (reisolated from rat livers after 3, 6, 9, 14 and 21 days, respectively); 7th and 8th lanes: CC531 cells (reisolated after 21 days and cultured* 

As shown for overview in **Table 2** (for details see **Figures 2**–**4**), knockdown of *igfbp3* or *igfbp7* was induced in cell lines with relevant expression only and caused significantly reduced proliferation rates (**Figure 2A–C**). Similarly, colony formation (**Figure 3A**–**C**) of CRC cells was diminished. Finally, cell migration was reduced in SW480 cells (**Figure 4B**), but not in CC531 (**Figure 4A**) and Caco2

Interestingly, different effects were noticed after *cldn1* or *cldn4* knockdown in CC531 cells. No significant effect on cell proliferation was observed, while a significant inhibition of colony formation and significant stimulation of cell migration resulted

**Target gene Cell proliferation Cell migration Colony formation**

*↓\* and ↑\* denote significant (p < 0.05) inhibition or stimulation of the investigated cell function (proliferation,* 

*Overview of the siRNA knockdown effects of insulin like growth factor binding proteins 3 and 7 (Igfbp3 and 7) and claudins 1 and 4 (cldn1 and 4) on cellular functions (cell proliferation, migration and colony formation)* 

*Igfbp3* (in SW480) ↓**\*** ↓**\*** ↓**\*** *Igfbp7* (in CC531 or Caco2) ↓**\*** ns ↓**\*** *Cldn1* (in CC531) ns ↑**\*** ↓**\*** *Cldn4* (in CC531) ns ↑**\*** ↓**\***

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**Table 2.**

*Effects of Igfbp7 or Igfbp3 knockdown on proliferation of colorectal cancer cells. (A) Reduced proliferation of rat CC531 colorectal cancer cells after si.Igfbp7 treatment. (B) Reduced proliferation of human SW480 colorectal cancer cells after si.Igfbp3 treatment. (C) Reduced proliferation of human Caco2 colorectal cancer cells after si.Igfbp7 treatment. Data (n = 3) are shown as means ± S.D. in percentage of nonsensetreated cells. Asterisk (\*) denotes a significant difference to controls (p < 0.05). Abbreviations: CF; colony formation.*

#### **Figure 3.**

*Effects of Igfbp7 or Igfbp3 knockdown on colony formation of colorectal cancer cells. (A) Inhibited colony formation of rat CC531 colorectal cancer cells after si.Igfbp7 treatment. (B) Inhibited colony formation of human SW480 colorectal cancer cells after si.Igfbp3 treatment. (C) Inhibited colony formation of human Caco2 colorectal cancer cells after si.Igfbp7 treatment. Data (n = 3) are shown as means ± S.D. in percentage of nonsense-treated cells. Asterisk (\*) denotes a significant difference to controls (p < 0.05). Abbreviations: CF; colony formation.*

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**Figure 4.**

*Experimental Results Help Shape the Development of Personalized Medicine...*

*Effects of Igfbp7 or Igfbp3 knockdown on migration of colorectal cancer cells. (A) Migration of rat CC531 colorectal cancer cells after si.Igfbp7 treatment. (B) Reduced migration of human SW480 colorectal cancer cells after si.Igfbp3 treatment. (C) Migration of human Caco2 colorectal cancer cells after si.Igfbp7 treatment. Data (n = 3) are shown as means ± S.D. in percentage of nonsense-treated cells. Asterisk (\*) denotes a significant* 

*difference to controls (p < 0.05). Abbreviations: CF; colony formation.*

*DOI: http://dx.doi.org/10.5772/intechopen.80752*

*Experimental Results Help Shape the Development of Personalized Medicine... DOI: http://dx.doi.org/10.5772/intechopen.80752*

#### **Figure 4.**

*Advances in the Molecular Understanding of Colorectal Cancer*

**24**

**Figure 3.**

*colony formation.*

*Effects of Igfbp7 or Igfbp3 knockdown on colony formation of colorectal cancer cells. (A) Inhibited colony formation of rat CC531 colorectal cancer cells after si.Igfbp7 treatment. (B) Inhibited colony formation of human SW480 colorectal cancer cells after si.Igfbp3 treatment. (C) Inhibited colony formation of human Caco2 colorectal cancer cells after si.Igfbp7 treatment. Data (n = 3) are shown as means ± S.D. in percentage of nonsense-treated cells. Asterisk (\*) denotes a significant difference to controls (p < 0.05). Abbreviations: CF;* 

*Effects of Igfbp7 or Igfbp3 knockdown on migration of colorectal cancer cells. (A) Migration of rat CC531 colorectal cancer cells after si.Igfbp7 treatment. (B) Reduced migration of human SW480 colorectal cancer cells after si.Igfbp3 treatment. (C) Migration of human Caco2 colorectal cancer cells after si.Igfbp7 treatment. Data (n = 3) are shown as means ± S.D. in percentage of nonsense-treated cells. Asterisk (\*) denotes a significant difference to controls (p < 0.05). Abbreviations: CF; colony formation.*
